# Cyclic nucleotide phosphodiesterases as drug targets

Michy Kelly, Viacheslav Nikolaev, Leila Gobejishvili, Claire Lugnier, Christian Hesslinger, Peter Nickolaus, David Kass, Walma Pereira de Vasconcelos, Rodolphe Fischmeister, Stefan Brocke, et al.

► To cite this version:

Michy Kelly, Viacheslav Nikolaev, Leila Gobejishvili, Claire Lugnier, Christian Hesslinger, et al. Cyclic nucleotide phosphodiesterases as drug targets. Pharmacological Reviews, 2025, pp.100042. 10.1016/j.pharmr.2025.100042. hal-04906722

HAL Id: hal-04906722

https://hal.science/hal-04906722v1

Submitted on 22 Jan 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
# Cyclic nucleotide phosphodiesterases as drug targets

## Authors list

Michy P. Kelly¹, Viacheslv O. Nikolaev², Leila Gobejishvili³, Claire Lugnier⁴, Christian Hesslinger⁵, Peter Nickolaus⁵, David A. Kass⁶, Walma Pereira De Vasconcelos⁷, Rodolphe Fischmeister⁷, Stefan Brocke⁸, Paul M. Epstein⁹, Gary Piazza¹⁰, Adam B. Keeton¹⁰, Gang Zhou¹¹, Mohammad Abdel-Halim¹², Ashraf H. Abadi¹², George S. Baillie¹³, Mark Giembycz¹⁴, Graeme Bolger¹⁵, Gretchen Snyder¹⁶, Kjetil Tasken¹⁷,¹⁸, Nathaniel E.B. Saidu¹⁷, Martina Schmidt¹⁹,²⁰, Manuela Zaccolo²¹, Ralph T. Schermuly²², Hengming Ke²³, Rick H. Cote²⁴, Soroush Mohammadi Jouabadi²⁵, Anton J.M. Roks²⁵.

*All authors contributed equally

## Affiliations:

1. Department of Neurobiology, Center for Research on Aging, University of Maryland School of Medicine, Baltimore, USA.

2. Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Germany.

3. Department of Physiology, School of Medicine, University of Louisville, Kentucky, USA

4. Translational CardioVascular Medicine, CRBS, UR 3074, 1 rue Eugène Boeckel, 67000 Strasbourg, France

5. Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach an der Riss, Germany

6. Division of Cardiology, Department of Medicine; Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

7. Université Paris-Saclay, Inserm, Signaling and Cardiovascular Pathophysiology, UMR-S 1180, Orsay, France

8. Department of Immunology, UConn Health, Farmington, CT, USA

9. Department of Cell Biology, UConn Health, Farmington, CT, USA

10. Department of Drug Discovery and Development, Harrison College of Pharmacy, Auburn University, Auburn, USA

11. Georgia Cancer Center, Augusta University, Augusta, USA

12. Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt

13. School of Cardiovascular and Metabolic Health, University of Glasgow, Glasgow, UK

14. Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

15. BZI Pharma LLC, Birmingham, AL 35203-1872, USA.

16. Molecular Neuropharmacology, Intra-Cellular Therapies Inc (ITI), New York, NY, USA

17. Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo Norway
18. Institute of Clinical Medicine, University of Oslo, Oslo, Norway

19. Department of Molecular Pharmacology, University of Groningen, Groningen, the Netherlands

20. Groningen Research Institute for Asthma and COPD, GRIAC, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands

21. Department of Physiology, Anatomy and Genetics and National Institute for Health and Care Research Oxford Biomedical Research Centre, University of Oxford, Oxford, UK

22. Department of internal Medicine, Justus Liebig University of Giessen, Giessen, Member of the German Center for Lung Research (DZL), Germany

23. Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, North Carolina, USA

24. Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA

25. Section of Vascular and Metabolic Disease, Department of Internal Medicine, Erasmus University Medical Center, Erasmus University Rotterdam, Rotterdam, The Netherlands

**Corresponding author:**

Dr. Anton J.M. Roks

Email: a.roks@erasmusmc.nl

Address: EE14, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands

Telephone number: 010 704 0704

**Keywords:** cyclic nucleotide phosphodiesterase, PDE inhibitors, disease, drug development, pathophysiology.

\#Pages: 151 (including references), \#Body word count:44,960 \#Figures: 18, \#Tables:3, \#Abstract word count: 225
Table of content

<table><tr><td>ABSTRACT</td></tr><tr><td>SIGNIFICANCE STATEMENT</td></tr></table>

## I. CHAPTER 1: GENERAL INTRODUCTION

1.1. Overview

1.2. Family organization & evolutionary perspective

1.2.1. PDE1 family

1.2.2. PDE2 family

1.2.3. PDE3 family

1.2.4. PDE4 family

1.2.5. PDE5 family

1.2.6. PDE6 family

1.2.7. PDE7 family

1.2.8. PDE8 family

1.2.9. PDE9 family

1.2.10. PDE10 family

1.2.11. PDE11 family

1.3. Structure-Activity relationship

1.3.1. Regulation of catalytic domain

1.3.2. Allosteric mechanisms for regulating cyclic nucleotide hydrolysis by PDEs

1.3.3. Peptide inhibitors, regulators, and activators

1.4. Role of PDEs in cyclic nucleotide compartmentalization

1.4.1. cAMP compartments: the birth of a new rationale

1.4.2. Organization in signalosomes and participation of PDE

1.4.3. Size matters, when measuring cAMP in nanodomains

## II. CHAPTER 2: PHYSIOLOGY AND CLINICAL DEVELOPMENT

2.1. Modulation of cardiac function by PDEs

2.1.1. Introduction (pathophysiology of the heart related to cyclic nucleotide Signalling

2.1.2. PDE regulation of cardiac function through cAMP

2.1.3. PDE regulation of cardiac function through cGMP

2.2. Smooth muscle cells, endothelial cells, and PDEs

2.2.1. Clinical and pathophysiological background of vascular disease

2.2.2. Role of PDEs in endothelial permeability and angiogenesis

2.2.3. Role of PDEs in arterial wall remodeling and vascular tone

2.3. PDE inhibitors for pulmonary diseases

2.3.1. Pulmonary hypertension

2.3.2. Role of PDEs in the pathophysiology and as drug targets in PH

2.3.3. Pulmonary fibrosis: epidemiology of pulmonary fibrosis

2.3.4. Role of PDEs in the pathophysiology and as drug targets in pulmonary fibrosis

2.3.5. Asthma and COPD

2.3.6. Hybrid Inhibitors for Asthma and COPD

2.3.7. Bifunctional Ligands for COPD

2.4. Liver disorders and PDEs

2.4.1. The role of cyclic nucleotides in liver fibrosis and cirrhosis pathophysiology

2.4.2. Role of cAMP-PDE

2.4.3. cGMP and dual substrate PDE

2.5. Neurodevelopmental Disorders
<table><tr><td>2.5.1.</td><td>PDE Isoforms, Cognitive impairment and Alzheimer</td></tr><tr><td>2.5.2.</td><td>PDE Isoforms, Dopamine Signaling, and Disease: Implications for Treatment</td></tr><tr><td>2.6.</td><td>Cancer and inflammation</td></tr><tr><td>2.6.1.</td><td>Effects in neoplastic cells</td></tr><tr><td>2.6.2.</td><td>Preclinical and clinical studies: cGMP PDE inhibition and anticancer activity</td></tr><tr><td>2.7.</td><td>The role of PDEs in immune regulation</td></tr><tr><td>2.7.1.</td><td>General background</td></tr><tr><td>2.7.2.</td><td>PDEs in the tumor immune microenvironment</td></tr><tr><td>2.7.3.</td><td>The role of PDEs in cells of the innate and adaptive immune systems</td></tr><tr><td>2.7.4.</td><td>The role of PDEs in specific immune cell types</td></tr></table>

III. CHAPTER 3: GENERAL PERSPECTIVES AND FUTURE DIRECTIONS

<table><tr><td>3.1.</td><td>Toward drug specificity</td></tr><tr><td>3.2.</td><td>Targeting specific driver mutations</td></tr><tr><td>3.3.</td><td>In Conclusion</td></tr></table>
**Abstract**

Cyclic nucleotides are synthesized by adenylyl and/or guanylyl cyclase, and downstream of this synthesis the cyclic nucleotide phosphodiesterase families (PDEs) specifically hydrolyze cyclic nucleotides. PDEs control cAMP and cGMP intracellular levels by mediating their quick return to the basal steady state levels. This often takes place in subcellular nanodomains. Thus, PDEs govern short-term protein phosphorylation, long-term protein expression, and even epigenetic mechanisms by modulating cyclic nucleotide levels. Consequently, their involvement in both health and disease is extensively investigated. PDE inhibition has emerged as a promising clinical intervention method, with ongoing developments aiming to enhance its efficacy and applicability. In this comprehensive review, we extensively look into the intricate landscape of PDEs biochemistry, exploring their diverse roles in various tissues. Furthermore, we outline the underlying mechanisms of PDEs in different pathophysiological conditions. Additionally, we review the application of PDE inhibition in related diseases, shedding light on current advancements and future prospects for clinical intervention.

**Significance Statement**

Regulating PDEs is a critical checkpoint for numerous (patho)physiological conditions. However, despite the development of several PDE inhibitors aimed at controlling over-activated PDEs, their applicability in clinical settings poses challenges. In this context, our focus is on pharmacodynamics and the structure-activity of PDEs, aiming to illustrate how selectivity and efficacy can be optimized. Additionally, we point to current preclinical and clinical evidence that depicts various optimization efforts and indications.
american college of gastroenterology (ACG), age-related cognitive decline (ACRD), adenylyl cyclases (ACs), alzheimer's disease (AD), aD-related dementias (ADRD), alcoholic hepatitis (AH), aryl hydrocarbon receptor (AhR), aryl interacting protein-like 1(AIPL1), a-kinase anchoring protein (AKAP), a kinase anchoring proteins (AKAPs), alcohol-associated liver disease (ALD), alcohol-associated steatohepatitis (ASH), airways smooth muscle (ASM), alcohol use disorder (AUD), amyloid beta (Aβ), bronchoalveolar lavage (BAL), basic fibroblast growth factor (bFGF), brain natriuretic peptide (BNP), cyclic adenosine mono phosphate (cAMP), calmodulin-PDE (CaM-PDE), cAMP binding domain (CBD), cyan fluorescent protein (CFP), cGMP-dependent kinase-1a (cGK-1a (also known as PKA1a)), cyclic guanosine mono phosphate (cGMP), cyclic GMP-stimulated PDE (cGS-PDE), central nervous system (CNS), chronic obstructive pulmonary disease (COPD), non-cyclooxygenase (COX), colorectal cancer (CRC), connecting tissue growth factor (CTGF), dopamine (DA), damage-associated molecular patterns (DAMPs), dendritic cell (DC), deubiquitinase-targeting chimeras (DUBTACS), early asthmatic response (EAR), endothelial cells (EC), extracellular matrix proteins (ECM), extracellular signal-regulated kinase (ERK), epithelial surface liquid (ESL), familial adenomatous polyposis (FAP), food and drug administration (FDA), forced expiratory volume in 1 second (FEV1), fibronectin (FN), Fluorescence or Förster Resonance Energy Transfer (FRET), forced vital capacity (FVC), guanylyl cyclase-1 (GC-1), global initiative for asthma (GINA), global initiative for obstructive lung diseases (GOLD), g-protein coupled receptor (GPCR), human chromosome location (hcl), huntington's disease (HD), heart failure (HF), human fetal lung fibroblasts (HFL-1), heart failure with preserved ejection fraction (HFpEF), heart failure and reduced ejection fraction (HFrEF), high frequency stimulation (HFS), head and neck squamous cell carcinoma (HNSCC), Hepatic stellate cells (HSC), hypertension with brachydactyly or Bilginturan syndrome (HTNB), human umbilical vein endothelial cell (HUVEC), isobutyl-methylxanthine (IBMX), inducible cAMP early repressor (ICER), immune checkpoint inhibition (ICI), Inhaled corticosteroid (ICS), immunoglobulin (Ig), interstitial lung diseases (ILDs), idiopathic pulmonary fibrosis (IPF), intra-cellular therapies Inc (ITI), kirsten rat sarcoma virus (KRAS), long-acting β2-adrenoceptor agonist (LABA), long-acting muscarinic receptor antagonist (LAMA), L-DOPA-induced dyskinesias (LIDs), long non-coding RNAs (lncRNA), long-term depression (LTD), long-term potentiation (LTP), left ventricular (LV), mild cognitive impairment (MCI), myeloid-derived suppressor cells (MDSCs), microRNAs (miRNA), myosin light chain kinase (MLCK), myosin light chain phosphatase (MLCP), matrix metalloproteinase (MMP), medium spiny neuron (MSN), metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), nuclear factor of activated T cells (NFAT), normal human lung fibroblasts (NHLFs), n-terminal hydrophobic membrane association regions (NHRs), natural killer (NK), nitric oxide (NO), natriuretic peptides (NPs), nonsteroidal anti-inflammatory drug (NSAID), non-small cell lung cancer (NSCLC), pulmonary arterial hypertension (PAH), per-Ant-Sim (PAS), human peripheral blood mononuclear cells (PBMCs), parkinson's disease (PD), phosphodiesterase (PDE), positron emission tomography (PET), prostaglandins (PG), prostaglandin E2 (PGE2), pulmonary hypertension (PH), protein Kinase A (PKA), protein kinase B (PKB), protein kinase G (PKG), popeye domain-containing (POPDC), progressive pulmonary fibrosis (PPF), protein-protein interactions (PPIs), phosphatases (PPs), proteolysis targeting chimeras (PROTACs), pentoxifylline (PTX), pulmonary vascular resistance (PVR), response Regulator Receiver (REC), reactive oxygen species (ROS), right ventricle (RV), type 2 ryanodine receptor (RyR2), short-acting β2-adrenoceptor agonist (SABA), senescence
associated secretory phenotype (SASP), small interfering RNA (siRNA), single nucleotide polymorphism (SNP), sarco-/endoplasmic reticulum (SR), thoracic aortic constriction (TAC), T cell receptor (TCR), transforming growth factor β1 (TGFβ1), tyrosine hydroxylase (TH), tumor microenvironment (TME), tumor necrosis factor (TNF), T regulatory cells (Treg), transient receptor potential channel 3 (TRPC3), upstream conserved regions 1 and 2 (UCR1 and UCR2), vascular endothelial growth factor (VEGF), vascular smooth muscle (VSMC), world health organization (WHO), yellow fluorescent protein (YFP), alpha smooth muscle actin (αSMA), β-adrenoceptor (β-ARs).
# CHAPTER 1: GENERAL INTRODUCTION

Downstream of transmembrane receptors, intracellular signaling plays a major role in governing normal and pathological cell responses. The intracellular second messenger cyclic nucleotide cascades, driven by cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP), are conserved among species and ubiquitously expressed throughout mammalian tissues. Their involvement in many disease processes has led to enzymes within these cascades being considered important drug targets. Earl W. Sutherland (Sutherland, 1972), Nobel laureate in 1971, showed how cAMP is a second messenger (Sutherland and Rall, 1960), is inactivated by phosphodiesterases (PDEs) by hydrolysis of 5'-adenosine monophosphate (5'-AMP) (Butcher and Sutherland, 1962). Ashman and colleagues showed the same for cGMP (Ashman et al., 1963).

PDEs act in the presence of H₂O and Mg²⁺, and hydrolyze the phosphate bond present 3' in both cAMP and cGMP, producing H⁺ (Keravis et al., 2005). Thus, PDEs control the intracellular levels of cAMP (cAMP-PDE) and cGMP (cGMP-PDE) by rapidly catalyzing their inactivation and returning levels to the basal or resting state. PDEs as a result control short-term protein phosphorylation, long-term protein expression and even epigenetic modifications (Abusnina et al., 2011). The role of cyclic nucleotides and PDEs in health and disease is widely investigated and has a long history (Figure 1). In this article, we review the role of PDEs in various tissues (Chapter 1), the utility of PDE inhibitors in the treatment of various diseases (Chapter 2) and discuss considerations for future clinical applications (Chapter 3).

## 1.1 | Overview

Cyclic nucleotide PDEs are encoded by 11 gene families (PDE1 to PDE11), giving rise a large and complex number of proteins. Each family includes 1 to 4 distinct genes (a total of 21 in mammals) that produce more than 100 different proteins or isoenzymes (for reviews, see (Ahmad et al., 2015; Azevedo et al., 2014; Conti and Beavo, 2007; Keravis and Lugnier, 2012; Maurice et al., 2014). PDEs were classified as a superfamily of metalo-phosphohydrolases and assigned the number EC 3.1.4.17.

PDEs generally exist as dimers. Each monomer of the dimer has a common structure composed of three distinct domains: i) the N-terminal regulatory domain; this characterizes each family and its variants; ii) the catalytic domain; this consists of about 340 amino acids and is relatively conserved among PDEs (~78% amino acid identity); and iii) the C-terminal domain, which can be prenylated or phosphorylated (Anant et al., 1992; Dousa, 1999; Qin et al., 1992) (Figure 2).

The multiplicity of biochemical and structural properties of PDEs, contributes to their tissue specificities, cellular and subcellular distributions. The possibility of designing (relatively) disease-specific pharmacotherapy is an attractive feature of PDE as a drug target, but also a challenge for pharmaceutical chemists (Figure 1).

## 1.2 | Family organization & evolutionary perspective

In the early years of PDE research (1980-1995), before an official nomenclature was established, PDEs were isolated from various tissues by chromatography. Their biochemical characteristics were determined according to the substrate hydrolyzed and how they were regulated (Keravis et al., 2005; Keravis et al., 1980; Thompson and Appleman, 1971) and PDEs were named accordingly, eg. cGMP-stimulated PDE (cGS-PDE) and cAMP-PDE, cGMP-inhibited PDE (cGI-PDE), (Lugnier and Schini, 1990); CaM-activated PDE (Lugnier et al., 1986); rolipram-inhibited PDE (ROI-PDE) (Komas et al., 1989) etc. To avoid confusion an official nomenclature system based on the human genome was developed in 1995 (Beavo, 1995), creating a unique descriptor for each
PDE. For example: PDE4D7 indicates a 3',5'-cyclic nucleotide PDE of the PDE4 gene family, gene D, splice variant 7. We now discuss the PDEs accordingly.

### 1.2.1 | PDE1 family

PDE1, initially named CaM-PDE, represents the sole family that is Ca²⁺-dependently regulated via CaM (a 16 kDa Ca²⁺-binding protein complexed with four Ca²⁺ ions). The PDE1 isoenzyme family is encoded by three genes: *PDE1A* (mapped on human chromosome 2q32), *PDE1B* (human chromosome location (hcl): 12q13) and *PDE1C* (hcl: 7p14.3). More than 10 human isoforms have been identified with molecular weights that vary from 58 to 86 kDa per monomer. The *N*-terminal regulatory domain contains two Ca²⁺/CaM-binding domains and two phosphorylation sites that modulate the biochemical activity of these enzymes (Figure 2). PDE1A and PDE1B preferentially hydrolyze cGMP, whereas PDE1C hydrolyzes cAMP and cGMP with similar *K*<sub>m</sub> values. Phosphorylation of PDE1A1 (59 kDa) and PDE1A2 (61 kDa) by PKA, and phosphorylation of PDE1B1 by CaM kinase II, decreases their sensitivity to Ca²⁺ and CaM and, thereby, reduces PDE1 activity (Zhao et al., 1997). PDE1 isoenzymes are mainly cytosolic, although PDE1A has been found in the nucleus where it contributes to the regulation of transcription factor activity and epigenetic control of gene transcription (Abusnina et al., 2011). Very few selective PDE1 inhibitors are available. To be effective, PDE1 inhibitors must inhibit both basal- and CaM-activated activity. Nimodipine was the first compound with this property, acting in the micromolar range (Epstein et al., 1982; Keravis and Lugnier, 2012). Dioclein followed, showing to relax human saphenous vein (Goncalves et al., 2009). Nowadays, IC86340 (PDE1A/PDE1C) and lenrispodun (ITI-214) are available as selective inhibitors with submicromolar potency (Maurice et al., 2014; Wennogle et al., 2017).

### 1.2.2 | PDE2 family

The PDE2 family, formerly cGS-PDE (Martins et al., 1982a; Yamamoto et al., 1983), consists of a single gene (hcl: 11q 13.4) that produces three splice variants of dual cAMP and cGMP hydrolyzing enzymes: cytosolic PDE2A1 (Sonnenburg et al., 1991) and membrane bound PDE2A2 and PDE2A3 (Rosman et al., 1997). The *N*-termini direct these iso-enzymes to their different subcellular locations. The *N*-terminal domain has two cGMP-binding domains, GAF-A and GAF-B (Figure 2). The GAF-A domain mediates PDE2 dimerization. GAF-B binds cGMP allosterically (1–5 µM), positively stimulating cAMP hydrolysis up to a 30-fold with *K*<sub>m</sub> values from 10–30 µM (Lugnier and Schini, 1990). EHNA (IC<sub>50</sub>= 2 µM) was the first PDE2 inhibitor (Duncan et al., 1982; Masood et al., 2009; Méry et al., 1995; Podzuweit et al., 1995). Later Bay 60-7550 (IC<sub>50</sub> = 4.7 nM) was discovered, effective only against cGMP-activated PDE2 (Boess et al., 2004; Masood et al., 2009). PDE2 inhibitor ND7001 was patented (WO2004041258-A2), and inhibits both basal- and cGMP-stimulated PDE2 (Masood et al., 2009), putatively the most effective tool to inspect the role of PDE2 in cellular signaling (Lueptow et al., 2016).

### 1.2.3 | PDE3 family

The PDE3 family, formerly cGI-PDE, is encoded by 2 genes: *PDE3A* (hcl: 12p12) and *PDE3B* (hcl: 11p15.1). Three variants are expressed for PDE3A: *PDE3A1* (136 kDa), *PDE3A2* (118 kDa) and *PDE3A3* (94 kDa), whereas only a single *PDE3B1* (137 kDa) variant has been identified, despite PDE3B of various sizes having been reported. PDE3 hydrolyses both cAMP and cGMP, and a unique 44-amino acid insert in the catalytic domain is present which is different between PDE3A and PDE3B (Ahmad et al., 2015) (Figure 2). The *V*<sub>max</sub> for cAMP hydrolysis is 10-fold higher than for cGMP hydrolysis. cGMP has a higher affinity for PDE3 than cAMP, and is a competitive inhibitor of cAMP hydrolysis (Lugnier, 2006). Accordingly, PDE3 participates in
cAMP/cGMP cross talk (Lugnier et al., 1999a). The PDE3 variants possess *N*-terminal hydrophobic membrane association regions (NHRs) allowing the targeting of PDE3 to various specific intracellular domains (Keravis and Lugnier, 2012). PDE3 is located in the cardiac sarcoplasmic reticulum (SR) (Lugnier et al., 1993), cardiac nuclear envelope, near nucleopore complexes (Lugnier et al., 1999b), and in liver Golgi endosomal fraction (Geoffroy et al., 2001). A PKA phosphorylation site is present in PDE3A1 and PDE3A2 for activation, acting as a negative feedback for cAMP signaling, whereas an Akt/protein kinase B (PKB) phosphorylation site is only present on PDE3A1 (Wechsler et al., 2002) to promote 14-3-3-protein binding and inhibit phosphatase-catalyzed inactivation (Palmer et al., 2007). PKB-dependent phosphorylation also activates PDE3B. PDE3A3 lacks PKA and PKB phosphorylation sites. PKC phosphorylates and activates PDE3A (Pozuelo Rubio et al., 2005).

The first potent and selective inhibitor described of PDE3 was cilostamide (Hidaka et al., 1979), and showed potential for application in cardiac disease. This led to the development of amrinone, milrinone and enoximone. Milrinone (IC₅₀ = 2.1 µM) was the first PDE3 inhibitor developed to improve myocardial contraction in heart failure, and safe for short term use, avoiding death by arrhythmia after chronic use (Lanfear et al., 2009). Another PDE3 inhibitor, cilostazol (IC₅₀ = 0.2 µM) has been approved by the United States Food and Drug Administration (FDA) for the treatment of intermittent claudication and is marketed as *Pletal*® (Real et al., 2018).

### 1.2.4 | PDE4 family

The cAMP-selective PDE4 family is arguably the most studied family. The more than 25 human isoforms (50-125 kDA) (Paes et al., 2021b) are encoded by four genes: *PDE4A* (hcl: 19p13.2), *PDE4B* (hcl: 1p31), *PDE4C* (hcl: 19p13.1) and *PDE4D* (hcl: 5p12). These genes feature different promoters and are subject to alternative mRNA splicing. PDE4 variants contain two unique stretches of amino acids called upstream conserved region (UCR) 1 and UCR2 (Beard et al., 2000) (Figure 2). The so-called “long” PDE4 isoforms contain both UCR1 and UCR2, whereas the “short” isoforms are *N*-terminally truncated and contain only UCR2, and even shorter (“super-short”) isoforms lack either a portion or the entirety of the UCR2 element (Kyurkchieva and Baillie, 2023; Paes et al., 2021b). Allosteric PDE4 activity regulation is detailed in Chapter 1.3.2. The catalytic region contains an extracellular signal-regulated kinase (ERK) phosphorylation site for activation of PDE4 short forms and inhibition of PDE4 long forms (Baillie et al., 2000; Houslay, 2001). Long-form PDE4 variants containing UCR1 and UCR2 can form dimers, which involves the UCRs (Bolger et al., 1993; Bolger et al., 2015). PDE4 variants are localized to specific subcellular nanodomains by A-kinase anchoring proteins (AKAP) (Dodge et al., 2001). PDE4s interact directly with many other intracellular proteins, thereby defining the PDE4 interactome.

Rolipram (IC₅₀ = 5 µM) and Ro 20-1724 (IC₅₀ = 18 µM) were the first inhibitors shown to be highly selective for PDE4 (Lugnier et al., 1986; Reeves et al., 1987). Thereafter, pharmaceutical companies synthesized many PDE4 inhibitors, particularly, as anti-inflammatory agents (Houslay et al., 2005; Warren et al., 2023). Some of these inhibitors have been approved for medicinal use and are described in Chapter 2.

### 1.2.5 | PDE5 family

PDE5 specifically hydrolyses cGMP and is encoded by one gene *PDE5A* (hcl: 4q27) with three variants being expressed: PDE5A1 (100 kDa), PDE5A2 (95 kDa) and PDE5A3 (95 kDa). Their *N*-termini contain tandem GAF-A and GAF-B domains (Figure 2). Only the GAF-A domain binds cGMP (Turko et al., 1998), promoting protein kinase G (PKG)-catalyzed PDE5 phosphorylation (Nakamura et al., 2018a). This prompts catalytic activity and increases cGMP-binding affinity converting PDE5 (see Chapter 2.1.3 for further details). Zaprinast (M&B 22948) was the first PDE5 inhibitor described (IC₅₀ = 0.4 µM) (Lugnier et al., 1986), followed by sildenafil (*Viagra*®), the first
clinically used oral PDE5 inhibitor ($IC_{50}$ = 4 nM) (Ballard et al., 1998; Boolell et al., 1996). Zaprinast also inhibits PDE9 with an $IC_{50}$ value of 35 µM (Fisher et al., 1998), and sildenafil's $IC_{50}$ is only 10–80-fold lower for PDE5 than for PDE1 and PDE6, respectively. More selective PDE5 inhibitors are vardenafil, tadalafil and avanafil (Andersson, 2018). However, tadalafil has an affinity for PDE11 that is about 20-times lower than for PDE5 (Weeks et al., 2009). Thus, there is still a need to develop more selective PDE5 inhibitors. The use of molecular fingerprint-based virtual screening protocols and structure-based pharmacophore development could facilitate the identification of more selective compounds (Kayik et al., 2017).

### 1.2.6 | PDE6 family

The cGMP-selective PDE6 family consists of three genes that encode catalytic subunit (PDE6A, PDE6B, and PDE6C) that are expressed at high concentrations in rod and cone photoreceptors in the retina [for recent review, see (Cote, 2021)]. PDE6A (also referred to as the α-subunit) and PDE6B (β-subunit) are localized to rod photoreceptor cells, whereas PDE6C (α'-subunit) is expressed in cone photoreceptor cells. PDE6 catalytic subunits (and other components of the visual signaling pathway) have also been identified in the pineal gland (Carcamo et al., 1995). The PDE6 family is unique in several respects: (1) rod PDE6 is the only PDE that can form a heterodimer ($\alpha\beta$); (2) enzyme regulation is mediated by binding of regulatory γ-subunits (PDE6G (rod, γ) and PDE6H (cone, γ') that inhibit catalysis of cGMP in the nonactivated state; (3) activation of the PDE6 holoenzyme ($\alpha\beta\gamma\gamma$ or $\alpha'\alpha'\gamma'\gamma'$) results upon binding of the photoreceptor G-protein α-subunit (GNAT1 in rods, GNAT2 in cones) and displacement of the γ-subunit from the enzyme active site; (4) activated PDE6 is the only PDE family that catalyzes cGMP hydrolysis at a diffusion-controlled rate; (5) the C-terminus of each catalytic subunit is prenylated, conferring tight association of PDE6 with the membrane; and (6) the expression and proper assembly of the PDE6 holoenzyme requires a photoreceptor-specific chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (Cote, 2021; Yadav and Artemyev, 2017). It is well-established that the rate-limiting step for the activation and deactivation of the visual signaling pathway in photoreceptors is controlled by the kinetics of PDE6 activation and inactivation, respectively (Arshavsky and Wensel, 2013; Pugh and Lamb, 2000). The rapid reduction in cGMP concentration in the photoreceptor cell upon PDE6 activation results in closure of cyclic nucleotide-gated ion channels and the generation of an electrical response.

Similar to PDE5, PDE6 catalytic subunits consists of two tandem GAF domains attached to the catalytic domain, and cGMP binding to the GAF-A domain contributes to the allosteric regulation of the holoenzyme (Kameni Tcheudji et al., 2001; Zhang et al., 2008c). The molecular organization of the PDE6 holoenzyme has revealed that the inhibitory γ-subunits bind to the catalytic dimer in an extended conformation that interacts with each of the GAF and catalytic domains (Gulati et al., 2019; Irwin et al., 2019). The pharmacological properties of PDE6 are similar in many respects to PDE5 (as described in the previous section), with zaprinast being notable for having 10-fold higher affinity for rod and cone PDE6 compared to PDE5 (Zhang et al., 2005c).

### 1.2.7 | PDE7 family

The PDE7 family specifically hydrolyses cAMP. There is no known regulatory domain in the N-terminal region (Figure 2). This family includes two genes *PDE7A* (hcl: 8q13) and *PDE7B* (hcl: 6q23-q24), with alternative splicing for *PDE7A* giving rise to PDE7A1 (57 kDa), PDE7A2 (50 kDa) and PDE7A3 (50 kDa). PDE7A3 lacks a part of the catalytic domain structure and retains the capacity of PDE7A1 to interact and inhibit the catalytic subunit of PKA. Four alternative splice *PDE7B* transcripts have been identified, of which PDE7B1 and PDE7B3 show two putative phosphorylation sites for PKA. Yet, no endogenous PDE7B proteins have been detected. IC242
was the first selective PDE7 inhibitor to be reported (IC₅₀= 0.84 µM) (Lee et al., 2002), and several new PDE7 inhibitors have been listed since (Zorn and Baillie, 2023).

### 1.2.8 | PDE8 family

The PDE8 isoenzyme family specifically hydrolyses cAMP with the highest affinity amongst all PDEs. It is encoded by *PDE8A* (hcl: 15q25.3) and *PDE8B* (hcl: 5q13.3). The primary structure of PDE8 includes *N*-terminal response regulator receiver (REC), Per-Ant-Sim (PAS) and three putative PKA and PKG phosphorylation sites (Figure 2). Various splice variants exist. PDE8A1, the longest (93 kDa) and most frequently expressed variant, contains REC and PAS domains. PDE8A2 lacks the PAS domain, whereas PDE8A3 and the truncated PDE8A4 and PDE8A5 lack both REC and PAS domains (Wang et al., 2001). PDE8B1 and PDE8B4 contain both REC and PAS domains, whereas PDE8B2 and PDE8B3 have a deletion in the PAS domain (Gamanuma et al., 2003). IκB proteins activate PDE8A1 through interaction with the PAS domain. Interestingly, PDE8A regulates the Raf-1 and ERK signaling network (Ahmad et al., 2015). PF-04957325 is a selective PDE8 inhibitor with possible application in airway disease and autoimmune encephalomyelitis (see Chapter 2.7.4) (Basole et al., 2022; Johnstone et al., 2018).

### 1.2.9 | PDE9 family

The PDE9 family specifically hydrolyses cGMP with the highest affinity amongst all PDE families. Twenty-one *N*-terminal mRNA variants can be encoded by a single gene, *PDE9A* (hcl: 21q22.3), along with three PDE9A protein isoforms that are much larger than the predicted molecular weight of these mRNA variants (named by molecular weight: PDE9X-100, PDE9X-120, and PDE9X-175; (Patel et al., 2018)). No regulatory function or phosphorylation of the *N*-terminal domain has been reported. PDE9A isoforms are differentially expressed and subcellularly localized in various tissues, and this changes with age (Patel et al., 2018; Wang et al., 2003b). PDE9A inhibitor BAY 73-6691 is 25-fold selectivity for the target over all other PDEs (Wunder et al., 2005). Orally available, brain-penetrant PDE9A inhibitors, BI-409306 and PF-04447943, have been developed for use in age-related cognitive decline (Chapter 2.5.1).

### 1.2.10 | PDE10 family

PDE10 represents a dual-substrate family of enzymes encoded by the gene, *PDE10A*. Human *PDE10A* maps to chromosome 6q26-27. There are 18 splice variants that can be derived from *PDE10A* (*PDE10A1*–*PDE10A19*) (MacMullen et al., 2016). The deduced amino acid sequence contains 779 amino acids (88 kDa), including two GAF domains in the *N*-terminal region (Fujishige et al., 2000) (Figure 2). In contrast to other PDEs, the GAF-A domain apparently binds only cAMP. Due to its kinetic properties for cAMP and cGMP hydrolysis, cGMP hydrolysis by PDE10 is potently inhibited by cAMP, and thus opposite to PDE3. Papaverine (IC₅₀= 36 nM) (Tian et al., 2011) and PF-2545920 are PD10A inhibitors, and were reported to cause seizures through neuronal excitability enhancement (Zhang et al., 2017c). Various possible clinical applications are highlighted through Chapter 2.

### 1.2.11 | PDE11 family

The dual-substrate PDE11 isoenzyme family has a catalytic site more similar to PDE5 than to PDE10A. Four *N*-terminal variants are encoded by the *PDE11A* gene, which maps to human chromosome 2 (2q31.2): PDE11A1 to PDE11A4. PDE11A1 (491 amino acids; predicted molecular mass 56 kDa) was cloned from human skeletal muscle and contains a partial GAF-B domain. PDE11A2 (65.8 kDa) and PDE11A3 (78 kDa) contain a complete GAF-B domain, with PDE11A3 also containing an incomplete GAF-A domain in the *N*-terminal region. PDE11A4 (100 kDa) is the longest protein within this family and including two full GAF domains and multiple
phosphorylation sites within the N-terminal region (Fawcett et al., 2000; Pilarzyk et al., 2022; Yuasa et al., 2000). The PDE11A4 GAF-A domain allosterically binds cGMP (Gross-Langenhoff et al., 2006). The degradation-resistant cGMP analog Rp-8pCPT-PET-cGMP binds the GAF-A domain to stimulate catalytic activity of PDE11A4, but cGMP binding does not (Jager et al., 2012). The GAF-B domain is involved in oligomerization of the enzyme (Weeks et al., 2007). Reports of PDE11A expression at the protein level are highly contradictory, probably due to antibody non-specificity and species differences (Kelly, 2015). Tissue and species differences in PDE11A isoforms have been abundantly reported (Kelly, 2015; Kelly, 2018b; Pilarzyk et al., 2022; Sbornova et al., 2023). The first published selective PDE11A inhibitors were identified using a, which showed moderate affinity for PDE11A (Ceyhan et al., 2012). With the help of yeast-based high-throughput assays that yielded inhibitors with IC₅₀ of 0.11 – 0.33 µM, the compounds BC11–28 and BC11–38 have been identified, showing highly selective inhibition (>350-fold selective for PDE11A vs. other PDE families). Improvement of potency and pharmacokinetic properties based on these scaffolds is ongoing (Mahmood et al., 2023).

**1.3 | Structure-Activity relationship**

As reviewed in section 1.2, PDEs are divided into a variable regulatory domain at the N-terminus and a conserved catalytic domain at the C-terminus, and specifically recognize substrates and inhibitors (Baillie et al., 2019a; Conti and Beavo, 2007; Francis et al., 2011; Maurice et al., 2014). In the present section, the structure-activity relationships and the exploitation thereof for the design of PDE subtype-selective inhibitors are reviewed.

**1.3.1 | Regulation of catalytic domain**

The first atomic-level structure of the PDE4B2B catalytic domain (Xu et al., 2000) is representative of all of the Class I PDE catalytic domains (i.e., those present in mammals and flies) (Ke and Wang, 2007). The catalytic domain (Pfam: PF00233) typically consists of 16 α-helices that bind two divalent metals, Zn²⁺ and (typically) Mg²⁺ that are essential for the catalysis of cAMP and cGMP (Figure 3). Zn²⁺ coordinates with His²³⁸, His²⁷⁴, Asp²⁷⁵, and Asp³⁹² of PDE4B2B, and 2 water molecules. Mg²⁺ chelates with Asp²⁷⁴ and 5 water molecules. The folding of the catalytic domain and metal interactions is conserved in all PDE families (Ke and Wang, 2006). Binding of substrate in the active site, a conserved hydrophobic pocket, is stabilized not only by the hydrogen bond with an invariant glutamine but also by interactions between the substrate's purine ring and several hydrophobic residues residing in the, so-called, "hydrophobic clamp" (Ke et al., 2011) that positions the cyclic monophosphate group for hydrolysis of the phosphodiester bond by a nucleophilic attack (Wang et al., 2007a). For example, Phe³⁷² stacks against the purine ring of cAMP in the structures of PDE4D2-AMP and D201N PDE4D2-cAMP on one side (Figure 4C), while Phe³⁴⁰ and Ile³³⁶ make hydrophobic interaction with the purine on the other side (Wang et al., 2007a). The highly conserved hydrophobic pocket is primarily responsible for the high affinity and selectivity of family-specific PDE inhibitors (Ke and Wang, 2007), as exemplified by the PDE4B2B - inhibitor NPV interaction that is called the "hydrophobic slot" (Hartley, 1964; Wang et al., 2007a). In contrast, several "sub-pockets" were proposed for binding of PDE family-selective inhibitors, as discussed in the later sections.

**1.3.2 | Allosteric mechanisms for regulating cyclic nucleotide hydrolysis by PDEs**

*General aspects*

Perhaps the best-studied structural elements hypothesized to regulate catalytic activity of PDEs are the flexible H-loop (including two short α-helices) and the flexible M-loop (the flexible region between α14 and α15) (for review see (Ke and Wang, 2007). Alignments of the catalytic domains of the entire superfamily suggest the involvement of these flexible regions in regulation of substrate
diffusion into the enzyme active site due to their vicinity to the entrance (Huang et al., 2015; Ke and Wang, 2007). However, the mechanisms for restricting substrate diffusion into the catalytic center differs between PDE families. Specific for PDE4, an α-helix downstream of the α16 helix in the catalytic domain putatively participates in phosphorylation-dependent auto-inhibition (Burgin et al., 2010b). The H- and M-loop can also undergo major conformational changes upon binding of PDE inhibitors to the active site, emphasizing their pharmacological importance in structure-aided drug design. In the following sections, we examine in detail the allosteric regulation of several PDE families with an emphasis on how knowledge of atomic-level PDE structures can inform and guide efforts to rationally design novel, family- and isoform-selective PDE inhibitors.

## PDE2

Structure studies (Martinez et al., 2002; Pandit et al., 2009) have revealed that cGMP binding at a flexible binding pocket within the GAF-B domain induces structural changes to enhance cGMP affinity. The x-ray structure of PDE2A (Pandit et al., 2009) shows that the two catalytic subunits cross over at the juncture of the GAF-B and catalytic domains (Figure 5). The H-loop (residues Gly⁷⁰² – Ser⁷²⁴ of PDE2A) from the opposite subunit is in proximity to, and restricts substrate access to, the active site of each monomer. Binding of the inhibitor BAY 60-7550 to the active site caused an outward movement (and change in conformation) of the H-loop (Zhu et al., 2013). It has been hypothesized that cGMP binding to the GAF-B domain induces conformational changes within GAF-B, which after propagation through the α-helix and the adjacent catalytic domain changes the H-loop conformation to enhance substrate entry and, thus, its catalysis (Pandit et al., 2009; Zhu et al., 2013). This thesis awaits confirmation.

The structure of PDE2 in complex with BAY 60-7550 (Figure 5C) identified a hydrophobic pocket consisting of Leu⁷⁰, Ile⁸⁶⁶, and the hydrophobic side chains of The⁸⁰⁵ and Asp⁸⁰⁸ (Figure 5C) (Zhu et al., 2013). This knowledge may enable the design of PDE2-selective inhibitors in the future, as evidenced by the subsequent discovery of a novel PDE2 inhibitor (Qiu et al., 2018).

PDE2A is widely expressed, and most abundant in the brain (Lakics et al., 2010a), implying application in CNS disorders, e.g. depression and anxiety (Masood et al., 2009; Xu et al., 2013; Zhang et al., 2015). Since past studies were hampered by the lack of selective and brain-penetrant compounds further inhibitor design is required.

## PDE5

There is ample information to support the idea that PDE5 forms a homodimer with the overall domain organization similar to those of PDE2 (Figure 5A) and PDE6 (Ahmed et al., 2021; Cote et al., 2022; Francis et al., 2006; Schultz, 2009). For the five PDE families containing two tandem GAF regulatory domains (GAF-A and GAF-B (Pfam PF01590); (Aravind and Ponting, 1997)), only one GAF domain per monomer binds cyclic nucleotides: cGMP binds to GAF-A in PDE5 PDE6, and PDE11 and to GAF-B in PDE2, while cAMP binds to GAF-B in PDE10 (Heikaus et al., 2009; Jager et al., 2012; Schultz, 2009; Zoraghi et al., 2004).

PDE5 catalytic activity is modulated by three allosteric mechanisms. First, PDE5 catalytic activity is stimulated upon binding of cGMP to noncatalytic sites localized to the GAF-A domain. The GAF-A binding site undergoes a major, local conformational change upon cGMP binding (Heikaus et al., 2008) that is allosterically communicated to the catalytic domain to cause an increase in cGMP hydrolysis. This allosteric effect is reciprocal in that occupancy of the PDE5 active site enhances the affinity of cGMP binding to GAF-A. In addition, cGMP binding to GAF-A stimulates phosphorylation of a serine residue in the N-terminal region that precedes the GAF-A domain; this effect is also reciprocal in that phosphorylation of PDE5 at this site enhances cGMP binding affinity to GAF-A and, in a cooperative manner, stimulates catalytic activity (for a comprehensive review,
see (Francis et al., 2011)). Delineation of the allosteric communication pathway from the regulatory to the catalytic domains of PDE5 awaits determination of the atomic structure of the full-length enzyme and identification of ligand-induced conformational changes.

The second intrinsic mechanism for allosteric regulation of PDE5 catalysis is observed in local conformational changes of the flexible elements around the active site (Figure 6). The most important of these is the H-loop (residues 661-678 of PDE5A1) that adopts different conformations upon binding different inhibitors (Wang et al., 2006). The ligand-free H-loop has a coiled conformation, and migrates dramatically to form a small 3₁₀ helix upon binding of IBMX or sildenafil (Figure 6A). Binding of the natural product, icarisid II (purified from the Chinese herb Ying Yang Hu which is known to promote sexual activity of goats) induced formation of two short anti-parallel β-strands in the H-loop (Figure 6A). In addition, sildenafil and vardenafil—two PDE5 inhibitors with similar molecular structures—induce completely different conformational changes in both the H-loop and the adjacent M-loop motif (residues 790-810; see Figure 5B, adapted from (Wang et al., 2008c)), possibly explaining their distinctive affinity for PDE5 and their physiological effects.

A third mechanism of regulating PDE5 activity has been identified for the allosteric inhibitor evodiamine, which upon binding to a site adjacent to the active site (Figure 7) induces conformational changes in the active site (Zhang et al., 2020c). Binding of evodiamine displaces several water molecules occupying the unliganded binding site and induces conformational changes to the H-loop at the active site (Figure 7C).

## PDE4

UCR1 and UCR2 participate in regulating cAMP hydrolysis in a coordinated manner. In long isoforms of PDE4, UCR1 and UCR2 interact to form a regulatory unit in which the C-terminal portion of UCR2 folds onto the catalytic domain and blocks substrate access to the active site (Burgin et al., 2010b; Cedervall et al., 2015). As mentioned above, occlusion of the active site can also occur when a C-terminal α-helix folds onto the active site (Burgin et al., 2010b).

The four genes that comprise the PDE4 family have very high (~78%) sequence identity within the 340 amino acids of the catalytic domain. This is also reflected in the structural superposition of the catalytic domains of PDE4D with PDE4A, PDE4B, and PDE4C (root-mean-squared deviations of 0.67, 0.73, and 0.64 Å for the Cα atoms; (Wang et al., 2007a). Consequently, most PDE4 inhibitors do not discriminate between the four members of this family in their affinity for binding to the catalytic pocket. After it was suggested that inhibition of PDE4D produces many of the side-effects of the PDE4 inhibitor rolipram, developing family-selective PDE4 inhibitors, especially against PDE4B, became an important medicinal chemistry objective (Azam and Tripuraneni, 2014). Moreover, attention has more recently turned towards the therapeutic potential of allosteric inhibitors that are more likely to bind specifically to a member of the PDE4 sub-family and may display an improved therapeutic ratio.

Two examples of modulators of PDE4 catalytic activity that are not simple competitive inhibitors at the enzyme active site are D 155871 (Burgin et al., 2010b) and zatolmilast (BPN 14770) (Gurney et al., 2019), both of which inhibit the long forms of PDE4D. Binding of D 155871 to the active site of PDE4D (Figure 8) induces dramatic migration of an α-helix of UCR2 from the back of the catalytic domain to cover the active site (Burgin et al., 2010b). However, it remains unclear if allosteric PDE4 inhibitors will have therapeutic applications.

## PDE9
The PDE9 catalytic subunit consists of a coiled N-terminal region and a conserved catalytic domain at the C-terminus and exists as a dimer. The crystal structure of PDE9 reveals that the catalytic sites in the two subunits have slightly different shapes, leading to different binding conformations of the same inhibitor in the dimer (Huang et al., 2015).

Examination of inhibitor binding suggests that a small “M-pocket” may determine the selectivity of PDE9 inhibitors (Huang et al., 2015)). The M-pocket is in an open conformation in PDE9, enabling large functional groups such as benzene to bind (Figure 9A). In contrast, sequence alignment of PDEs in the vicinity of the M-pocket (Figure 9C) illustrate that Phe⁴⁴¹ and Ala⁴⁵² of PDE9 (that serve as gates for the M-pocket) are substituted with bulky side chains in PDE5 (Leu⁸⁰⁴ and Met⁸¹⁶) that would allow only small functional groups to penetrate into the M-pocket, as shown for the ethoxy group in the PDE5-sildenafil crystal structure (Wang et al., 2008c). In the case of the PDE8A1 structure (Wang et al., 2008b), the M-pocket is too small to accommodate binding of the PDE9 inhibitor, C33, providing a structural basis for the observed selectivity of C33 over PDE5 and PDE8 (Huang et al., 2015). In summary, the M-pocket of PDE9 may be an excellent target for structure-aided design of highly selective PDE9 inhibitors.

### 1.3.3 | Peptide inhibitors, regulators, and activators

In addition to cyclic nucleotide binding, posttranslational modifications, and the various pharmacological PDE inhibitors that have been characterized, other methods of altering PDE activity have been used as research tools, namely: i) techniques for PDE depletion, ii) activators or methods that upregulate PDE protein, and iii) techniques that displace a defined PDE “pool” from one specific nano-domain within a cell.

#### Depletion of endogenously expressed PDEs: RNA and DNA-based mechanisms

To define the role of specific PDEs, overexpression by constructs encoding full length PDE proteins or, conversely, gene silencing using anti-sense or small interfering (si) RNA oligonucleotides has been employed. Detrimental to interpretation, overexpression can lead to aberrant cellular localization of the enzyme and, as is apparent from earlier sections of this review (see sections 1.2.1; 1.2.11), the localization of a cAMP-PDE is central to its function. Antisense oligonucleotides offered the first method to specifically target PDE isoforms (Epstein, 1998) and this approach has also been used to target different PDE families concomitantly for therapeutic benefit (Fortin et al., 2009). Soon, the development of siRNA sequences to evaluate the functional relevance of a variety of PDEs followed, as exemplified by PDE2, PDE4D, PDE7 and PDE8 (Hiramoto et al., 2014; Li et al., 2011; Pekkinen et al., 2008).

Another method for PDE silencing that has come to the fore recently is CRISPR/Cas9. It has been used to characterize the differential role of PDE2A isoforms and PDE3A in shaping cAMP dynamics in neonatal and adult rat cardiomyocytes following β-adrenoceptor stimulation (Skryabin et al., 2023). The ability to pinpoint isoforms rather than sub-families or families may lead to the development of therapies that result in fewer side-effects. This is particularly relevant for PDE4D isoforms in Alzheimer's disease (AD) were a body of work using knock-out animals (Li et al., 2011), dominant negatives (Bolger et al., 2020), siRNA (Li et al., 2011) and PDE4D-selective inhibitors (Ricciarelli et al., 2017) have identified the subfamily as having a crucial role. Recently CRISPR/Cas9 genome editing has determined that specific silencing of the long PDE4D isoforms, PDE4D3, D5, D7 and D9, confers protection against β-amyloid-induced reductions in neuronal plasticity (Paes et al., 2023).

MicroRNAs (miRNA) are endogenous regulators of protein expression. Specific miRNA have been identified for PDE4 in synovial fibroblasts modulating pro-inflammatory processes (Wade et al., 2019), PDE3 in cerebral microvascular endothelial cells affecting cognitive decline in cerebral
small vessel disease (Yasmeen et al., 2019) and PDE1 in lung fibroblasts where the miRNA is
protective against lung fibrosis (Ren et al., 2017). miRNAs are, themselves, known to be regulated
by long non-coding RNAs (lncRNA) and lncRNA GAS5 "sponges" miRNA that prevents
translation of *PDE4B2* to increase expression of the PDE, preventing the accumulation of lipid in
cell models of non-alcoholic fatty liver disease (Xu et al., 2022).

PDE (in)activation by manipulation at the protein level: proteolysis targeting chimeras
(PROTACs)

Besides targeting DNA and RNA, there is a novel way to specifically reduce the activity of active
enzymes in a cell via targeted protein degradation. This involves a small molecule that can link the
enzyme of interest to the ubiquitin proteasome system by anchoring a ubiquitin E3 ligase close to
the target. These small-sized molecules, of which the pharmacokinetic properties can be optimized
for use *in vivo*, partly independently of Lipinski's rule of five, have been termed proteolysis
targeting chimeras (PROTACs) and are now in phase II clinical trials for oncology (Wang et al.,
2023). PROTACs appear to be ideal as PDE inhibitors (Konstantinidou et al., 2019). Firstly because
rather than relying on the constant presence of high concentrations, a reason why inhibitors cause side
effects, to warrant occupation of the active site, PROTACS de-activate the PDE in one event.
Secondly, protein degradation attenuates the enzymatic and non-enzymatic functions, allowing also
the inhibition of PDEs as scaffolds for protein interactions (Susuki-Miyata et al., 2015). Finally, as
the PROTAC molecule is formed of three integral units (warhead, linker and E3 recruiter molecule),
enhanced selectivity and potency can be engineered into the structural design via molecular
modeling, providing better opportunity for isoform specificity.

PROTACs that evoke degradation of the KRAS (Kirsten Rat Sarcoma Virus)-shuttling PDE6δ, a PDE6 subunit without enzymatic function, results in mislocalization of KRAS preventing its activation (Cheng et al., 2020; Teng et al., 2022; Zhang et al., 2005a). This has been a promising strategy to treat KRAS mutation-related cancer. “SNIPER” protein erasers demonstrated that a PDE4 inhibitor could be used as warhead to co-localize E3 ubiquitin ligases for PDE4 degradation (Ohoka et al., 2017). The work might inspire the development of PROTACS for the other families.

PDE (in)activation by manipulation at the protein level: Disruptors of PDE compartmentalization

Cellular expression of cAMP-PDEs is often low but highly localized in order to shape cAMP in
nanodomains (Baillie, 2009). Peptide disruptors that relocate cAMP-PDEs allow researchers to
define the role of specific isoforms (reviewed in (Blair and Baillie, 2019; Lee et al., 2013)). As
single isoforms can exist in more than one cellular locale and have multiple functions depending
on proximity to various cAMP effector proteins (e.g. PDE4D5 (Wills et al., 2016)), displacement
is the only method that takes compartmentalization into account. This benefit was demonstrated
recently when revealing the role of PDE4-Popeye domain-containing 1 interaction in nanodomain
calcium transient regulation for sino-atrial pace-making (Tibbo et al., 2022). The use of a pan-PDE4
inhibitor would not have been as due to pools of various PDE4 subfamilies and isoforms associated
with cardiac calcium handling (Maurice et al., 2014).

The use of catalytically dead, dominant-negative PDEs that displace endogenously active forms is
applicable in transfected cell lines (McCahill et al., 2005) and whole organism models of disease
(Bolger et al., 2020; McGirr et al., 2016). Although this approach involves PDE displacement, it
also relies on overexpression, so it is likely all complexes containing the PDE of interest being
overexpressed as a dominant negative will be disrupted concomitantly, limiting the deconvolution
of data pertaining to compartmentalized responses. In the case of PDE11A, compartmentalization
can be changed by disrupting homodimerization via the expression of its isolated GAF-B domain
or by phosphorylation of select residues in its regulatory N-terminal domain, both of which are
sufficient to alter memory formation in mice (see 2.5.10 for more detail; Pathak et al., 2017; Pilarzyk et al., 2023; Pilarzyk et al., 2022)

*PDE (in)activation by manipulation at the protein level: Molecules that enable increases in PDE activity*

Recently, Mironid developed small molecules that activate PDE4 via allosteric binding to the UCR 1/2 regions to lock the long-form dimer in the “open” conformation (Omar et al., 2019). Activation of PDE4 long-forms in this way is sufficient to counteract chronic cAMP elevation. Application in polycystic kidney disease and prostate cancer have been proposed for these compounds (Gulliver et al., 2023b; Hansen et al., 2022). This activation mechanism has been observed historically with lipids (Grange et al., 2000) and peptides (Wang et al., 2015a), and now the small molecules have opened the door for the development of therapies that require PDE activation. Another technologically advanced way to enhance the protein stability of active enzymes is by using the converse approach to PROTACs. Targeted protein stabilization using deubiquitinase-targeting chimeras (DUBTACS) can stabilize target proteins by preventing them engaging with the proteosome (Henning et al., 2022). This relatively new approach for selective PDE isoform activation holds promise for the future.

**1.4 | Role of PDEs in cyclic nucleotide compartmentalization**

**1.4.1 | cAMP compartments: the birth of a new rationale**

The canonical model stating that cAMP signaling links G-protein coupled receptor (GPCR) ligand binding to cAMP generation, PKA activation, and substrate phosphorylation in a linear cascade from the plasma membrane to the terminal intracellular effector cannot explain the functional versatility of cAMP within one cell type. This enigma persisted for decades, until an alternative model developed over the last twenty years: the paradigm of compartmentalization. This spatial confinement proves critical for hormonal specificity, with $G_s$-coupled receptor activation generating spatially distinct cAMP pools that, in turn, activate defined subsets of the effector enzyme PKA.

Selective PKA activation hinges on the immobilization of the kinase to specific subcellular locations through interaction with AKAPs, tethering the enzyme in proximity to specific phosphorylation targets (Scott et al., 2013). AKAPs nucleate signaling hubs, or signalosomes, through protein-protein interactions (PPIs) that include varying assortments of signaling molecules such as GPCR, adenylyl cyclases, phosphatases (PPs), and cAMP-PDEs. Each signalosome results from a unique combination of signaling components, facilitating specific regulation of distinct cellular functions at distinct locations. This paradigm started from studies on cardiac β-adrenoceptors, and has since spread to virtually all cell types (Zaccolo et al., 2021).

Early imaging studies demonstrated the critical role of cAMP-hydrolyzing enzymes in limiting the spatial propagation of the second messenger as inhibition of PDEs disrupted the local cAMP gradients (Chen et al., 2008; Feinstein et al., 2012; Guellich et al., 2014; Iancu et al., 2007; Leroy et al., 2008; Mika et al., 2012; Terrin et al., 2006; Zaccolo and Pozzan, 2002) (Jurevicius and Fischmeister, 1996). The advent of genetically encoded probes for real-time cAMP monitoring was a breakthrough in demonstrating the steep intracellular cAMP gradients that create nanodomains (Nikolaev et al., 2004; Ponsioen et al., 2004; Zaccolo et al., 2000; Zaccolo and Pozzan, 2002). These probes typically consist of a cAMP-binding domain (CBD) sandwiched between two fluorescent protein spectral variants, enabling Förster resonance energy transfer (FRET) imaging. The altered FRET signaling that occurs upon cAMP binding is detectable with a conventional optical microscope. The first reported probe consisted of a PKA regulatory subunit fused to a cyan fluorescent protein and a PKA catalytic subunit fused to a yellow fluorescent protein (YFP),
allowing measurement of cAMP binding to the PKA holoenzyme (Zaccolo and Pozzan, 2002). On application of noradrenaline to cardiac myocytes expressing the probe, the elicited cAMP response was not homogeneous across the cell but was localized to subcellular compartments where PKA is anchored to AKAPs. In agreement, a probe variant lacking the domain necessary for AKAPs anchoring resulted in the sensor being unable to pick up any significant cAMP signal. Studies using these biosensors demonstrated that different GPCRs couple with distinct adenylyl cyclases, generating specific local cAMP pools that activate a limited subset of effectors, thus creating functional diversity between GPCRs (Di Benedetto et al., 2008).

Further compartmentalization mechanisms include physical barriers (Richards et al., 2016), buffering (Bock et al., 2020a), phase separation of the regulatory subunit of PKA (Zhang et al., 2020a), and generation of cAMP from internalized GPCRs (Lohse et al., 2023).

### 1.4.2 | Organization in signalosomes and participation of PDE

Recent mounting evidence that $G_s$PCRs can initiate cAMP production post-internalization introduces a novel and intriguing aspect to the cAMP compartmentalization model (Calebiro et al., 2009; Ferrandon et al., 2009; Irannejad et al., 2013). Traditionally, internalization of GPCRs upon ligand binding has been considered as a mechanism that leads to receptor desensitization, offering protection against excessive stimulation. However, recent studies combining real-time cAMP imaging with observations of GPCR trafficking events in intact cells, challenged this notion as they demonstrated sustained cAMP signaling from $G_s$PCRs embedded in internalized vesicles (Calebiro et al., 2010; Ferrandon et al., 2009; Lan et al., 2012; Pavlos and Friedman, 2017). Internalized GPCRs were shown to encounter G proteins on internal membranes, such as endosomes or Golgi-associated vesicles, and create a fully functional signaling complex initiating a subsequent wave of signaling by triggering the generation of localized pools of second messenger. Alternatively, a substantial pool of some GPCRs, such as $\beta_1$-adrenoceptor, can be permanently located at intracellular membranes, as, for example, in the SR of cardiac myocytes to locally regulate contractility and relaxation (Lin et al., 2023b).

The complexity of the PDE system allows for a further highly sophisticated regulation of local cAMP levels. The number of possible permutations in combinations of PDE isoform, location, activity regulation by Ca2+, cGMP or other mechanisms is extensive, and can all determine the subcellular topography of cAMP nanodomains (Beltejar et al., 2017; Subramaniam et al., 2023) (Figure 10). The resulting dynamic cellular landscape of cAMP gradients remains largely to be defined in its details as is any remodeling that occurs in pathological conditions.

### 1.4.3 | Size matters, when measuring cAMP in nanodomains

The organizational complexity of PDEs and their interactions to generate local steep cAMP gradients proves highly effective for achieving hormonal specificity. Compartmentalized cAMP in cardiac myocytes, for example, allows $\beta$-adrenoceptors to increase PKA-dependent phosphorylation of phospholamban, while activation of the prostaglandin receptor does not, despite generating a similar overall cAMP amount (Hayes et al., 1980). Targeted FRET reporters further demonstrated that distinct cAMP signals and heterogeneous cAMP responses are generated by activation of different cardiac GPCRs with positive inotropic effects (Di Benedetto et al., 2008), By contrast, a homogeneous cAMP increase throughout the cell, achieved through pharmacological PDE inhibition, results in a diminished inotropic response (Surdo et al., 2017). This emphasizes the functional relevance of this spatial organization.

As an alternative to nanodomain-targeted sensors, recent developments using integrated analysis of PDE isoform-selective interactomes and PDE family-dependent phosphoproteomes allowed the identification of multiple novel, non-obvious cAMP subcellular nanodomains (Subramaniam et al.,
2023). Early imaging experiments estimated the size of cAMP subcellular compartments to be in the micrometer range (Zaccolo and Pozzan, 2002). Recent studies, however, revealed that the cAMP subcellular domains can be as small as tens of nanometers (Anton et al., 2022a; Surdo et al., 2017). FRET reporters that include spacers with known length (nano-rulers) revealed cAMP compartment sizes as small as 10 nm, ruling out the necessity for a physical barrier to achieve cAMP compartmentalization (Anton et al., 2022a). Experiments with cAMP reporters fused directly to PDEs (Herget et al., 2008) demonstrated that PDEs can create regions of low cAMP concentration, obliterating PKA activation within their immediate surroundings. The action radius varies based on the enzymatic properties of the involved PDE isoform. For instance, PDE4A1, a relatively high-affinity (low $K_m$) but low-turnover (low $V_{max}$) enzyme (Bender and Beavo, 2006), creates a domain with low cAMP with a 10 nm radius, while PDE2A3, a lower affinity but faster enzyme, can deplete cAMP within a radius exceeding 30 nm (Bock et al., 2020a). Recent development of nanodomain-targeted cAMP sensors provided more detailed insights into cAMP signaling (Allen and Zhang, 2006; DiPilato et al., 2004; Lefkimmiatis et al., 2013; Liu et al., 2011; Pendin et al., 2017; Surdo et al., 2017). Sensors targeted to plasma membrane nanodomains (Bastug-Ozel et al., 2019; Perera et al., 2015) and SR domains in close proximity to the ryanodine receptor (Berisha et al., 2021) and SR $Ca^{2+}$ ATPase (Sprenger et al., 2015) could reveal that cardiac hypertrophy and heart failure lead to dramatic alteration of subcellular localization of multiple PDEs, resulting in changes of contractility, relaxation and cardiac arrhythmias. Therefore, better understanding and specific targeting of such nanodomain remodeling may pave the way to new approaches for cardiovascular therapies. Possible approaches that have been proposed include overexpression of distinct PDE isoforms (Karam et al., 2020), their specific knockdown (Skryabin et al., 2023) or other means aimed at restoration of proper nanodomain architecture. Obviously, the future of thorough unravelling of cAMP-PDE signaling depends on the development of sophisticated, high resolution sensors.
# CHAPTER 2: PHYSIOLOGY AND CLINICAL DEVELOPMENT

## 2.1 | Modulation of cardiac function by PDEs

### 2.1.1 | Introduction: (patho)physiology of the heart related to cyclic nucleotide signaling

Heart failure and reduced ejection fraction (HFrEF) represents a persistent clinical syndrome characterized by the gradual decline of cardiac function. Ultimately, the heart's ability to pump blood efficiently becomes inadequate to meet the body's oxygen demands, resulting in organ failure and, in severe cases, death. HFrEF can be caused by various factors, including arrhythmias, cardiomyopathies, coronary artery disease, congenital heart defects, infections, hypertension, valve problems, and the cardiotoxicity of certain anticancer drugs (McDonagh et al., 2022). Regardless of its origin, reduced cardiac function triggers the activation of neuro-hormonal systems and the development of cardiac hypertrophy to normalize ventricular wall stress (Hartupee and Mann, 2017). However, this compensated state is usually short-lived and gradually progresses toward chamber enlargement, eventually leading to HFrEF.

In normal physiological conditions cAMP and cGMP have contrasting roles in cardiac function. β-adrenoceptor-stimulated cAMP release increases cardiac output through PKA. Conversely, nitric oxide (NO) or natriuretic peptide (NP) -induced cGMP can exert both synergistic and opposing effects to cAMP. In HF, decreased cardiac function chronically elevates sympathetic catecholamines (Cohn et al., 1984). This initiates a pathophysiological 'vicious circle' of excessive β-adrenoceptor stimulation, explaining the beneficial effects of β-blockers in HFrEF (El-Armouche and Eschenhagen, 2009). Continuous cAMP-PKA signaling triggers the maladaptive remodeling leading to HFrEF, featuring hypertrophy, cardiomyocyte death, and fibrosis. Conversely, NO and NPs are anti-hypertrophic and anti-fibrotic. Therefore, NP-, NO- and cGMP-increasing drugs enhance conventional treatments for patients with HFrEF (Armstrong et al., 2020; McMurray et al., 2014; Petraina et al., 2022).

PDE control of cyclic nucleotides in nanodomains with PKA-AKAP is pivotal in cardiac function regulation (Chapter 1.4) (Anton et al., 2022b; Bock et al., 2020b; Kokkonen and Kass, 2017). The nanodomain organization undergoes significant remodeling in cases of pathological hypertrophy and HF, believed to contribute to heart function deterioration. In HFrEF, changes in PDE expression, activity, and subcellular localization alter cAMP and cGMP signaling, and is associated with modulated β-adrenoceptor signaling, decreased NO bioavailability and impaired NP signaling (Dickey et al., 2012; Katz et al., 2005; Lohse et al., 2003; Nikolaev et al., 2010). PDE3 and PDE5 inhibitors have shown detrimental effects in HFrEF patients (Packer et al., 1991), and lack of efficacy in heart failure with preserved ejection fraction (HFpEF) (Redfield et al., 2013). However, ongoing research suggests that specific PDEs could be potential therapeutic targets to prevent cardiac remodeling and HF (Chen and Yan, 2021; Preedy, 2020).

PDE1, PDE2, PDE3, PDE4, PDE5, PDE8, PDE9, and PDE10 are expressed in the heart, and all have a functional role. Much of the work and details of how these PDEs influence the normal and diseased heart can be found in several recent reviews (Kamel et al., 2023; Preedy, 2020). Here, we focus on recent advances (Figure 11).

### 2.1.2 | PDE regulation of cardiac function through cAMP

#### PDE1

Of the PDE1 isoforms only PDE1A and PDE1C are expressed in the heart. PDE1A dominates in the myocardium of the rat and mouse, and preferentially hydrolyzes cGMP (Miller et al., 2011;
Miller et al., 2009a). PDE1C has balanced selectivity for cGMP and cAMP in cell-free conditions, but in myocytes and intact hearts, it primarily impacts cAMP (Hashimoto et al., 2018; Knight et al., 2016b). In large mammal and human heart PDE1C is more prominent, and constitutively expressed, providing most of the basal cGMP and cAMP hydrolysis activity measured in soluble cardiomyocyte fractions (Muller et al., 2021; Vandeput et al., 2007). PDE1C was further shown to reside in a complex with the adenosine A₂A receptor (coupled to cAMP formation) and the transient receptor potential channel 3 (TRPC3) that mediates Ca²⁺/CaM-dependent activation of PDE1C (Zhang et al., 2018b).

Both PDE1A and PDE1C expression rise in human and animal models of HF (Knight et al., 2016b; Miller et al., 2009a; Wu et al., 2017). Genetic deletion of PDE1C in mice generates no basal phenotype but is protective against pressure overload hypertrophy and fibrosis via cAMP-PKA and phosphatidylinositol 3-kinase/Akt-dependent pathways (Knight et al., 2016b). Fibrosis was also reduced. Fibroblasts do not express PDE1C, suggesting a paracrine mechanism. 1-2 week treatment with IC 86340 or vinpocetin attenuated heart disease caused by angiotensin II stimulation (Wu et al., 2017), CryAB<sup>R12G</sup>-induced proteinopathy (Zhang et al., 2019), and doxorubicin toxicity (Zhang et al., 2018b). This benefit coupled to cAMP-PKA signaling, implicating activation of Akt, the proteosome, and anti-apoptotic pathways, respectively.

In both conscious dogs and intact rabbits, selective PDE1 inhibition (with lenrispodun (ITI-214)) results in enhanced contractility and relaxation, reduced arterial resistance, and an elevated heart rate (Hashimoto et al., 2018). The latter involves β-adrenoceptors, whereas enhanced contraction-relaxation implicated PDE1-adenosine A₂ receptor coupling (Hashimoto et al., 2018). In isolated myocytes PDE1 inhibition only augmented voltage-gated calcium conductance, through Cav₁.₂ channels, but did not increase phosphorylation of phospholamban, troponin I, or myosin binding protein C, or raise sarcoplasmic reticular Ca²⁺ load (Muller et al., 2021). In contrast, PDE3 inhibition augments Cav₁.₂, phospholamban and MyBPC phosphorylation induced by β-adrenoceptor stimulation (Mika et al., 2013; Muller et al., 2021). Compared to PDE3 inhibition, suppressing PDE1 led to a smaller increase in intracellular calcium transients, and correspondingly less arrhythmia. These findings spawned the only clinical test of a PDE1 inhibitor in human HF (Gilotra et al., 2021), a single dose placebo randomized protocol in which lenrispodun increased ventricular power index, cardiac output, and heart rate, and lowered vascular resistance.

## PDE2

PDE2 activity and expression is increased in human myocardium from patients with end-stage HF (Mehel et al., 2013). This is also seen in isoprenaline-induced HF in rat (Kaumann et al., 2009; Mehel et al., 2013). PDE2 hydrolyzes cAMP in the vicinity of SERCA2a in hypertrophied cardiomyocytes (Sprenger et al., 2015). PDE2 increase results in reduced β-adrenoceptor-mediated hypertrophic remodeling induced by noradrenaline and phenylephrine (Mehel et al., 2013). Thus, PDE2 activation might be cardioprotective, as was confirmed in cardiac-specific PDE2-overexpressing mice for catecholamine-induced ventricular tachycardia and for cardiac dysfunction after myocardial infarction (Vettel et al., 2017). Conversely, PDE2 inhibition with BAY 60-7550 increased susceptibility to arrhythmias after reperfusion injury of isolated mouse hearts (Wagner et al., 2021). In this same study, the increase in PDE2 expression specifically in the heart prevented the incidence of depolarizations induced by isoprenaline, a pathogenic cause of arrhythmia (Wagner et al., 2021). Accordingly, gene therapy with PDE2A overexpression was shown to limit cardiac adverse left ventricle remodeling, dysfunction and arrhythmias induced by catecholamines. Hence, strategies that increase PDE2A activity could prevent progression towards HF (Kamel et al., 2023).

PDE2A is a cGMP-activated PDE (Martins et al., 1982b), which might account for its protection against HF (Numata and Takimoto, 2022). Activation of the particulate guanylyl cyclase localized
near PDE2A (Castro et al., 2006a; Castro et al., 2006b) results in a NP/BNP/cGMP-triggered
defense mechanism during cardiac stress, in particular during excessive β-adrenoceptor-mediated
drive. Interestingly, sacubitril, a neutral endopeptidase inhibitor that elevates NPs, improves
classical treatments for HF (McMurray et al., 2014), and NPs exert antiarrhythmic effects via PDE2
(Cachorro et al., 2023).

In paradox, some studies have shown detrimental effect of PDE2 activation. PDE2 upregulation
was found to be pro-hypertrophic as the PDE2 inhibitor BAY 60-7550 antagonized cardiomyocyte
growth via PKA phosphorylation of nuclear factor of activated T cells (NFAT), preventing the
activation of the hypertrophic gene program (Zoccarato et al., 2015), and protected against
apoptosis by promoting mitochondrial elongation (Monterisi et al., 2017). PDE2 activity and
expression were upregulated in hypertrophied mouse myocytes after pressure overload and
isoprenaline, and BAY 60-7550 decreased left ventricular (LV) hypertrophy, LV dilation,
contractility and fibrosis (Baliga et al., 2018). In addition, BAY 60-7550 or overexpression of
catalytically inactive forms of PDE2 led to a restoration in the modulation of noradrenaline release
in the stellate ganglion by BNP, which might further protect against HF (Liu et al., 2018).

The described paradox may be attributed to differences in the PDE2 isoforms expressed, the
constructs used, or the cellular localization of the heterologous PDE2. More generally, the
discrepancies may depend on the etiology of the HF. Further studies are needed to clarify whether
inhibition or activation of the enzyme is preferred, implicating this difference in etiology.

PDE3

PDE3 is expressed in the myocardium of many species, and particularly abundant in large mammals including humans. Thus, PDE3 inhibitors have been developed for the treatment of HF. In congestive HF, PDE3 inhibitors in the short term induce a positive inotropic response and vasodilation (Movsesian et al., 2011). However, PDE3 inhibitors increase the mortality incidence of patients as a result of arrhythmias and sudden death (Packer et al., 1991). Although not fully elucidated, the mechanisms involved herein might implicate increased intracellular calcium concentrations due to chronically increased cAMP (Packer et al., 1991). In rodent models, pimobendan induced significant diastolic dysfunction with an increase in type I collagen deposition (Nakata et al., 2019). PDE3 inhibition might also be detrimental in HF through its effect on apoptosis. *In vitro*, PDE3A has been implicated in the activation of ICER (inducible cAMP early repressor), which is a transcriptional repressor of the anti-apoptotic molecule Bcl-2. The inhibition of PDE3A leads to an increase in cAMP levels and consequent activation of PKA, which increases the ICER protein, resulting in cardiomyocyte apoptosis (Yan et al., 2007).

Conversely, transgenic mice overexpressing the Pde3a1 isoform specifically in the myocardium
showed a cardioprotective effect in a ischemia and reperfusion model, with a reduction in the
number of apoptotic cells and the size of the infarcted area in these transgenic animals. These
beneficial effects were associated with a reduction in the cAMP/PKA/ICER signaling pathway and
an increase in the Bcl-2 protein. These data suggest a therapeutic potential of PDE3A1 activation
to prevent the deleterious effects of HF (Oikawa et al., 2013). Interestingly, activating mutations in
PDE3, responsible for a rare disease characterized by the combination of brachydactyly and
hypertension, confers cardioprotection despite the increase in afterload (Ercu et al., 2022). This
latter observation supports the postulate reported earlier (Karam et al., 2020), that increasing rather
than decreasing the activity of PDEs in cardiac tissue is beneficial. Despite the clinical limitations
and unfavorable findings in rodent models, PDE3 inhibitors had positive effects on isoprenaline-
induced myocardial injury in rats (Nakata et al., 2019), and showed favorable results on LV
remodeling in mouse thoracic aortic constriction (TAC) (Polidovitch et al., 2019). With Pde3a and
Pde3b gene KO mice PDE3A was found to be responsible for the beneficial milrinone effects in
TAC (Polidovitch et al., 2019). Perhaps the adverse effects of PDE3 inhibition do not involve PDE3A, although this remains controversial (Movsesian, 2003a).

In models of myocardial ischemia and reperfusion in dogs, a bolus injection of PDE3 inhibitor 30 minutes before coronary occlusion decreased the infarcted zone through the PKA/MAPK p38 pathway (Sanada et al., 2004; Sanada et al., 2001). This was likely due to PDE3B inhibition, as in *in vivo* and Langendorff-perfused heart models of acute ischemia and reperfusion using *Pde3a*- and *Pde3b*-deficient mice, the size of the infarcted area was reduced only in the latter (Chung et al., 2015). Thus, despite the adverse effects of chronic PDE3 inhibitors in HF, their pre-ischemia, single bolus use protects the myocardium against ischemic injuries (Sanada et al., 2001).

## PDE4

Numerous studies have shown the involvement of PDE4 in cardiac pathophysiology in animal models, suggesting the negative impact of its absence. Accordingly, the involvement of PDE4B has been shown in tachycardia, involving Cav1.2 channel current amplitude elevation, and in HF of various etiology, where PDE4B is decreased and its overexpression attenuates HF (Abi-Gerges et al., 2009; Karam et al., 2020; Leroy et al., 2011; Mika et al., 2019). In addition to PDE4B, *Pde4d*-deficient mice showed an increase in ventricular tachycardia when subjected to physical training and developed cardiomyopathy with age (Lehnart et al., 2005). PDE4D resides in close spatial relationship the type 2 ryanodine receptor (RyR2), and its disappearance increases RyR2 phosphorylation by PKA, resulting in increased Ca²⁺ leakage from the SR prompting arrhythmias (Lehnart et al., 2005). PDE4D was shown to be decreased in the myocardium in humans with idiopathic cardiomyopathy (Richter et al., 2011a). PDE4D5 was shown to counteract hypertrophic events in neonatal cardiomyocytes (Berthouze-Duquesnes et al., 2013). In this study, PDE4D5 forms a complex with β-arrestin 2 and its disruption leads to a change from non-hypertrophic β₂-adrenoceptor signaling to hypertrophic β₁-adrenoceptor signaling involving Epac1.

PDE4 is also expressed in human atrial myocytes, where it contributes to cAMP hydrolysis by controlling Ca²⁺ influx through Cav1.2 channels under basal conditions and under β-adrenoceptor stimulation (Molina et al., 2012). Inhibition of PDE4 in these atrial cells leads to an increase in the frequency of Ca²⁺ sparks and waves, leading to arrhythmias. In alignment with these findings, PDE4 activity has been shown to be reduced in patients with atrial fibrillation, a condition in which there is an alteration in the cAMP signaling pathway and Ca²⁺ dynamics (Molina et al., 2012). This highlights the involvement of this family of PDEs, not only at ventricular level but also in human atrial myocardium, in the control of cAMP levels, both under basal conditions and under adrenergic stimulation, thus protecting against the development of arrhythmias.

## PDE8

The importance of PDE8 in cardiac pathophysiology has yet to be fully explored. In *Pde8a* isoform KO mouse β-adrenoceptor-stimulated Ca²⁺ transients, *I*<sub>Ca,L</sub> and Ca²⁺ sparks in ventricular cardiomyocytes are increased (Patrucco et al., 2010). More recent work has demonstrated PDE8A and PDE8B in the human atrium, and a the role of PDE8B in persistent atrial fibrillation. PDE8B is located in the plasma membrane in the atrial myocytes of humans with paroxystic atrial fibrillation, where it controls cAMP levels. Its expression is increased in these patients and reduces cAMP levels in the vicinity of the Cav1.2 channel, leading to a reduction in *I*<sub>Ca,L</sub> current (Grammatika Pavlidou et al., 2023). This is the first evidence of a role for PDE8 as a regulator of L-type voltage-gated Ca²⁺ channels in human atrial myocytes.

### 2.1.3 | PDE regulation of cardiac function through cGMP

*PDE5A and PDE9A*
The cGMP-PDE PDE5A and PDE9A are expressed in the cardiomyocytes at low levels, and localized resp. at Z-discs, and at T-tubular structures and mitochondria. (Nagayama et al., 2008) (Lee et al., 2015). PDE5 modulates NO-stimulated guanylyl cyclase-1 (GC-1)-derived cGMP, and PDE9 shapes NP-guanylyl cyclase A-derived cGMP signaling (Lee et al., 2015; Takimoto et al., 2005). The result of cGMP signaling in the heart as shaped by PDE5 is well-studied and involves a number of downstream targets that affect hypertrophy, anti-oxidant defense, mitochondrial respiration, angiogenesis and proteostasis, that lead to protection against ischemic damage and hypertrophy, as carefully reviewed elsewhere (Samidurai et al., 2023). In intact hearts subjected to pathological pressure-overload, both PDE5A and PDE9A inhibition similarly reduced hypertrophy, fibrosis, and improved cardiac function. However, if animals were concomitantly administered with L-NAME to inhibit NO synthase, then only the PDE9A inhibitor conferred these effects. Removal of ovaries in females reduces estrogen-coupled NOS activation. When this was done, PDE5A blockade also became ineffective (Fukuma et al., 2020; Sasaki et al., 2014), whereas PDE9A inhibition still protected the myocardium (Mishra et al., 2021).

Oxidative stress differentially modifies the regulatory function of PDE5 vs. PDE9. Oxidation of cGMP-dependent kinase-1α (cGK-1α, also known as PKG1α) at two cysteine residues in the homodimer N-terminus (C⁴²-C⁴²) alters the localization of cGK-1α to assume a more diffuse cytosolic pattern (Nakamura et al., 2015; Nakamura et al., 2018b). Preventing this oxidation by expressing a C42S mutation in cGK-1α itself reduced the maladaptive response to pressure overload associated with maintenance of the kinase to the sarcolemmal membrane ((Nakamura et al., 2015). However, the outer membrane is not where PDE5A co-localizes, and likely as a result, PDE5A inhibition was ineffective in mice harboring this same cGK1α C42S mutation (Nakamura et al., 2018b), as was also the case for sGC activator (Nakamura et al., 2018b). By contrast, cell-studies indicate this does not apply to PDE9A inhibition that remains effective in this setting (Dunkerly-Eyring et al., 2022). The therapeutic implication is that inhibiting PDE9A is more likely to be impactful over PDE5A to activate cGK1α under conditions of oxidative stress, a common feature in many heart diseases.

Both PDE5A and PDE9A protein expression are increased in human HF (Besler et al., 2021; Lee et al., 2015; Pokreisz et al., 2009; Shan et al., 2012b), and their inhibition has been found protective in heart disease of various etiologies (Kamel et al., 2023). The common denominator herein is activation of cGK-1. Inhibition of PDE5A suppresses NFAT signaling that, in turn, is coupled to phosphorylation of the transient receptor canonical channel type 3 and 6 (Kiso et al., 2013; Koitabashi et al., 2010), interaction with regulator of G-coupled signaling protein Type 2 and Type 4 to counter Gq-coupled agonist stimulation (e.g. by angiotensin II) (Nishida et al., 2010; Takimoto et al., 2009), phosphorylation of tuberin (TSC2) at S¹³⁶⁵ (S¹³⁶⁴ in humans) to suppress activation of mTORC1 complex signaling (Ranek et al., 2019), and of carboxy terminus heart shock cognate interacting protein (CHIP, also *Stub1*) at S²⁰ (S¹⁹ in humans) to enhance protein quality control (Ranek et al., 2020). PDE9A inhibition achieves a number of similar downstream effects, although not as many have been tested to date.

PDE9A, but not PDE5A, localizes to mitochondria and modulates fatty acid oxidation (Mishra et al., 2021). PDE9A inhibition induced a thermogenic program in brown and white adipocytes (fat browning) both *in vitro* and in a model of severe, diet-induced obesity with and without pressure-overload stress on the heart. Chronic PDE9A inhibition reduced fat mass with no change in lean mass, improved heart function, and reduced liver steatosis. These changes required activation of the transcriptional regulator PPARα, a master controller of fat metabolism genes. Interestingly, PDE9A inhibition only caused these changes in males and ovariectomized females but had not impact in intact females. The likely cause is that estrogen suppresses PPARα transcriptional control over fat
catabolism genes. The exact link between mitochondrial PDE9A, PPARα and fat metabolism remains to be determined.

While many animal studies using PDE5A inhibition have shown benefits for various myocardial diseases, this has not been translated into the clinic (Borlaug et al., 2015; Cooper et al., 2022). Both positive and negative placebo controlled trials are available, and recent meta-analyses further emphasize the contradicting findings regarding a potential application in coronary disease or heart failure (Cao et al.; Samidurai et al., 2023; Soulaidopoulos et al.). Reasons are uncertain, but PDE5A inhibitors are widely used to treat pulmonary hypertension (PH) and erectile dysfunction, but HF indications remain elusive. PDE9A inhibition has been studied in a sheep model of acute heart failure, and alone or in combination with a neprilysin inhibitor (to further enhance cGMP levels that PDE9A would in turn regulate), improves heart and renal function (Scott et al., 2023; Scott et al., 2019). Clinical trials of PDE9A inhibition in HF are ongoing, and whether the promising animal data ultimately translate to humans for this PDE remains to be seen.

## PDE10A

Expression of the dual substrate PDE, PDE10A (Chen et al., 2020), increases in mouse and human HF myocardium. PDE10A inhibitor TP-10 pathological hypertrophy, fibrosis, and chamber dysfunction in various pressure overload models. Mice with genetic deletion of *Pde10a* displayed reduced pathological responses and less mortality after pressure overload stress. Although cAMP and cGMP are both increased, though their relative importance for PDE10 inhibition effects is unknown. Interestingly, inhibition of PDE10A ameliorated cardiac damage resulting from doxorubicin toxicity while simultaneously suppressing tumor growth in a breast cancer model (Chen et al., 2023). Thus, PDE10A is interesting in the new field of cardio-oncology.

## 2.2 | Smooth muscle cells, endothelial cells, and PDEs

### 2.2.1 | Clinical and pathophysiological background of vascular disease

Cardiovascular diseases account for around 30% of disability and mortality in economically developed countries, representing the largest health care problem according to the World Health Organization (WHO) (Pencina et al., 2019). Vascular aging and disease are one of the main drivers (Abdellatif et al., 2023). Vascular disease can be roughly divided into three categories of obstructive arterial disease, i.e., atherosclerosis leading to infarction, non-obstructive vascular aging, and aneurysms. Due to improved prevention and treatment of infarctions, vascular aging has increased (Jouabadi et al., 2023), which has led to increase of diastolic compared to systolic heart failure, and of dementia.

Vascular smooth muscle cells (VSMC) and endothelial cells (EC) are the main cell types involved in vascular homeostasis and disease. This involves the following functions: vascular tone and compliance regulation, angiogenesis, regulation of endothelial permeability and of blood coagulation. Pericytes, fibroblasts, adipocytes and resident macrophages play an auxiliary role in these functions. VSMC are the engine of the artery wall as they contract and relax. Contraction of VSMC depends on cytoplasmic $Ca^{2+}$ increase, followed by CaM-mediated myosin light chain kinase (MLCK) phosphorylation, and subsequent phosphorylation-induced actin-myosin fibre shortening. Dephosphorylation of actin-myosin is executed by myosin light chain phosphatase (MLCP) at low $Ca^{2+}$ causing relaxation. Rho kinase activation supports contraction by Ser-phosphorylation-induced inhibition of MLCP, thereby increasing $Ca^{2+}$ sensitivity of the VSMC. Herein, cAMP-PKA and cGMP-PKG signaling inhibit phosphorylation-induced MLCP deactivation and Rho kinase, respectively (see (Ito et al., 2022) for detailed review). Further, cAMP lowers $Ca^{2+}$ through modulation of L-type calcium channel opening (Lincoln and Cornwell, 1991;
Majed and Khalil, 2012). VSMC can also migrate, redifferentiate into myofibroblasts, or both, which leads to vessel hypertrophy and fibrosis. In the microvasculature, the VSMC are present as pericytes, which are in contact with the EC, regulating, for example, the blood brain barrier.

EC are the regulators of the vessel wall (Deanfield et al., 2007). They form a smooth layer covering the luminal side of the blood vessel. They have an anti-thrombotic and anti-inflammatory activity, suppress VMSC proliferation and provide an important barrier function. EC also release signaling factors to VSMC that change vascular tone, and mediate angiogenesis through the process of sprouting, with tip and stalk cells guiding neovascularization. Tonus regulation and vascular remodeling are regulated by VSMC guided by stimuli derived from the circulation or neurotransmitters released by sympathetic neurons. VSMC are also regulated indirectly via the release of vasoactive signaling factors from EC that respond to circulating substances, flow changes or hypoxia. Hypoxia is also the main stimulus for angiogenesis.

Vascular disease is characterized by the disturbed communication between EC and VSMC, low grade inflammation, endothelial permeability, and oxidative stress. Cellular senescence its associated secretory phenotype (SASP) is believed to play an important role herein (Clayton et al., 2023; Garrido et al., 2022; Jouabadi et al., 2023). In summary, inflammation, senescence, apoptosis, increased EC permeability, decreased EC anti-thrombotic and angiogenic function, VSMC proliferation, migration and fibrosis jointly contribute to vascular disease and related morbidities, such as HF and dementia. We discuss here the roles of cyclic nucleotide PDEs in these processes, focussing on VSMC and EC (Figure 12).

### 2.2.2| Role of PDEs in endothelial permeability and angiogenesis

#### EC effects through cAMP

The elevation of cytosolic and membrane cAMP resp. decreases (cytosolic cAMP) and increases endothelial (membrane cAMP) permeability (Sayner et al., 2006). The required local gradient is created through the intervention of cAMP-PDEs (Feinstein et al., 2012). Various complementary studies (Diebold et al., 2009; Seybold et al., 2005; Surapisitchat et al., 2007) have shown that cGMP levels steer endothelial PDE2 and PDE3 to alternatively regulate permeability by cAMP. Low levels of cGMP or PDE3A inhibition, which maintains high cAMP levels, decrease thrombin-induced permeability. As cGMP increases PDE2A activation bypasses PDE3A inhibition, thus switching from higher to lower cAMP levels, decreasing EC barrier function. Presumably this involves membrane cAMP levels. Endothelial PDE4 regulates microvascular permeability. Rolipram and roflumilast have been shown to attenuate vascular leakage in models of inflammation and sepsis (Schick and Schlegel, 2022). Further details describing downstream signaling and microdomains in endothelial permeability have been recently reviewed (Vina et al., 2021).

Important for angiogenesis, cAMP inhibits EC proliferation and migration (Netherton and Maurice, 2005). cAMP blocks proliferation through vascular endothelial growth factor (VEGF) signaling and a Rac1-mediated increase of NADPH-oxidase-produced reactive oxygen species (ROS) (Sadek et al., 2020). Accordingly, PDE2, 3 and 4 inhibition inhibits VEGF-induced proliferation and migration of cultured human EC, and angiogenesis in the chick chorioallantoic membrane model (Netherton and Maurice, 2005). Inhibition of these PDEs might also account for the anti-angiogenic effect of curcumin (Abusnina et al., 2015). Downstream of VEGF, PDE2 inhibition involves the cyclin A - p27<sup>kip1</sup>, whereas combined PDE2 and 4 inhibition targets the cyclin D1 - p21<sup>waf1/cip1</sup> cell cycle pathway (Favot et al., 2004). In contrast, microvascular EC adhesion depends on cAMP-EPAC, and is regulated by PDE3B or PDE4D (Netherton et al., 2007). Furthermore, a role for pericyte PDE3A in cAMP regulation relevant for angiogenesis and permeability was proposed (Spiranec et al., 2018). Interestingly, PDE3 or 4 inhibition improved eNOS activity and NO-mediated tube formation in cultured human aortic EC through cAMP/PKA and phosphatidylinositol
3-kinase/Akt (Hashimoto et al., 2006). The result of the conflict with the anti-angiogenic effects of cAMP and of PDE2, 3 and 4 inhibition is unclear. Unfortunately, *in vivo* experiments in mammal angiogenesis models with PDE2, 3 and 4 inhibitors are lacking. In addition, the role of other cAMP-selective PDE subtypes in the endothelium is unknown.

### EC effects through cGMP

cGMP is pro-angiogenic, and PDE1 and PDE5 emerge when cultured bovine EC pass from a quiescent to a proliferative phenotype (Keravis et al., 2000). sildenafil increases *in vitro* tube formation and *in vivo* angiogenesis in a rat embolic stroke model, improving neurological recovery in the latter (Zhang et al., 2005b; Zhang et al., 2003). Sildenafil protects against renal microvascular degeneration in the mouse streptozotocin diabetes model (Pofi et al., 2017). Sildenafil and tadalafil were also reported to increase endothelial progenitor cell number in healthy humans and patients with erectile dysfunction (Bocchio et al., 2008; Foresta et al., 2009). The ongoing discussion about the identity and origin of endothelial progenitor cells and the general lack of clinical trials showing a positive effect of cell therapy make predictions of the therapeutic relevance of such observations challenging (Chambers et al., 2021).

The role of cGMP in endothelial permeability is unclear; an increase, decrease or no effect have all been reported (Bodiga et al., 2020; Bohara et al., 2014; Lakshminarayanan et al., 2000; Rentsendorj et al., 2008; Yin et al., 2014). Differences in EC origin, permeability stimulus and measurement methods, and data interpretation may underlie the confounding results. With respect to interpretation, it is sometimes overlooked that NO increases permeability through catenins and not PKG (Marin et al., 2012). The role of cGMP-PDEs in regulating permeability has not been reported. Activation of pericytes leads to endothelial cell leakage, worsens NO-mediated vasodilation and is anti-angiogenic. This is prevented by PDE5 inhibitor sildenafil in a pericyte-EC co-culture model (Majumder et al., 2007).

In summary, inhibition of PDE2, 3 and 4 might mitigate pathogenesis that involves increased endothelial permeability and angiogenesis. PDE5 inhibition seems applicable in conditions where angiogenesis is favorable. The specific role of PDE1 or other cGMP-PDE has not been evaluated.

### 2.2.3| Role of PDEs in arterial wall remodeling and vascular tone

#### Remodeling effects through cAMP

Anti-proliferative effects in VSMC have been described for cAMP. Genetic knockout of *PDE1C* attenuates injury-induced neointima formation, involving cAMP/PKA and platelet-derived growth factor receptor β (Cai et al., 2015). Furthermore, PDE1C was shown to be involved in VSMC migration through a concerted interplay between adenylate cyclase 8 and orai1 $Ca^{2+}$ channels (Brzezinska and Maurice, 2019; Brzezinska et al., 2021). PDE3 inhibition decreased neointima formation after arterial injury in rat (Indolfi et al., 1997; Inoue et al., 2000; Ishizaka et al., 1999), but had no effect on atherosclerosis in ApoE-KO mouse (Umebayashi et al., 2018). Further development as an anti-restenosis therapy has not been pursued, possibly due to competition of cytostatin-eluting stents. Nevertheless, vascular aging-related intimal thickening and arterial stiffening might be considered as a future application.

Aneurysm formation is a disease with often dramatic consequences. Early investigations demonstrated that theophylline, a non-selective PDE inhibitor, induces aneurysms in chick embryos (Gilbert et al., 1977). In contrast, deletion of *Pde1c* and inhibition of PDE1, PDE3 or PDE4 with IC86340, cilostazol or rolipram, respectively, was shown to be protective in abdominal aneurysm models (Gao et al., 2022; Umebayashi et al., 2018; Varona et al., 2021; Zhang et al., 2021; Zhang et al., 2011b). Cilostazol effects were associated with the reduced inflammation, ROS levels and
matrix metalloproteinases 2 and 9 levels (Umebayashi et al., 2018; Zhang et al., 2011b). Of note, Furthermore, it remains unclear if rolipram mediates its effects by inhibiting PDE4B in infiltrating inflammatory cells or PDE4D in VSMC (Gao et al., 2022; Varona et al., 2021). In summary, inhibition of cAMP-PDEs is a viable concept for the prevention of aneurysms.

### Remodeling effects through cGMP

Dual PDE1/PDE5 inhibition with SCH51866 had an antiplatelet and anti-hypertrophic effect in a hypertensive rat angioplasty model, whereas the selective PDE5 inhibitor, E4021, only decreased platelet adhesion (Vemulapalli et al., 1996). Zaprinast, which also targets PDE9 and 11, was reported to reduce neointima formation (Keswani et al., 2009). Vardenafil inhibited fibroblasts-to-myofibroblast transdifferentiation in a mouse model of focal segmental glomerulosclerosis (Hu et al., 2022), highlighting possibilities for antifibrotic treatment. Selective PDE1 inhibition has emerged as a target for diseases related to VSMC and adventitial fibroblasts (Roks, 2022; Yan, 2015; Zhou et al., 2010). Genetic knockout of *PDE1C* or inhibition of PDE1 with IC86340 attenuated injury-induced neointima formation. However, the mechanism of action of cGMP is unclear given that, unlike cAMP (see above), PKG does not regulate platelet-derived growth factor receptor β (Cai et al., 2015). Similar to PDE3 inhibition, development of PDE1 and PDE5 inhibitors as clinical drugs against in-stent restenosis might prove complicated, whereas attenuation of vascular aging might be a possible indication. Indeed, in a mouse model of accelerated aging, chronic sildenafil treatment significantly improved vasomotor function (Golshiri et al., 2020).

With respect to aneurysms, several cases of aortic dissection or subarachnoid hemorrhage have been reported in patients with erectile function taking sildenafil. Although this was attributable to an effect on VSMC, mechanistic evidence has not been reported (De-Giorgio et al., 2011; Edwin et al., 2009; Tiryakioglu et al., 2009). It is also unclear if the protective effect of PDE1 inhibition against arterial wall thinning described above for cAMP, also involves cGMP (Zhang et al., 2021). Currently, it is not understood how both cyclic nucleotides interact to regulate arterial wall thickening (neointima formation) and thinning (aneurysms).

### Regulation of vascular tone by cAMP

PDE2 inhibition was shown to unmask or improve cAMP-mediated relaxation of the pulmonary artery and aorta of rats exposed to hypoxia to induce pulmonary hypertension (Bubb et al., 2014). PDE3A is known to be involved in brachydactyly-short stature-hypertension, a rare inherited disease also known as Bilginturan syndrome (Bilginturan et al., 1973). Due to a diverse pallet of gain-of-function mutations in these patients, PDE3A activity in VSMC increases, causing hypertension (Ercu et al., 2023). Patients respond to most standard antihypertensive treatments, except for inhibitors of the renin-angiotensin system, because this system is not activated. Left untreated, patients die from stroke at around the age of 50 years. Due to the detrimental cardiac effects, chronic PDE3 inhibition is not considered to be a logical alternative when standard antihypertensive treatment is effective.

Together with PDE10A, PDE3A is a target for papaverine (Li et al., 2023), a drug used to stop vasospasms in claudication. Based on their vasodilator and anti-thrombotic actions, the PDE3 inhibitors, cilostazol and milrinone, are prescribed for intermittent claudication; their potential utility in vasospasm after cerebral hemorrhage is under clinical evaluation (Lakhal et al., 2021; Saber et al., 2018).

### Regulation of vascular tone by cGMP

PDE5 inhibition has been considered to improve clinical outcomes after stroke. A meta-analysis of oral sildenafil effects after subarachnoid hemorrhage suggested improved long-term anatomical and
functional outcomes (Faropoulos et al., 2023). This is in contradiction with the alleged increased
risk for stroke observed in patients with erectile dysfunction mentioned above. Explorative
pharmacoepidemiological research might be necessary before initiating an intervention study.

In rats, under normoxic conditions, relaxations of the pulmonary artery induced by atrial natriuretic
peptide (ANP) and NO are modified by the cAMP-PDE PDE2, whereas this was restricted to ANP
in vessels harvested from hypoxic animals (Bubb et al., 2014). In the aorta, PDE2 inhibition
increased NO-mediated relaxation, and this was also lost after exposure to hypoxia. Thus, the
regulation of ANP/pGC/cGMP signaling by PDE2 present under healthy conditions appears to
depend on the type of blood vessels or, perhaps, the hemodynamic conditions to which it is exposed.
Hypoxia is known promote vasoconstriction (Haynes et al., 1996) and, apparently, this centers
around the regulation of particulate guanylyl cyclase-generated cGMP. The NO/sGC/cGMP
regulation by PDE2 is abolished by hypoxia. Almost complete loss of PDE2 transcripts and protein
was observed after hypoxia, whereas paradoxically cytosolic PDE2 activity was still measurable
(Bubb et al., 2014). Perhaps a differential involvement of membrane- vs. cytosolic-located PDE2
might explain this discrepancy. Microdomain regulation of PDE2 has been demonstrated in
myocardial cells (Castro et al., 2006a) and in stellate neurons of spontaneously hypertensive rats
(Li et al., 2022), but remains to be elucidated in vascular cells.

The role of PDE1 and PDE5 in blood pressure and vasomotor function has been well-investigated, has been comprehensively reviewed recently (Roks, 2022), and is summarized here. Whereas PDE1 is Ca²⁺/CaM-dependent, PDE5 is activated by PKA- and PKG-mediated phosphorylation. Activation of PDE5 is cGMP-dependent and suppressed by NO inhibition (Teixeira et al., 2006; Wyatt et al., 1998). In contrast to PDE5, PDE1 has cAMP-metabolizing activity (Francis et al., 2011). Thus, PDE1 and PDE5 are respectively active during contractile versus relaxing conditions, and their (patho)physiological role can be expected to essentially differ.

Various genetic models implicated PDE1A in VSMC myosin-actin regulation, which involves MLCK phosphorylation, and blood pressure (Nagel et al., 2006; Wang et al., 2017b). In contrast *Pde1c* deletion has no blood pressure effect in young adult mice (Ahmad et al., 2015; Knight et al., 2016a; Zhang et al., 2018b). PDE1 or PDE5 inhibition leads to modest blood pressure lowering in healthy animals and humans (Gilotra et al., 2021; Herrmann et al., 2000; Jüttner, 2022; Laursen et al., 2017; Miller et al., 2009b) (Dey et al., 2020) Hashimoto et al., 2018). The PDE1-selective inhibitors, Lu AF41228 and Lu AF58027, produced concentration-related relaxations of isolated rat mesenteric arteries in an NO- and cAMP-dependent manner (Laursen et al., 2017). The PDE1 inhibitor, lenrispodun, improved NO-mediated vasodilation and appears to be dominant over PDE5 inhibition in mouse aorta with aged VSMC (Ataei Ataabadi et al., 2021).

In physiological vs. pathological conditions expression of PDE1 and PDE5 is differentially affected (Chen et al., 2018; Rybalkin et al., 1997; Rybalkin et al., 2002; Yan et al., 1996; Zhang et al., 2021). Recently, it was found that in the aorta of mice with aged VSMC, PDE1, rather than PDE5, impairs NO-cGMP-mediated signaling (Ataei Ataabadi et al., 2021). When NO-cGMP-PKG signaling is optimal, PDE5 represents a negative feedback mechanism to oppose relaxation. Conversely, under disease conditions where NO-cGMP signaling is lowered and Ca²⁺-induced constriction may be increased, PDE1 may further augment Ca²⁺ sensitivity by inactivating cGMP and cAMP. Thus, PDE1 appears to be the disease associated PDE in VSMC (Roks, 2022). Indeed, in mouse models of accelerated aging lenrispodun countered vascular aging more effectively than sildenafil (Golshiri et al., 2020; Golshiri et al., 2021b). In addition, PDE1, but not PDE5, appears to play a role in the regulation of vascular smooth muscle cell senescence (Bautista Nino et al., 2015; Zhang et al., 2021). PDE5 expression was reduced in *in vitro* aged VSMC (Wyatt et al., 1998), whereas PDE1 levels were increased in senescent human VSMC, aged mouse aorta and VSMC of mouse aortic aneurysms (Bautista Nino et al., 2015; Zhang et al., 2021). Also, PDE1 inhibition attenuated VSMC
senescence (Bautista Nino et al., 2015; Zhang et al., 2021). This effect might be explained by the cAMP-mediated activation of sirtuin-1, which is an important in nutrient sensing – energy metabolism, during PDE1 inhibition (Zhang et al., 2021). The role of cGMP still needs to be further interrogated especially given that soluble guanylyl cyclase activators have anti-senescent effects in the aorta of mice with accelerated aging (Ataei Ataabadi et al., 2022). These observations for PDE1 vs. PDE5 lead to a paradigm shift in their role in healthy and aged vascular tissue.

Of potential clinical relevance are epidemiological studies that have found single nucleotide polymorphisms (SNPs) in the *PDE1A* gene that are associated with diastolic blood pressure, mean arterial pressure and common carotid intima-media thickness (Bautista Nino et al., 2015; Tragante et al., 2014). *PDE1C* polymorphisms did not associate with vascular aging variables. It is unknown if this is due lack of functional mutations. Further discussion can be found elsewhere (Ataei Ataabadi et al., 2020; Golshiri et al., 2019; Roks, 2022).

PDE1 has also been proposed to play a role in nitrate tolerance during treatment of recurrent angina pectoris (Kim et al., 2001). However, these experiments employed vinpocetine, which is not selective for PDE1 (Dunkern and Hatzelmann, 2007). The impact of PDE1 on vasodilation or blood pressure in relation to cAMP has not been elucidated. The discrete effect of PDE1 inhibition on blood pressure might be explained by opposing vasorelaxation vs. positive inotropic effects (Gilotra et al., 2021). In conclusion, the balance of evidence suggests that inhibition of PDE1 or PDE5 may not be optimal approach to treat hypertension. Nevertheless, a role for these PDEs in aging-related loss of vasodilation capacity warrants further inspection.

## 2.3 | PDE inhibitions for pulmonary diseases

PDE inhibition is a prominent target in pulmonary disease. The groups of pulmonary diseases that are target for PDE intervention is diverse, involving different etiologies that implicate pulmonary, vascular and inflammatory cell types, and the cardiovascular system. It comprises pulmonary hypertension, pulmonary fibrosis, chronic obstructive pulmonary disease and asthma. These diseases will now be reviewed in this order.

### 2.3.1 | Pulmonary hypertension

Pulmonary hypertension (PH) is a debilitating and potentially fatal cardiovascular disorder (Schermuly et al., 2011) in which PDEs play a pivotal role (Ahmad et al., 2015). This section explores the diverse roles of PDEs in the pathogenesis and treatment of PH.

#### Pathophysiology of PH

PH is a complex cardiovascular disorder characterized by elevated pulmonary arterial pressure (Schermuly et al., 2011). It increases afterload on the cardiac right ventricle (RV). If left untreated, it can result in right ventricular dysfunction and ultimately HF (Schermuly et al., 2011). The adaptation of the RV to the increased afterload is crucial for survival (Tello et al., 2023). PH can be classified into five main groups, each with distinct etiologies and pathophysiological mechanisms (Humbert et al., 2022).

#### Subtypes and classification of PH

Pulmonary arterial hypertension (PAH; Group 1) is the best-known subtype, and is characterized by pathological changes in the small pulmonary arteries. Changes include vasoconstriction, vascular remodeling, and endothelial dysfunction (Schermuly et al., 2011). PAH is often idiopathic, but can also be caused by connective tissue diseases, congenital heart diseases, or chemical poisoning (D'Alto and Mahadevan, 2012; Montani et al., 2013; Zanatta et al., 2019). Mutations in
the *BMPR2* and other genes have been implicated in the development of heritable PAH (Evans et al., 2016; Morrell et al., 2019).

PH associated with left-sided heart diseases such as HF, valvular diseases, or LV dysfunction is classed as Group 2 PH (Vachieri et al., 2019). Increased left atrial pressure is transmitted backward to the pulmonary circulation, and can lead to pulmonary vascular abnormalities resulting in increased pulmonary vascular resistance (PVR) (Rosenkranz et al., 2016).

PH associated with lung diseases (particularly COPD and interstitial lung diseases) is classed as Group 3 PH (Gredic et al., 2021; Humbert et al., 2022). Hypoxia and inflammation are key factors contributing to vascular changes in this group (Ye et al., 2023).

Chronic thromboembolic PH (Group 4) is a unique form of PH caused by chronic pulmonary thromboembolism, where blood clots obstruct the pulmonary arteries (Ghofrani et al., 2021; Pepke-Zaba et al., 2017). Although normally addressed with pulmonary thromboendarterectomy, some patients require additional therapy or lung transplantation (Ghofrani et al., 2021).

PH with unclear and/or multifactorial mechanisms (Group 5) encompasses a heterogeneous collection of PH conditions that result from hematological or systemic disorders, or that have no clear cause (Al-Qadi et al., 2020; Lahm and Chakinala, 2013).

## Key mechanisms contributing to PH

One of the hallmark features of PH is increased pulmonary vasoconstriction and endothelial dysfunction (Evans et al., 2021; Hopkins and Stickland, 2023; Kurakula et al., 2021). Endothelial dysfunction disrupts the balance between vasodilators (e.g., NO and prostacyclin) and vasoconstrictors (e.g., endothelin-1, 5-hydroxytryptamine, and thromboxane) (Huertas et al., 2018). Endothelin-1, in particular, plays a central role in promoting vasoconstriction and vascular remodeling in PH, both of which increase PVR (Chester and Yacoub, 2014).

Vascular remodeling in PH involves thickening of the pulmonary artery walls due to cellular proliferation and hypertrophy (Leopold and Maron, 2016; Shimoda, 2020; Shimoda and Laurie, 2013), involving all layers. This structural alteration narrows the vascular lumen. Endothelial dysfunction contributes to inflammation and thrombosis within the pulmonary arteries (Evans et al., 2021; Kurakula et al., 2021). Chronic inflammation is increasingly recognized as a key player in the pathogenesis of PH, especially in Group 3 and 5 (Frid et al., 2020; Yoo and Marin, 2022). Inflammatory cells, growth factors, cytokines, and signaling pathways such as the platelet derived growth factor-β pathway are implicated in the vascular remodeling that increases PVR (Liu et al., 2022; Shimoda, 2020; Wang et al., 2022) (Leopold and Maron, 2016).

Understanding the pathophysiology of PH is crucial for developing effective treatments. This section will further delve into the role of PDEs in the multiple pathways and mechanisms that contribute and serve as therapeutic targets in PH.

## The importance of cAMP and cGMP in pulmonary vascular physiology and PH

In the context of PH, cAMP and cGMP play a central role in regulating pulmonary vascular tone, cellular proliferation, and inflammation and are important downstream mediators of NO, natriuretic peptides, prostacyclin, and other hormones (Bobin et al., 2016; Chen et al., 2013). The cyclic nucleotides exert vasodilatory effects in the pulmonary circulation in the same manner as described in Chapter 2.2 for arteries in general (Bobin et al., 2016; Lincoln and Cornwell, 1991; Majed and Khalil, 2012)(Ghofrani et al., 2002b; Klinger and Kadowitz, 2017). Furthermore, inhibition of VSMC proliferation by cAMP is critical in vascular remodeling seen in PH (Growcott et al., 2006). cGMP inhibits platelet activation and aggregation, further preventing thrombosis in the pulmonary
arteries, a common complication of PH (Degjoni et al., 2022; Wen et al., 2018). In PH, reduced levels of cAMP and cGMP due to increased PDE activity contribute to enhanced vasoconstriction, abnormal cellular proliferation, and inflammation, all of which are hallmark features of the disease (Bubb et al., 2014; Muraki et al., 2019). Hence, targeting PDEs to restore cyclic nucleotide levels represents a promising therapeutic approach in PH (Wilkins et al., 2008). The next section reviews the role of PDE subtypes and their potential as drug targets in PH.

### 2.3.2 Role of PDEs in the pathophysiology and as drug targets in PH

#### cAMP-PDEs in PH

##### PDE3

PDE3 plays a pivotal role in vascular tone regulation and cell proliferation in PH via cAMP (Chen et al., 2009a; Thelitz et al., 2004; Tilley and Maurice, 2002). PDE3 counteracts the vasodilation, contributing to vasoconstriction and increased PVR (Busch et al., 2010; Dillard et al., 2020; Jeffery and Wanstall, 1998). By blocking PDE3 activity, the levels of cAMP are preserved, leading to enhanced vasodilation and inhibition of VSMC proliferation (Dony et al., 2008; Murray et al., 2002). PDE3 inhibitors such as milrinone and cilostazol have shown promise in PH in experimental models and early-phase clinical trials (Chen et al., 1997; James et al., 2016). Milrinone, originally developed as an inotropic agent for heart failure, has been repurposed for PH therapy (Bassler et al., 2006; James et al., 2016). However, its use is limited by its short half-life and the need for continuous intravenous infusion. Cilostazol is used primarily for its antiplatelet and vasodilatory effects in peripheral arterial disease (Manolis et al., 2022). Some studies have explored its potential in PH (Chang et al., 2008; Ito et al., 2021), but more research on its efficacy and safety in patients with PH is required.

##### PDE4

PDE4 has been implicated in the pathophysiology of PH (Dony et al., 2008; Izikki et al., 2009). One of the distinguishing features of PH is chronic inflammation within the pulmonary vasculature (Pugliese et al., 2015). In this context, PDE4 plays a crucial role (Li et al., 2018). PDE4 isoforms are abundantly expressed in various immune cells, including macrophages and lymphocytes (Schick and Schlegel, 2022). Reduced cAMP levels result in heightened inflammation and immune cell activation (Raker et al., 2016), identifying PDE4 inhibitors as potential therapeutic agents in PH (Du et al., 2023). PDE4 increases intracellular cAMP levels in immune cells, leading to the downregulation of pro-inflammatory cytokines and reduced immune cell activation (Li et al., 2018). This can complement the effects of existing PH therapies that primarily focus on vasodilation and vascular remodeling (Meloche et al., 2013). PDE4 inhibitors have been explored in experimental PH (Izikki et al., 2009) where improves pulmonary hemodynamics, reduces vascular remodeling, and attenuates inflammation (De Franceschi et al., 2008; Izikki et al., 2009; Seimetz et al., 2015). PDE4 inhibitors require careful dosing to avoid gastrointestinal side effects (Kumar et al., 2013; Li et al., 2018). Therefore, challenges remain in optimizing dosing regimens, addressing potential side effects, and conducting larger-scale clinical trials to establish safety and efficacy in patients with PH.

#### cGMP-PDEs

##### PDE5

PDE5 is particularly significant in the pathophysiology and treatment of PH (Barnes et al., 2019). PDE5 is highly expressed in the pulmonary vasculature, making it a central player in regulating cGMP levels in the lung (Tang et al., 2017; Wilkins et al., 2008). By hydrolyzing cGMP PDE5 contributes to the vasoconstriction and vascular remodeling seen in PH (Chen et al., 2013; Tang et
al., 2017). PDE5 is strongly upregulated in the medial layer in lungs (Wharton et al., 2005) as well as in the hypertrophied RV of patients with PAH (Nagendran et al., 2007). Sildenafil (Revatio®) and tadalafil (Adcirca®) have proven their efficacy in placebo-controlled multicenter clinical trials (Barnes et al., 2019; Galie et al., 2009; Galie et al., 2005; Ghofrani et al., 2004a; Montani et al., 2009), improving exercise capacity, hemodynamics, and quality of life (Ghofrani et al., 2004b; Michelakis et al., 2003; Pepke-Zaba et al., 2008). Tadalafil, with its longer half-life (Forgue et al., 2006), offers the convenience of once-daily dosing. Some patients may develop resistance or suboptimal responses on PDE5 inhibitors over time (Barnes et al., 2019; Garcia et al., 2023; Unegbu et al., 2017), and research to enhance their effectiveness is ongoing. Combination therapy with other PH-targeted agents, such as prostacyclin analogs or endothelin receptor antagonists, is being investigated to address the multifactorial nature of PH and provide a more comprehensive treatment strategy (Humbert and Ghofrani, 2016). In this regard, it was shown experimentally that subthreshold doses of specific PDE inhibitors enhanced the pulmonary vasodilatory response to nebulized prostanoids (Schermuly et al., 1999; Schermuly et al., 2001). Clinically, PDE inhibition by sildenafil (Ghofrani et al., 2003) or the PDE3/4 dual-selective inhibitor tolafentrine (Ghofrani et al., 2002) amplified the pulmonary vasodilatory response to the inhaled prostacyclin analog iloprost and provided the basis for the implementation of combination therapy for PAH. This combination strategy aims to address vasoconstriction, vascular remodeling, inflammation, and maintenance of gas exchange (Ruopp and Cockrill, 2022). The combination of the PDE5 inhibitors sildenafil or tadalafil with endothelin receptor antagonists and/or prostacyclin analogs is the gold standard for the treatment of PAH and is included in the European PH treatment guidelines (Humbert et al., 2022).

Advantages of PDE5 inhibition in PH management include oral administration, relatively few side effects, and improvements in exercise capacity and quality of life (Buckley et al., 2010). Limitations include response variability (patient and PH subtype-dependent) the potential for drug interactions and the need for careful monitoring of adverse effects, such as hypotension and visual disturbances (Rashid, 2005). Patient stratification based on underlying causes and pathophysiology is therefore very important (Humbert et al., 2022).

Ongoing research is exploring novel PDE5 inhibitors, alternative dosing regimens, and innovative drug delivery methods (Rashid et al., 2017). Furthermore, a deeper understanding of the heterogeneity of PH, clinical presentation, and the genetic factors influencing PDE5 responsiveness is guiding personalized treatment approaches (Savale et al., 2018; Wilkins, 2021). Also challenges in combination therapy are being addressed (Burks et al., 2018; Dos Santos Fernandes et al., 2017; Lajoie et al., 2017), as well as interactions with drugs given for other indications (Dos Santos Fernandes et al., 2017; Lajoie et al., 2017).

## Dual-substrate PDEs in PH

### PDE1

PDE1 isoforms have garnered attention for their involvement in PH pathogenesis (Schermuly et al., 2007). Elevated PDE1 activity has been observed in PH in animal models and patients (Crosswhite and Sun, 2013; Murray et al., 2007; Schermuly et al., 2007). This heightened activity leads to reduced levels of both cAMP and cGMP, contributing to vasoconstriction, enhanced proliferation of VSMCs, and inflammation (Crosswhite and Sun, 2013; Murray et al., 2007; Schermuly et al., 2007). Importantly, the breakdown of the second messengers limits the efficacy of prostacyclin and NO. By blocking PDE1 activity, it is possible to increase intracellular levels of cAMP and cGMP, promoting vasodilation and inhibiting VSMC proliferation (Murray et al., 2007; Schermuly et al., 2007). In experimental models of PH PDE1 inhibitors improved pulmonary hemodynamics, reduced PVR, and attenuated vascular remodeling (Crosswhite and Sun, 2013; Evgenov et al., 2006;
Schermuly et al., 2007). These findings support the idea that PDE1 inhibitors could be a valuable addition to the PH treatment arsenal.

## PDE2

Although less extensively studied than other PDE isoforms in PH, cellular and functional studies have shown that PDE2 inhibition elicits pulmonary vasodilation, prevents pulmonary vascular remodeling, and reduces right ventricular hypertrophy in experimental PH (Bubb et al., 2014). As PDE2 is also highly expressed in the failing heart (Bobin et al., 2016), further investigations into the mechanisms and effects of PDE2 dysregulation in the RV could offer valuable insights for the development of novel treatment strategies aimed at restoring cyclic nucleotide balance and ameliorating the vascular abnormalities associated with this debilitating disease.

## PDE10

PDE10 is present in the pulmonary vasculature (Tian et al., 2011). In experimental models of PH, PDE10 inhibitors have been shown to increase cAMP levels, promote vasodilation, and reduce pulmonary vascular remodeling (Huang et al., 2019b; Tian et al., 2011). These findings suggest that PDE10 inhibition could offer a novel therapeutic approach for PH. The translation of preclinical findings into clinical applications is an ongoing process. The long-term safety and tolerability of PDE10 inhibitors need to be thoroughly evaluated in clinical trials, particularly considering the strong expression of PDE10 in the brain. Clinical trials evaluating the safety and efficacy of PDE10 inhibitors in schizophrenia are underway and may provide relevant information to guide clinical development in PH.

### 2.3.3 | *Pulmonary fibrosis: epidemiology of pulmonary fibrosis*

Progressive pulmonary fibrosis (PPF) and idiopathic pulmonary fibrosis (IPF) are interstitial lung diseases (ILDs) of known and unknown origin, respectively, and are characterized by progressive fibrosis of the pulmonary interstitium leading to an impairment of lung function and finally to death (Cottin et al., 2019; Kolb and Vasakova, 2019; Raghu et al., 2014; Raghu et al., 2022). Up to 40% of patients with ILDs may develop a progressing fibrotic phenotype which is associated with high mortality, with median post-diagnosis survival in patients with IPF estimated at 2–5 years (Raghu et al., 2014). Progression of fibrosing ILD is reflected especially in decline in pulmonary function, decrease in exercise capacity, deterioration in quality of life, worsening of cough and dyspnea, acute exacerbations, and increase of morphologic abnormalities (Cottin et al., 2019; Kolb and Vasakova, 2019). In patients with IPF, decline of forced vital capacity (FVC), the maximum amount of air one can forcibly exhale from the lungs after fully inhaling, is a well-established predictor of mortality, and some blood biomarkers including the pneumyocyte type 2-regeneration marker KL-6, surfactant protein (SP)-D and extracellular matrix remodeling enzyme matrix metalloproteinase (MMP)-7 have been shown to be prognostic for disease progression (Karampitsakos et al., 2023). Currently, the only approved treatments to slow disease progression in IPF are nintedanib, a tyrosine kinase inhibitor, which is also indicated for PF-ILD, and pirfenidone, a pyridone with an unknown mechanism of action (Richeldi et al., 2018). However, the medical need in IPF and other progressive fibrosing ILDs remains high, with lung transplantation representing the only potentially curative treatment for IPF.

#### Pathophysiology of pulmonary fibrosis

With respect to IPF pathophysiology, there has been a paradigm shift in recent years from a chronic inflammatory disorder to a primarily fibrotic disease. The current hypothesis of disease pathogenesis involves sustained alveolar epithelial micro-injury, followed by a disordered repair, and wound healing response. This is characterized by uncontrolled activation of lung fibroblasts
and differentiation to myofibroblasts, resulting in excessive extracellular matrix deposition, scarring of lung parenchyma, leading to loss of pulmonary function (Sgalla et al., 2018; Spagnolo et al., 2018). The wound-healing process includes an inflammatory phase, with the involvement of inflammatory cells and increased levels of cytokines and growth factors, creating a biochemical environment supporting chronic tissue remodeling. Nintedanib and pirfenidone, have displayed antifibrotic and anti-inflammatory activity (Heukels et al., 2019), but are only slowing down the decline in FVC. Both compounds evoke significant side effects, leaving room for a better tolerated and efficacious medication. PDE4 inhibition offers this via anti-inflammatory and anti-fibrotic effects of cAMP (Richeldi et al., 2022). Increase in cGMP levels by inhibition of PDE5 is ineffective, eg. when sildenafil is given on top of nintedanib, in IPF (Kolb et al., 2018).

### 2.3.4 | Role of PDEs in the pathophysiology and as drug targets in pulmonary fibrosis

#### cAMP-PDEs: PDE4

In pulmonary fibrosis (PF), the possible application of PDE targeting is centered around PDE4. Very scarce evidence is available with respect to other PDEs although PDE1, PDE5 and PDE9 have been mentioned as possible targets (Balaha et al., 2023; Ren et al., 2017; Wu et al., 2020; Yildirim et al., 2010). PDE4 has traditionally been implicated in the regulation of inflammation and the modulation of immune-competent cells, and data for the three selective pan-PDE4 inhibitors (active on PDE4A-D) currently marketed (roflumilast, apremilast and crisaborole) support a beneficial role for PDE4 inhibition in inflammatory and/or autoimmune diseases (Hatzelmann et al., 2010; Li et al., 2018; Sakkas et al., 2017).

Important for PF, in bleomycin-induced PF in rat rolipram initially was shown to inhibit fibrotic score, content of fibrosis marker hydroxyproline, and of the inflammatory marker, serum TNF-α (Pan et al., 2009). A second early study in mice and rats showed that oral roflumilast was active both in preventive and in therapeutic protocols (Cortijo et al., 2009). Cilomilast was shown to inhibit late-stage lung fibrosis and tended to reduce collagen content in the mouse bleomycin model (Udalov et al., 2010). In a murine model of lung fibrosis targeting type II alveolar epithelial cells, roflumilast lowered lung hydroxyproline content and mRNA expression of TNF-α, fibronectin (FN), and connecting tissue growth factor (CTGF) (Sisson et al., 2018). Again, roflumilast was active both in a preventive and in a therapeutic regimen, and under the latter conditions appeared to be therapeutically equi-effective with pirfenidone and nintedanib. Furthermore, in a mouse model of chronic graft-versus-host disease, lung fibrosis was attenuated by roflumilast (Kim et al., 2016).

PDE4 inhibitors might act indirectly via inhibition of pro-inflammatory cells (like alveolar macrophages) and their mediators, and/or directly on fibrotic cell types. In human embryonal fibroblast models PDE4 and prostaglandin E₂ (PGE₂), which increases cAMP, importantly interact: rolipram and cilomilast inhibited FN-induced chemotaxis and contraction of collagen gels, an effect that involved PGE₂ (Kohyama et al., 2002). The inhibition of the fibroblast functions by cilomilast could be modulated by cytokines like IL-1β or IL-4. In addition, TGF-β1-stimulated FN release was inhibited by a PDE4 inhibitor, paralleled by stimulation of PGE₂ release as a positive feedback mechanism (Togo et al., 2009). Roflumilast N-oxide, the active metabolite of roflumilast, in the presence of PGE₂ was shown to inhibit intercellular adhesion molecule-1 and eotaxin release stimulated by TNFα, proliferation stimulated by basic fibroblast growth factor (bFGF) plus IL-1β, as well as TGFβ1-induced α-SMA, CTGF, and FN mRNA expression in the presence of IL-1β (Sabatini et al., 2010). In normal human lung fibroblasts, TGF-β-induced fibroblast-to-myofibroblast conversion assessed by α-SMA expression was shown to be inhibited by piclamilast in the presence of PGE₂ (Dunkern et al., 2007). In subsequent papers, the same investigators showed the inhibition of IL-1β plus bFGF-stimulated fibroblast proliferation by piclamilast and the importance of COX-2 and PGE₂ (Selige et al., 2010). The importance of a cAMP trigger for the
modulation of fibroblast functions by PDE4 inhibition was corroborated by the inhibition by
roflumilast of TGF-β1-induced CTGF mRNA and α-SMA protein expression, and FN in the
presence of the long-acting β2-adrenoceptor agonist, indacaterol (Tannheimer et al., 2012).
Moreover, the inhibition by rolipram of another interesting aspect of fibrosis, epithelial-
mesenchymal transition, was shown in the TGF-β1-stimulated A549 human alveolar epithelial cell
line (Kolosionek et al., 2009). Thus, a multitude of in vitro studies indicate that PDE4 inhibitors
can directly inhibit various cAMP-dependent fibroblast functions. Upregulation of PDE4 activity
by cytokines such as IL-1β may further enhance this role.

There is also evidence for a role of isoform-selective PDE4 inhibition. By using PDE4 subtype-
specific siRNA, the involvement of PDE4B and PDE4A in the attenuation of IL-1β plus bFGF-
stimulated fibroblast proliferation, as well as the involvement of PDE4B and PDE4D in TGF-β-
induced α-SMA expression, were shown (Selige et al., 2011). In most of the fibrosis-relevant cell
types, like fibroblasts, macrophages and epithelial cells, the PDE4B seems to have a more
prominent role than other PDE4 subtypes (Hatzelmann et al., 2010), which was confirmed by the
inhibition of cytokine release, proliferation, fibroblast-to-myofibroblast transition and expression
of extracellular matrix proteins by BI 1015550 (nerandomilast), a preferential PDE4B inhibitor with
9-fold selectivity for PDE4B vs PDE4D (Herrmann et al., 2022). In a Phase II study in IPF patients,
BI 1015550, while showing acceptable tolerability and safety, stabilized lung function in both
patients with and without antifibrotic background therapy over 12 weeks, and reduced disease-
relevant blood biomarkers indicative of effects on the epithelium, fibrosis and inflammation
(Richeldi et al., 2022). BI 1015550 is currently in Phase III clinical studies for IPF and PPF
(NCT05321069, NCT05321082).

2.3.5 | Asthma and COPD

Asthma and COPD are pulmonary disease in which the interaction of the inflammatory system with lung tissue plays an important role, and current therapies are show important parallels. Although PDE inhibition is not yet a clinical standard in these diseases, developments are ongoing, and are especially centers around the cAMP-PDE PDE4. It is noteworthy that cigarette smoking, an important risk factor for asthma and COPD exacerbation, can alter the expression and function of PDE4 in the airways, as observed in cell culture, tissue and animal models, and in humans (Barber et al., 2004; Milara et al., 2012; Singh et al., 2009; Zuo et al., 2020; Zuo et al., 2018). The physiological consequences of altered PDE4 activity are not well defined. However, in COPD and other obstructive lung diseases one might predict that an intervention that lowers cAMP could enhance pulmonary inflammation. These finding may have clinical relevance given that a genome-wide association study of a cohort of Korean individuals identified a SNP in *PDE4D*, rs16878037, which was significantly associated with a susceptibility to non-emphysematous COPD (Yoon et al., 2014). In the following sections we will review the relationship between asthma, COPD, PDE4 and the potential role of other PDE subtypes.

Asthma: epidemiology and pathophysiology

Asthma afflicts > 350 million people globally and represents one of the most common, non-
communicable diseases with a prevalence that is predicted to increase to 450 million by 2025 (Vos,
2017). Mortality from asthma is low, but the burden it inflicts on society is considerable in terms
of morbidity, quality of life and associated economic costs (Dharmage et al., 2019; Reddel et al.,
2019). Asthma is a heterogeneous disease with many phenotypes/endotypes that do not respond
equally to current drug interventions (Wenzel, 2012). In many cases (~50%), asthma has an allergic
basis that is characterized by recurrent airways obstruction, airways hyper-responsiveness (AHR),
airway inflammation (driven by Th2 cytokines) and airway remodeling (Woodruff et al., 2009).
When an allergen (e.g., house dust mite, grass pollen, cat dander) first enters the body, it is digested
into small peptides by professional antigen-presenting cells (typically dendritic cells). These
peptides are transported to the cell's surface by major histocompatibility complex molecules and
presented to naïve T-lymphocytes. In individuals predisposed to developing asthma, the T-
lymphocytes cause B-lymphocytes to differentiate into plasma cells and generate allergen-specific
immunoglobulin (Ig) E. The IgE bind to high affinity receptors (FcεRI) on the surface of mast cells
and basophils, priming them to recognize the allergen the next time it enters the body (Gauvreau et
al., 2015). When this occurs, mast cells degranulate and release histamine, proteases and
chemotactic factors; arachidonic acid is also metabolized resulting in the *de novo* synthesis
cysteinyl leukotrienes and prostaglandins (PG) (Gauvreau et al., 2015). Collectively, these
mediators produce the early asthmatic response by binding to their cognate GPCRs on airways
smooth muscle (ASM) to cause rapid (within minutes), transient (1-3 h) bronchoconstriction (Liu
et al., 1991). Approximately 60% of adults and 80% of children that experience an EAR will, 3 to
8 h later, undergo a late asthmatic response (Diamant et al., 2013). This secondary
bronchoconstriction is also caused by leukotrienes and histamine released by pro-inflammatory
cells (typically eosinophils, basophils and, less frequently, neutrophils) that are attracted to the
lungs by other Th2-derived cytokines, including IL-5, IL-9, IL-13, IL17 and IL-22 (Davis et al.,
2009; Hamilton et al., 1998; Taylor et al., 1991). Of note, these inflammatory mediators are
pleiotropic and can promote eosinophilopoiesis (IL-5), mast cell development (IL-9), goblet cell
hyperplasia (IL-13), AHR (IL-13) and certain facets of airway remodeling (IL-17, IL-22)
(Margelidon-Cozzolino et al., 2022) (see section 2.3.4.a).

Despite the heterogeneity of disease, the recommended treatment options for mild-to-moderate
asthma are uniform. Combination of an inhaled corticosteroid (ICS) and the long-acting β₂-
adrenoceptor agonist (LABA), formoterol is the “preferred” approach, and when suboptimal or in
severe disease, a long-acting muscarinic receptor antagonist (LAMA) should be considered (Reddel
et al., 2019). Still, many patients with severe asthma who suffer frequent exacerbations (i.e., an
acute and rapid worsening of symptoms that is beyond the normal day-to-day variation of the
disease) cannot be controlled despite the availability of biologicals. PDE inhibitors may prove
beneficial as add-on therapies in these cases and remain in clinical development.

COPD: epidemiology and pathophysiology

Chronic obstructive pulmonary disease is a leading cause of morbidity and mortality globally that imposes a significant social and economic burden (Mirza et al., 2018; Reddel et al., 2019; Vogelmeier et al., 2017). COPD is a generic term that describes a heterogeneity of phenotypes or endotypes (Barnes, 2019; Miravitlles et al., 2013; Vestbo, 2014) where chronic bronchitis, cough, idiopathic sputum production, airway wall thickening, mucus hypersecretion and destruction of alveolar septa (i.e., emphysema) are present to a greater or lesser extent (Barnes, 2004; 2008; Krzyzanowski et al., 2005; McDonough et al., 2011); together, it is these pathologies that contribute to the persistent, partially irreversible, and usually progressive decline in lung function that defines COPD (Hogg and Timens, 2009; Mirza et al., 2018; Postma and Timens, 2006; van den Berge et al., 2011; Vogelmeier et al., 2017).

Inflammatory stimuli are key causal factors in COPD. In the West, cigarette smoking is a main
cause, whereas in relatively low-income countries job-related exposure to dust, fumes and smoke
through the burning of biomass fuels (e.g., wood, charcoal, animal waste) contribute significantly
(Annesi-Maesano, 2017; Krzyzanowski et al., 2005; Ling et al., 2011). The number of deaths from
COPD is projected to increase because of higher rates of cigarette smoking in the developing world
and an aging global population in general.

ROS produced either by inflammatory cells or inhaled directly into the airways are believed to play
a central role in COPD pathogenesis (Barnes, 2004; 2009; 2013; 2018; Kirkham and Barnes, 2013).
ROS can activate transcription factors (e.g., nuclear factor-κB) that increase pro-inflammatory signals including IL-4, IL-6, IL-8 and TNFα (Barnes, 2004; 2008; 2009; 2018; McDonough et al., 2011). These gene products themselves can then activate feed-forward loops of transcription to amplify and perpetuate the inflammatory response. ROS can also reduce the integrity of the epithelium, thus enabling unwanted inflammatory cell recruitment into the airway lumen (Barnes, 2004; 2008; 2009; 2018; McDonough et al., 2011). Collectively, chronic ROS exposure is key in COPD.

Treatment of COPD is, for the most part, restricted to bronchodilators. Specifically, a LAMA, LABA or combination thereof are recommended to relief symptoms. These drugs can effectively reduce airflow obstruction and dynamic hyperinflation (i.e., the abnormal retention of air in the lungs after exhalation), which often improves exercise tolerance (Mirza et al., 2018; Vogelmeier et al., 2017). In patients with more severe disease, anti-inflammatory therapy is indicated with an ICS and/or an oral PDE4 inhibitor being the only drugs currently available (Mirza et al., 2018; Vogelmeier et al., 2017). However, responsiveness to ICS monotherapy and to PDE4 inhibitors is restricted to sub-groups, as reviewed elsewhere (Briggs et al., 2010; Giembycz and Maurice, 2014; Giembycz and Newton, 2014; Hurst et al., 2010; Maurice et al., 2014; Mirza et al., 2018; Vogelmeier et al., 2017). Exacerbations of COPD are of major clinical concern and a primary therapeutic objective because they are difficult to control, contribute significantly to the decline in lung function and are the major cause of premature mortality (Kurai et al., 2013; Mallia and Johnston, 2006; Viniol and Vogelmeier, 2018). Exacerbations are often precipitated by a bacterial and/or viral infection and, thus, have an inflammatory basis (Kurai et al., 2013; Mallia and Johnston, 2006; Ni et al., 2015; Viniol and Vogelmeier, 2018). This may explain why ICS and PDE4 inhibitors can be of benefit in subjects with COPD in whom airways inflammation is discernible

*The role of PDE inhibition in asthma: cAMP-PDE PDE4.*

PDE4 is present in most immune and structural cells in the airways and regulates a variety of inflammatory processes (Giembycz, 1992; Schudt et al., 1995; Torphy, 1998; Torphy and Undem, 1991). As from the late 1980s a huge effort ensued in academia and industry alike to rigorously define PDE4 as a viable therapeutic target in asthma (Giembycz, 1992; Schudt et al., 1995; Torphy, 1998; Torphy and Undem, 1991). Despite initial optimism, results of clinical studies have been uniformly disappointing and all PDE4 inhibitors have been discontinued for clinical development (Giembycz, 2008). The high rate of attrition is attributable to a low therapeutic ratio because of inhibition of PDE4 in non-target tissues with nausea and vomiting being the most severe, dose-limiting adverse effects. Thus, understanding the molecular basis of emesis became a priority. It was known that cAMP could enhance noradrenaline-containing neuronal activity within the highly vascularized area postrema in the brainstem, which is linked, causally, to emesis (Carpenter et al., 1988). This was confirmed by delivering PDE4 inhibitors directly into the area postrema of ferrets by intracerebroventricular injection (Robichaud et al., 1999). PDE4 inhibitor-induced emesis was inhibited by the α₂-adrenoceptor agonist clonidine (Robichaud et al., 2001). Since the α₂-adrenoceptor is negatively coupled to adenylyl cyclase, the emetic response was attributed to the elevation of cAMP in noradrenergic neurones (Robichaud et al., 2001). Indeed, the tolerability of PDE4 inhibitors can be improved by limiting brain penetration (Aoki et al., 2001).

The attention was then focused on the question if emesis could be linked to the inhibition of a particular PDE4 isoform. A behavioral correlate of vomiting was developed in mice carrying targeted deletions of the genes encoding *Pde4b* and *Pde4d* (Robichaud et al., 2002). Mice (and rodents in general) are anatomically constrained and cannot vomit (Borison et al., 1981; Hanson, 2003; Horn et al., 2013). Instead, the murine model of emesis relies on the ability of α₂-adrenoceptor agonists to promote anesthesia by reducing the cAMP content in noradrenergic fibres within the brainstem, which can be attenuated by PDE4 inhibitors (Giembycz, 2002; Robichaud et al., 2002).
Robichaud et al., (2002) found that the duration of α₂-adrenoceptor-mediated anesthesia was significantly attenuated in mice lacking *Pde4d* but not *Pde4b*, implicating PDE4D in the emetic response. Nevertheless both PDE4D and PDE4B can be found in various relevant brain regions (Cherry and Davis, 1999; Lamontagne et al., 2001; Pérez-Torres et al., 2000; Takahashi et al., 1999a), and inhibitors of PDE4B, that are at least 80-fold selective over PDE4D, do not display a superior therapeutic index (Naganuma et al., 2009; Suzuki et al., 2013). Moreover, so-called, negative allosteric PDE4D inhibitors have been described, which *preferentially* partition into the brain and, therefore, will reach the area postrema (Burgin et al., 2010c). These compounds, of which D159687 and zatolmilast are examples, display a unique mechanism of action in that they block cAMP hydrolysis by modifying the dimeric structure of PDE4D rather than by simply competing with substrate at the catalytic site (Burgin et al., 2010c; Gurney et al., 2011; Houslay and Adams, 2010). Based on the aforementioned results obtained in *Pde4d* knockout mice, these compounds should promote emesis. However, paradoxically, they have significantly reduced emetic liability (Burgin et al., 2010c; Gurney et al., 2011; Zhang et al., 2017b). Thus, the assumption that inhibitors with weak activity against PDE4D relative to the other isoenzymes should be less emetogenic is likely misplaced. Indeed, several companies including Tetra Therapeutics (a subsidiary of Shionogi & Co., Ltd.) are purposefully developing selective PDE4D inhibitors (e.g., zatolmilast) with Fragile X syndrome and AD being primary indications. Clinical trials of these compounds are ongoing (e.g., NCT05367960, NCT03817684), and it will be instructive, from the perspective of developing new PDE4 inhibitors for asthma and COPD, if the improved therapeutic ratios found in animal models translate into improved safety and tolerability in humans.

### Role of PDE inhibitors in COPD: PDE4.

The abject failure of PDE4 inhibitors as an asthma therapy, prompted the pharmaceutical industry to repurpose this drug class for COPD. Cilomilast (Ariflo®, SB-207499) was the first PDE4 inhibitor to progress to phase III clinical trials (Giembycz, 2001; 2006; Kroegel and Foerster, 2007; Rennard et al., 2008; Schachter, 2006). Clinical development was based on a conceptually robust hypothesis, abundant preclinical data and the encouraging results of phase II trials (Torphy et al., 1999) but the cilomilast Phase III clinical development program had disappointing results (Giembycz, 2001; 2006; Kroegel and Foerster, 2007; Rennard et al., 2008; Schachter, 2006). As in asthma, dose-limiting adverse events remained a major cause for concern. The development of cilomilast was discontinued in 2007.

Other repurposed PDE4 inhibitors were roflumilast (Daxas®, Daliresp®, Byk 2869). Based on international, multicenter trials, the European Medicines Agency Committee for Medicinal Products for Human Use approved the use of roflumilast for the "maintenance treatment of severe COPD associated with chronic bronchitis in adult patients with a history of frequent exacerbations as add-on to bronchodilator treatment" in 2010 (Calverley et al., 2009; Calverley et al., 2007; Fabbri et al., 2009; Giembycz and Field, 2010; Gross et al., 2010; Rabe et al., 2005; Wedzicha et al., 2016). Generally, roflumilast reduced the frequency of exacerbations. This effect was more pronounced in patients that displayed a severe, bronchitic phenotype, implicating anti-inflammatory activity (Giembycz and Newton, 2014; Miravitlles et al., 2013). The most common adverse effects experienced were gastrointestinal discomfort and headache (presumably due to cerebrovascular vasodilation) (Calverley et al., 2009; Calverley et al., 2007; Fabbri et al., 2009; Rabe et al., 2005), which is similar to all other PDE4 inhibitors. Currently, roflumilast is the only PDE4 inhibitor approved for COPD and offers physicians an add-on treatment option for patients with more severe disease in whom traditional bronchodilator and glucocorticoid therapies are sub-optimal.

Despite the emetic liability of PDE4 inhibitors, interest in these compounds as therapeutics for respiratory diseases continues with tanimilast (CHF-6001) being the most advanced candidate in clinical development. Tanimilast is a sub-nanomolar inhibitor without isoform preference (Armani
et al., 2014), and has been optimised for inhaled delivery as a dry powder to limit systemic exposure and associated adverse effects. In cell-based assays and pre-clinical models of airway inflammation, it displays pleiotropic anti-inflammatory activity (Facchinetti et al., 2021; Fioni et al., 2018; Schioppa et al., 2022), and administered topically to ferrets it does not induce nausea or vomiting, which likely reflects low systemic exposure and limited blood-brain barrier passage (Villetti et al., 2015). In contrast, GSK 256066, another highly potent PDE4 inhibitor (Tralau-Stewart et al., 2011) that was used as a systemically delivered comparator, was equally effective at blunting pulmonary eosinophil recruitment yet produced clear behavioural signs of nausea (Villetti et al., 2015). Studies in subjects with COPD have shown that at steady state (after 800 µg or 1600 µg inhaled b.i.d. for 32 days), the concentration of tanimilast in sputum was approximately 2000-fold higher than in plasma indicating high pulmonary retention and low systemic exposure (Singh et al., 2019). Moreover, in the PIONEER (new Phosphodiesterase Inhibitor with Optimal anti-iNflammatory Effect dosE Response in COPD patients) Phase IIb trial, which involved 1130 participants with COPD, tanimilast was well-tolerated with a similar incidence of adverse events across four increasing doubling doses (Singh et al., 2020a).

On the basis of successful safety, tolerability and preliminary efficacy studies, two 52 week phase III clinical trials (PILASTER [a new inhaled Phosphodiesterase Inhibitor Evaluated on moderate/severe exacerbationS on top of maintenance Triple therapy in COPD Patients] and PILLAR [a new inhaled Phosphodiesterase Inhibitor given on top of maintenance trip therapy in COPD Patients evaluation on moderate/severe exacerbations]) have been initiated to determine if tanimilast can reduce the frequency of exacerbations in a population of patients with severe, bronchitic COPD who are still symptomatic despite treatment with triple therapy (i.e., an ICS, LABA and LAMA) (Facchinetti et al., 2021), thus addressing a high clinical need.

*Scientific rationale for adding-on a PDE4 inhibitor to combination therapy.*

Glucocorticoid monotherapy is poorly effective in COPD and can be contraindicated due to an increased risk of pneumonia and tuberculosis (Brassard et al., 2011; Ernst et al., 2007). ICS-containing combination therapy is more effective than simple bronchodilators in reducing COPD exacerbations (Ding et al., 2022), suggesting utility in individuals with severe, bronchitic COPD. Data that support this hypothesis and rationalise the PILASTER and PILLAR trials (Facchinetti et al., 2021) can be derived from the earlier roflumilast clinical studies that revealed that roflumilast reduces the rate of exacerbations in individuals with severe COPD and on ICS, whereas no such benefit was derived if ICS use was excluded (Rennard et al., 2011). Lung function in COPD patients of the bronchitic phenotype was also improved by roflumilast, regardless of co-existing emphysema, and this was greater if they had received concomitant ICS rather than placebo (Rennard et al., 2011). Collectively, these data imply that an ICS and roflumilast in combination have superior therapeutic activity than either drug alone.

Glucocorticoids suppress inflammation by modulating the expression of hundreds of cytokines, chemokines and growth factors, mainly via gene induction (transactivation) (Newton, 2014). There is compelling *in vitro* evidence that cAMP-elevating agents can interact with glucocorticoids in an additive or cooperative manner herein (Giembycz and Maurice, 2014; Giembycz and Newton, 2011; 2014; 2015; Rangarajan et al., 1992). This may help to explain how adding-on a LABA and a PDE4 inhibitor to an ICS reduces inflammation and improves lung function in individuals with asthma and COPD. Indeed, an increase in cAMP can augment glucocorticoid-induced gene expression changes in several cell types including the airway epithelium (BinMahfouz et al., 2015; Giembycz et al., 2008; Greer et al., 2013; Joshi et al., 2015; Kaur et al., 2008; Moodley et al., 2013; Mostafa et al., 2021; Newton and Giembycz, 2016; Reddy et al., 2020; Rider et al., 2018; Turner et al., 2020; Wilson et al., 2009). In many cases, the cAMP-elevating agent alone is without effect, but interacts with the glucocorticoid in a positive, cooperative fashion to enhance gene transcription.
This implies that a LABA or PDE4 inhibitor is “steroid-sparing” because the glucocorticoid can now produce a given level of gene induction at a significantly lower concentration (Joshi et al., 2015; Kaur et al., 2008). Indeed, in airway epithelial cells, roflumilast potentiated the ability of the LABA, formoterol, to enhance the expression of a panel of glucocorticoid-inducible gene that may have anti-inflammatory activity in COPD (Moodley et al., 2013). These results highlight a non-bronchodilator role of LABAs. Adding on a PDE4 inhibitor could potentiate β₂-adrenoceptor-mediated cAMP formation and, thereby, further enhance the genomic response. This interaction could be particularly relevant in pro-inflammatory or immune cells where β₂-adrenoceptors are expressed in low abundance or are poorly coupled to adenylyl cyclase. In this situation, a cell type that is poorly responsive to a LABA (e.g., an eosinophil) (Muñoz et al., 1995; Rabe et al., 1993), could be sensitized by a PDE4 inhibitor allowing a cAMP signal to be generated of sufficient magnitude to enhance glucocorticoid-induced gene expression. Collectively, these results provide a mechanistic basis for the superior efficacy of ICS/LABA/PDE4 inhibitor triple combination therapy on COPD exacerbations reported throughout the roflumilast Phase III clinical development program (Rennard et al., 2011). By extension, adding-on a PDE4 inhibitor to ICS/LABA combination therapy in individuals with difficult-to-treat asthma might also afford additional benefit.

Another interesting interaction to mention is the fact that β₂-Adrenoceptor agonists and other cAMP-elevating agents can also increase the expression of various PDE4 isoforms. Typically, these are transcriptional responses and occur in airway immune and structural cells alike including ASM (Hu et al., 2008; Le Jeune et al., 2002), monocytes (Manning et al., 1996; Torphy et al., 1992; Torphy et al., 1995), T-lymphocytes (Erdogan and Houslay, 1997b; Seybold et al., 1998), neutrophils (Ortiz et al., 2000) and endothelial cells (Zhu et al., 2004). The functional implications of these gene expression changes may be significant because SABAs and LABAs, which are consumed by individuals with asthma and COPD on a long-term basis, could attenuate signaling mediated by all GPCRs that stimulate adenylyl cyclase (Giembycz, 1996). In this context, the concurrent use of a PDE4 inhibitor can be rationalised because heterologous GPCR desensitization would be mitigated.

### PDE inhibition beyond PDE4

Based on an analysis of a panel of putative anti-inflammatory genes (e.g., *RGS2*, *CD200*, *CRISPLD2*), the efficacy of triple combination therapy appears to be improved if both PDE3 and PDE4 are blocked (BinMahfouz et al., 2015). This needs to be confirmed *in vivo* in individuals with COPD. Differences in the expression of transcripts that encode other PDEs including *PDE1A*, *PDE6A*, *PDE7A* and *PDE11A* have been found in nasal and bronchial epithelial cells obtained from current smokers when compared to never smokers (Zuo et al., 2020). These effects on mRNA expression were not verified at the level of protein and so the (patho)physiological relevance, if any, is not understood. Nevertheless, *Pde11a* has been genetically linked to inflammatory pulmonary conditions including asthma and symptomatic tuberculosis (Bazhin et al., 2010; DeWan et al., 2010; Oki et al., 2011; Witwicka et al., 2007; Zhu et al., 2019b).

### 2.3.6 | Hybrid Inhibitors for Asthma and COPD

Polypharmacology is a branch of pharmacology that is dedicated to understanding the mechanism of action of compounds that interact with more than one molecular target in a disease network (Jalencas and Mestres, 2013). This discipline addresses the likelihood that improved clinical outcomes can be realised over the traditional ‘one drug, one target’ concept of therapeutics (Morphy and Rankovic, 2005). Several polypharmacological approaches are possible including the administration of (i) two or more drugs separately or together in a single formulation; (ii) two or more pro-drugs formulated as a single chemical entity that are released at the desired site of action
by enzymatic cleavage, and (iii) a single chemical entity that interacts with two or more targets, simultaneously (Morphy and Rankovic, 2005). Compounds that fall into this latter category include bifunctional ligands (see last section of this Chapter) and hybrid PDE inhibitors that contain a single “promiscuous” pharmacophore that blocks the catalytic sites of two or more PDE isoforms. Of the 11 PDE families described, the simultaneous inhibition of PDE4 and either PDE1, PDE3 or PDE7 may provide the means to further enhance clinical efficacy (Giembycz, 2005b; Giembycz and Newton, 2011; Zuo et al., 2019). Indeed, expanding on the discussion in the preceding section, the discovery and utility of multi-component, syncretic drugs provides a theoretical means to treat various asthma and COPD phenotypes/endotypes given that additive and/or synergistic outcomes can be produced when multiple PDEs are inhibited concurrently (Keith et al., 2005).

*Inhibitors of PDE4 and PDE1.* Remodeling of the airways is a characteristic feature of obstructive lung diseases (Aoshiba and Nagai, 2004; Hogg, 2004; Hogg et al., 2004; Hossain and Heard, 1970; Jeffery, 2001; Lazaar and Panettieri, 2003; Wenzel, 2003). Hypertrophy and hyperplasia of airway myocytes play an important role herein (Benayoun et al., 2003; Ebina et al., 1993; Hossain and Heard, 1970; Woodruff et al., 2004)(Aoshiba and Nagai, 2004; Hogg et al., 2004; Hossain and Heard, 1970; Jeffery, 2001). In both asthma and COPD, the process of remodeling thickens and increases the volume of the respiratory tract wall, which contributes to AHR (Hogg, 1996; Martin et al., 2000; Postma and Kerstjens, 1998; Wiggs et al., 1990).

PDE1 is highly expressed in vascular smooth muscle where it has been implicated in the control of proliferation (Chapter 2.2) (Rybalkin et al., 1997; Rybalkin et al., 2002). PDE1 is also highly expressed in ASM (Giembycz and Barnes, 1991; Torphy et al., 1993) where it may regulate the same function. On this basis, one might speculate that a dual inhibitor of PDE1 and PDE4 could retard remodeling and, at the same time, suppress inflammation. Data that support this idea include the ability of the PDE1/PDE4 inhibitor, KF-19514, to suppress inflammation and airway remodeling in a murine model of chronic asthma (Fujimura et al., 1998; Kita et al., 2009; Manabe et al., 2000; Manabe et al., 1997). However, a review of the literature suggests that this potential therapeutic opportunity has not gained traction.

*Inhibitors of PDE4 and PDE3.* PDE3 inhibitors are effective bronchodilators in humans (Bardin et al., 1998; Brunnée et al., 1992; Fujimura et al., 1997; Fujimura et al., 1995; Leeman et al., 1987; Myou et al., 2003; Myou et al., 1999; Singh et al., 2020b). This property led to the theory that compounds that block PDE3 and PDE4 at a similar dose could have a polypharmacological advantage over a selective PDE4 inhibitor by producing both ASM relaxation (PDE3-dependent) and anti-inflammatory activity (PDE4-dependent). In addition, many pro-inflammatory and immune cells also express PDE3 (Banner and Press, 2009; Torphy, 1998) and, in many cases, the anti-inflammatory effects of concurrent inhibition of PDE3 and PDE4 is superior to those of PDE4 alone. For example, *in vitro* studies have shown that while PDE3 inhibitors have little or no effect on T-cell proliferation or on IL-2 generation, they enhance the repressive effect of a PDE4 inhibitor (Giembycz et al., 1996; Robicsek et al., 1991). Similar data have been reported for the inhibition of pro-inflammatory responses in human cell types important for obstructive lung disease (Blease et al., 1998; Gantner et al., 1999; Schudt et al., 1995; Selige et al., 2010; Wright et al., 1998). There is also evidence garnered from allergic mouse models of asthma that inhibitors of PDE3 may be anti-inflammatory *per se*, and that both Pde3A and Pde3b play regulatory roles (Beute et al., 2020; Beute et al., 2018).

Based upon encouraging pre-clinical data, several hybrid PDE3/PDE4 inhibitors were developed and evaluated in humans including zardaverine, benzafentrine, tolafentrine and pumafentrine but all were discontinued because of lack of efficacy, a poor adverse effect profile or limited duration of action (Banner and Press, 2009). Nevertheless, interest in the PDE3/PDE4 inhibitor concept has endured with at least two compounds currently in clinical development for asthma and/or COPD:
ensifentrine (aka RPL 554) and, what appears to be a structurally related compound, TQC-3721 (Yang et al., 2023).

Ensifentrine is a long-acting, well-tolerated inhalation bronchodilator (Boswell-Smith et al., 2006; Calzetta et al., 2013) that improves forced expiratory volume in 1 second ($FEV_1$) (Donohue et al., 2023). Ensifentrine has progressed to phase III clinical evaluation and in 2023 the results of the two ENHANCE (Ensifentrine as a Novel inHAled Nebulized COPD thErapy) trials were reported (Anzueto et al., 2023). Both trials, performed in moderate-to-severe COPD patients, showed improved $FEV_1$, and exacerbation rates were also reduced by 40% although it is unclear if this was due to the inhibition of PDE4 (Singh, 2023). The role of PDE4 herein is under debate (Franciosi et al., 2013; Singh, 2023).

The referral to ensifentrine as a hybrid PDE3/PDE4 inhibitor is disputable. Ensifentrine is >3440× more potent against PDE3 than PDE4 (Boswell-Smith et al., 2006), which is comparable to, or even greater than, the selectivity of compounds that are classified as selective PDE3 inhibitors including cilostazol, cilostamide and milrinone (Sudo et al., 2000). This implies that inhibition of PDE3 will predominate. Potentially, a single dose of ensifentrine of 0.018 mg/kg exceeds the dosage required to abolish PDE3 activity (Franciosi et al., 2013). Using the technique of bronchosorption (Leaker et al., 2015) it has been estimated that the concentration of the LABA, salmeterol, in the ESL of normal healthy subjects 1 h after inhalation of a 50 µg dose was ~80 nM (Sadiq et al., 2021). Assuming remotely similar pulmonary pharmacokinetics, the concentration of ensifentrine in ESL after inhalation of a 3 mg dose (used in the ENHANCE trial) could be ~5 µM. Indeed, both compounds have comparable molecular weights (~450 Da), clog D values (~1.9 at pH = 7; calculated using ACD/Labs software; www.acdlabs.com), high lung retention and low systemic exposure (Bäckström et al., 2016; Sadiq et al., 2021; Zuiker, 2016). Thus, inhalation of a 3 mg dose of ensifentrine could be sufficient to inhibit PDE4 in airway epithelia and inflammatory cells in BAL fluid by >70%. Of the limited studies reported to date, the $IC_{50}$ of ensifentrine for suppressing cytokine release (e.g., GM-CSF, MCP-1, TNF$\alpha$) from human airway epithelial cells and monocytes is in the low micromolar range (Boswell-Smith et al., 2006; Turner et al., 2020). Likewise, the threshold concentration for ensifentrine to increase *global* cAMP in human airway epithelial cells is reported to be ~1 µM (Turner et al., 2020).

Ensifentrine relaxed ACh-contracted human ASM ($EC_{50}$ ~10 µM) and inhibited PDE3 ($IC_{50}$ = 0.4 nM) with potencies that differed by ~25,000-fold, a paradox not observed for anti-inflammatory effects (Boswell-Smith et al., 2006; Calzetta et al., 2013; Turner et al., 2020). Functional antagonism of smooth muscle tone might explain this phenomenon. Functional antagonism of Ach in ASM evoked by endogenous spasmogens has been described, being more pronounced with ACh (and related agonists including MCh and CCh) than with histamine, 5-hydroxytryptamine and leukotriene $D_4$ (Roffel et al., 1995; Russell, 1984; Torphy, 1984; Torphy et al., 1983; van den Brink, 1973). For example, the $EC_{50}$ of isoprenaline for relaxing bovine tracheal smooth muscle contracted with 10 nM (~$EC_{20}$), 100 nM (~$EC_{70}$) and 10 µM metacholine ($EC_{100}$) was 0.65 nM, 81 nM and 3.16 µM, respectively (a maximum difference of >4800-fold) (Roffel et al., 1995). Similar data have been reported for the selective PDE3 inhibitor, siguazodan (SK&F 94836), on metacholine-contracted canine ASM (Torphy et al., 1988). Logic dictates that ensifentrine, which like isoprenaline and siguazodan relaxes ASM by a cAMP-dependent mechanism, would be affected similarly both *in vitro* and *in vivo*. Indeed, the potency of ensifentrine for inhibiting electrical field stimulation-induced twitch responses of human medium size bronchi, progressively decreased with increasing frequency of nerve stimulation (Calzetta et al., 2015). Thus, functional antagonism may help explain the erroneous description of ensifentrine as a hybrid PDE3/PDE4 inhibitor because the biochemical and functional outcomes of a bronchodilator cannot easily be compared.
From a safety perspective, inhibition of PDE3 is of concern given the well-documented cardiovascular toxicity of this class of drugs and the fact that subjects with COPD will require long-term therapy over many months or years. While PDE3 inhibitors were developed to treat dilated cardiomyopathy, chronic dosing increased mortality (Amsallem et al., 2005; Movsesian, 2003b). This could be problematic because ~20% of people with COPD have right side heart failure that is often secondary to PH (de Miguel Díez et al., 2013; Naeije, 2003). Chronic PDE3 inhibition could, therefore, be contraindicated even for a compound given by inhalation and so pulmonary retention will be critical. In this respect, the peak plasma concentration of ensifentrine in 13 subjects with allergic asthma after inhalation of 0.018 mg/kg (o.d. for 6 days) was ~2 ng/ml (4.2 nM) (Zuiker, 2016). This dose equates to 1.26 mg/70 kg individual, which is 42% of the 3 mg dose assessed in the two ENHANCE clinical trials (Anzueto et al., 2023). Adverse events were reported to be mild, although a reduction in blood pressure and a [compensatory] increase in heart rate were noted. The study investigators attributed these cardiovascular events to PDE3 inhibition in the vasculature, which was consistent with an increase incidence of headache and dizziness in 4 and 3 of the 13 subjects, respectively (Zuiker, 2016).

c. *Inhibitors of PDE4 and PDE7*. PDE7 is ubiquitously expressed in human immune and pro-inflammatory cells (Smith et al., 2003). Selective PDE7 inhibitors could represent an alternative class of anti-inflammatory drugs with an improved adverse-effect profile over compounds that block PDE3 and/or PDE4 (Giembycz and Maurice, 2014; Giembycz and Smith, 2006a; Giembycz and Smith, 2006b; Jankowska et al., 2017b). It took 20 years to develop selective inhibitors after the discovery of PDE7 (Goto et al., 2009; Jones et al., 2007; Kadoshima-Yamaoka et al., 2009a; Kadoshima-Yamaoka et al., 2009b; Kadoshima-Yamaoka et al., 2009d; Michaeli et al., 1993; Nakata et al., 2002; Smith et al., 2004b; Yang et al., 2003). Of some interest, the PDE7A inhibitor BRL 50481 failed to attenuate human T-cell proliferation *per se*, yet it significantly enhanced the anti-mitogenic activity of the PDE4 inhibitor, rolipram (Smith et al., 2004b). LPS-induced TNFα generation from human monocytes and lung macrophages was regulated similarly (Smith et al., 2004b). This profile of activity was replicated with the dual PDE4/PDE7 inhibitor BC54, which inhibited TNF-α and IL-12 production from U-937 monocytic cells and Jurkat T-cells, respectively and was more effective than rolipram, alone (de Medeiros et al., 2017). Collectively, these data are reminiscent of the behaviour of PDE3 inhibitors (Giembycz, 2005a; Vijayakrishnan et al., 2007). In one investigation, YM-393059, which is 45-fold more selective for PDE7 over PDE4, demonstrated efficacy in preclinical models of inflammation with a reduced emetic liability (Yamamoto et al., 2006a; Yamamoto et al., 2006b). Likewise, mice subjected to cigarette smoke-induced pulmonary inflammation were protected by prior endo-tracheal administration of antisense oligonucleotides directed against *Pde4b*, *Pde4d* and *Pde7a*, and that this intervention was superior to classical pharmacotherapy with roflumilast (Fortin et al., 2009).

d. *PDE3/PDE4 inhibitors and a LAMA*. Several patents have been filed describing the utility of combining ensifentrine with a LAMA (Walker et al., 2017; 2019). The inventions claim that a low concentration of a muscarinic receptor antagonist (e.g., glycopyrronium) interacts synergistically with ensifentrine to relax medium and small human bronchi *in vitro* (Calzetta et al., 2015; Calzetta et al., 2013). This unexpected effect led to the proposal that in subjects with COPD, antagonising the effects of endogenously-released ACh from parasympathetic nerve fibres in the lung with a LAMA and raising the cAMP content in ASM with ensifentrine could produce added clinical benefit by reducing dynamic hyper-inflation (Calzetta et al., 2015).

### 2.3.7 | Bifunctional Ligands for COPD

An alternative to a hybrid inhibitor is a compound that contains two pharmacophores joined covalently by a rationally designed and inert "spacer" (Phillips and Salmon, 2012; Shonberg et al., 2011). In the context of respiratory diseases, these, so-called, bifunctional ligands, have several
advantages over their mono-functional parent compounds because of their relatively high molecular weight (often > 1000 Da). This physical property often translates into enhanced pulmonary retention, low oral bioavailability and reduced systemic exposure (Phillips and Salmon, 2012). The development of bifunctional ligands is also simplified because two pharmacophores in the same compound will have matched pharmacokinetics and identical deposition characteristics (Phillips and Salmon, 2012). Several bifunctional ligands containing a PDE4 inhibitor have been synthesised. The most common “partners” for a PDE4 inhibitor have included a LAMA and a LABA in an attempt to harness both anti-inflammatory and bronchodilator activity at a similar dose (Giembycz and Maurice, 2014). The first example of a “Muscarinic receptor Antagonist-PDE4 Inhibitor (i.e., a MAPI) was the 4,6-diaminopyrimidine derivative, UCB-101333-3 (Provins et al., 2006). In mice, the inhaled compound attenuated cigarette smoke-induced pulmonary neutrophilia and keratinocyte chemoattractant levels in BAL fluid and protected against heavy metal-induced emphysema (Provins et al., 2007). Since that original report, the interest in developing MAPIs for COPD has continued including compound 10f from Cheisi Farmaceutici (Rizzi et al., 2023). This ligand is a fusion of tanimilast with a muscarinic receptor antagonist based on a phenylglycine scaffold. *In vitro* assays indicated that 10f is a balanced molecule with an affinity and inhibitory potency at the muscarinic M₃ receptor and PDE4B, respectively of ~1 nM (Rizzi et al., 2023). In rodents the inhaled compound displayed adequate lung retention and limited systemic exposure, and retained the pharmacological activity balance. Thus, 10f inhibited CCh-induced bronchoconstriction and ovalbumin-induced pulmonary eosinophilia in sensitized and challenged rats at the same dose and with an acceptable duration of action (Rizzi et al., 2023).

Another means to achieve anti-inflammatory and bronchodilator activity resp. in a single molecule is to couple pharmacophores that display PDE4 inhibitory activity and β₂-adrenoceptor agonism, as conceptually demonstrated with combined roflumilast and salmeterol treatment (Fabbri et al., 2009). Several bifunctional ligands have been described in which the head group of formoterol or salmeterol was fused to roflumilast or a phthalazone-based PDE4 inhibitor by a simple butyl spacer (Liu et al., 2013; Shan et al., 2012a). However, the activities of these compounds are unbalanced in that they are more potent (440–2500-fold) β₂-adrenoceptor agonists than inhibitors of PDE4. Improvements were achieved by modifying the spacer (to hexyloxyphenyl propanol or hexane) but β₂-adrenoceptor agonism remained the dominant activity (Huang et al., 2014; Liu et al., 2013). Gilead Sciences has also reported the discovery of bifunctional LABA/PDE4 inhibitors for COPD, which have been optimised for inhaled delivery (Baker et al., 2011). GS-5759 is a compound that has equal affinity (~1 nM) for PDE4B and the human β₂-adrenoceptor, thus representing a significantly improved ligand (Tralau-Stewart et al., 2011). In addition, the possibility existed that both pharmacophores in GS-5759 could interact in immune and pro-inflammatory cells in a synergistic manner and suppress inflammation to greater extent than a PDE4 inhibitor alone (Joshi et al., 2017). In vitro, GS-5759 was shown to be active in a panel of assays where it inhibited the release of superoxide from human neutrophils, TNFα, IL-6 and CCL3 from human monocytes and ET-1, CCL5, CXCL10 and GM-CSF from human lung fibroblasts (Tannheimer et al., 2014); it also up-regulated the expression of a plethora of genes (e.g., *DUSP1*, *CD200*, *CRISPLD2*, *CDKN1C*, *FGFR2*) in the BEAS-2B human airway epithelial cell line, which, if reproduced *in vivo*, could impart anti-inflammatory activity in COPD (Joshi et al., 2017). GS-5759 was also active in several pre-clinical models of COPD (Salmon et al., 2014). Significantly, no emesis was produced in ferrets at doses effective for pulmonary protection in rat (Salmon et al., 2014). Despite these encouraging data, a primary therapeutic target of GS-5759 is the ASM, which likely displays a large β₂-adrenoceptor reserve for β2A (Giembycz, 2009). This will probably render GS-5759 unbalanced because its potency to affect bronchodilation will be greater, may be considerably, than its affinity for the β₂-adrenoceptor *and* for inhibition of PDE4 at once (Giembycz, 2009; Joshi et al., 2017). The only way to overcome this limitation is to either increase the potency of the pharmacophore
that inhibits PDE4 or reduce the affinity of the β2A orthostere for the β2-adrenoceptor. The presence of spare receptors represents a challenge for the development of bifunctional ligands in general because receptor density and/or coupling efficiency to downstream effectors will differ between these targets.

An unexpected finding of these investigations was that GS-5759 had 35-fold higher affinity for the β₂-adrenoceptor than did β2A (Joshi et al., 2017). This pharmacological behaviour has been reported previously for the bifunctional LABA/LAMA, THRX 198321 (Steinfeld et al., 2011) and may also apply to the MAPI, 10f (Rizzi et al., 2023). Mechanistically, the enhanced affinity of these compounds for the β₂-adrenoceptor may be due to positive allosterism (Rizzi et al., 2023; Steinfeld et al., 2011) or “forced proximity” binding (Hughes et al., 2011; Joshi et al., 2017; Valant et al., 2012; Vauquelin and Charlton, 2013). Regardless, the fact remains that the properties of bifunctional ligands are often distinct from their monofunctional parent compounds, which could reveal new opportunities for drug discovery.

## 2.4 | Liver disorders and PDEs

### 2.4.1 | The role of cyclic nucleotides in liver fibrosis and cirrhosis pathophysiology

Chronic liver disease results from ongoing hepatocyte damage caused by factors such as viruses, alcohol, and poor nutrition. It can lead to fibrosis, cirrhosis, and hepatocellular cancer, a major cause of morbidity and mortality (Rich, 2024). Affecting over a billion people globally, chronic liver disease causes more than a million deaths from cirrhosis each year (Disease et al., 2018). The most common causes are viral hepatitis, alcohol use, and metabolic dysfunction (Leszczynska et al., 2023). Following injury, hepatocytes release various chemokines and damage-associated molecular patterns (DAMPs), which attract and activate immune cells and hepatic stellate cells in the liver. Hepatic stellate cells (HSC) have very important functions in the liver including the storage of vitamin A, antigen presentation and wound healing. However, during persistent liver injury, HSC undergo activation and become proliferative, migratory myofibroblasts (Wang and Friedman, 2023). HSC-myofibroblasts are the main source of extracellular matrix proteins (ECM) in fibrotic liver. Excessive accumulation of ECM in the liver tissue results in deterioration of liver function which can lead to cirrhosis and liver failure.

cAMP and cGMP signaling have been studied in various liver cell functions, including hepatocytes, macrophages, T cells and hepatic stellate cells (Elnagdy et al., 2020; Elnagdy et al., 2023; Wahlang et al., 2018). The first studies related to alcohol-associated liver disease (ALD) reported a lower level of cAMP in peripheral blood mononuclear cells of alcoholic hepatitis (AH) patients with immune dysfunction (Barlas et al., 1983) and alcohol use disorders (Diamond et al., 1987). Effects of chronic alcohol exposure on cAMP levels were later shown in human and murine monocytes and macrophages including liver resident macrophages or Kupffer cells (Gobejishvili et al., 2006). Importantly, this decrease in cAMP and its signaling was identified as a critical mechanism of macrophage “priming” to produce increased levels of TNFα in response to endotoxin (Gobejishvili et al., 2008; Gobejishvili et al., 2006).

Persistent hepatocyte injury and inflammation will result in the activation of HSC in the liver and their trans-differentiation to myofibroblasts (the main producers of extracellular matrix proteins). Because trans-differentiation of HSCs plays a key role in the development of liver fibrosis, targeting HSC activation has become a focal point in treating liver fibrosis (Li et al., 2008). Upon activation, HSCs express alpha smooth muscle actin (αSMA) and produce ECM proteins like collagens and fibronectin. Perhaps the most profibrogenic cytokine leading to HSC activation is transforming growth factor β1 (TGFβ1). cAMP elevating agents and agonists have been shown to inhibit TGFβ1-
induced expression of αSMA and collagen in different cell types (Cortijo et al., 2009; Desmouliere et al., 1999; Garrison et al., 2013; Houglum et al., 1997; Insel et al., 2012; Liu et al., 2006). Indeed, EPAC1 has been identified as a critical regulator of TGFβ1 signaling in various tissue fibroblasts (Insel et al., 2012; Yokoyama et al., 2008), including in HSCs. Recent studies showed that TGFβ1 decreases cAMP and EPAC1 levels in HSCs, which contributes to their activation (Elnagdy et al., 2023; Schippers et al., 2017).

### 2.4.2 | Role of cAMP-PDE

Since inflammation plays a major role in chronic liver disease, anti-inflammatory strategies using PDE inhibitors have been utilized to evaluate their beneficial effect on liver injury. Moreover, inflammation and dysregulated hepatocyte function in experimental models of liver injury and fibrosis have been shown to be accompanied by increased expression of PDE enzymes in the liver (Avila et al., 2016; Essam et al., 2019; Gobejishvili et al., 2013). A pathogenic role of PDE enzymes in the development of liver injury has been confirmed by demonstrating that PDE inhibitors alleviate liver damage and inflammation (El-Deen et al., 2020; Elnagdy et al., 2023; Essam et al., 2019; Gobejishvili et al., 2013; Ma et al., 2022; Rodriguez et al., 2019; Tao et al., 2023). More specifically, the effect of cAMP in macrophage priming was shown to be mediated by increased activity and expression of PDE4, specifically PDE4B (Gobejishvili et al., 2008). PDE4 inhibition resulted in a significant decrease in endotoxin-induced inflammatory cytokine production by human peripheral blood mononuclear cells (PBMCs) from patients with alcohol-associated hepatitis and monocytes/macrophages of human and murine origin (Gobejishvili et al., 2011; Gobejishvili et al., 2013; Gobejishvili et al., 2008; Rodriguez et al., 2019). Importantly, studies using gene knockout mice identified PDE4B as an essential player in endotoxin-mediated production of TNFα (Jin and Conti, 2002; Jin et al., 2005).

However, the role of PDE4-regulated cAMP signaling is not limited to immune cells and their inflammatory responses. Importantly, increased expression of PDE4 enzymes has been associated with both spontaneous and TGFβ1-induced activation of HSCs (Elnagdy et al., 2023; Gobejishvili et al., 2013). Moreover, a recent work demonstrated that PDE4A, B and D are upregulated in the livers of patients with metabolic dysfunction-associated steatotic liver disease (MASLD), and their levels are positively correlated with TGFβ1 (Elnagdy et al., 2023). Importantly, PDE4 enzymes are also expressed in activated HSCs/myofibroblasts in human and mouse livers (Elnagdy et al., 2023). PDE4 inhibition significantly attenuated cytoskeleton remodeling and HSC migration both *in vitro* and *in vivo* leading to a reduced collagen deposition and fibrosis (Elnagdy et al., 2023).

In addition to extracellular matrix remodeling, cAMP signaling plays a significant role in glucose and lipid metabolism in hepatocytes (reviewed in (Wahlang et al., 2018)). In relevance to ALD, studies have shown that alcohol attenuates cAMP/PKA signaling in hepatocytes which results in a decrease in the gene (Cpt1a) encoding carnitine palmitoyltransferase 1A and fatty acid β-oxidation (Elnagdy et al., 2020). Inhibition of PDE4, and specifically PDE4B, prevents alcohol-mediated decrease in cAMP signaling and Cpt1 expression and prevents alcohol-induced lipid accumulation in the liver (Avila et al., 2016; Ma et al., 2022). An effect of PDE4 inhibition on alcohol-induced ER stress has also been shown (Rodriguez et al., 2019). Notably, PDE4 inhibition decreases an alcohol-mediated increase in JNK activation and hepatocyte death (Rodriguez et al., 2019). More recent studies showed that overexpression of PDE4D in the liver led to the development of MASLD in mice, which was attenuated by a PDE4 inhibitor (Tao et al., 2023; Tao et al., 2022b).

### 2.4.3 | cGMP and dual substrate PDEs

Besides PDE4, recent papers have demonstrated a role of PDE9 and 10 in liver and lung fibrosis as well as diet-induced obesity (Ceddia et al., 2021; Li et al., 2023; Mishra et al., 2021; Wu et al., 2021) indicating that cGMP signaling is also critical in tissue fibrogenesis. Indeed, a recent study
reported increased hepatic levels of cGMP in patients and mice with alcohol-associated steatohepatitis (ASH) (Montoya-Durango et al., 2023). Moreover, these changes were associated with significant alterations in various cAMP- and cGMP-selective PDEs in the liver highlighting the potential role of PDE enzymes in the pathogenesis on ASH. Given a crucial role of NO-cGMP signaling in the regulation of hepatic sinusoids and portal pressure, perhaps the most significant findings presented in this recent study, were increased levels of PDE1A, PDE4A, PDE4D, and PDE5A (Montoya-Durango et al., 2023). Indeed these enzymes have been shown to modulate vascular tone, remodeling and exchange via both cAMP and cGMP (Houslay et al., 2007; Netherton et al., 2007). Increased levels of soluble guanylyl cyclase and PDE5 in cirrhotic livers have been reported in another human study (Kreisel et al., 2021). In a normal liver, PDE5 protein is highly expressed in perisinusoidal cells with a very weak expression in hepatocytes. In cirrhotic livers, PDE5 expression increases in fibrous septa, and perisinusoidal cells throughout the parenchyma (Kreisel et al., 2021). PDE5 inhibitors have shown anti-inflammatory and antifibrotic properties in several studies, as well improvement in portal hypertension (Brusilovskaya et al., 2020; Choi et al., 2009; Deibert et al., 2018; Knorr et al., 2008; Kreisel et al., 2020; Schaffner et al., 2018). Notably, a recent study reported the age-dependent sexual dimorphism in the vascular PDE expression patterns (Wang et al., 2021). This is the first study to highlight sexual dimorphism in PDE expression. Future studies are needed to examine whether there are sex-dependent differences in PDE expression in the liver and whether it contributes to the susceptibility of females to certain types of liver injury.

Interestingly, differential beneficial effects of various PDE inhibitors on a high fat-induced MASLD model in rats has been reported (El-Deen et al., 2020). Specifically, when administered in a treatment paradigm, the authors observed that pentoxifylline (PTX), a broad spectrum PDE inhibitor, had the strongest effect on oxidative stress markers, steatosis, inflammation, and liver injury when compared to cilostazol and sildenafil (El-Deen et al., 2020). Notably, PTX (alone and in combination with other drugs) has been widely used to treat MASLD and ALD in humans (Alam et al., 2017; Cioboata et al., 2017; Culafic et al., 2020; Louvet et al., 2018; Marot et al., 2020; Smart et al., 2013; Szabo et al., 2022; Teschke, 2018; Zein et al., 2012; Zein et al., 2011). However, although several studies have been reported significant beneficial effects, including anti-fibrotic and anti-oxidative stress (Fouda et al., 2021; Kedarisetty et al., 2021; Sridharan et al., 2018; Zein et al., 2012; Zein et al., 2011), the use of PTX has been debated (Van Wagner et al., 2011). PTX therapy has shown mixed results in trials in patients with AH (Akriviadis et al., 2000; Duan et al., 2023; Kedarisetty et al., 2021; Louvet et al., 2018; Philips et al., 2022; Thursz et al., 2015), with the large STOPAH trial reporting lack of efficacy in reducing mortality (Thursz et al., 2015). Subjects with alcohol use disorder (AUD) are reported to be less compliant with many medical regimens. PTX is a non-selective and weak PDE inhibitor and requires three times a day (t.i.d.) dosing. Patients report significant gastrointestinal upset which causes noncompliance. Compliance with PTX in the most recent large AH trial was 49.4% ± 40.2% versus placebo 65.5% ± 35.3% (p = 0.06) with nausea being the primary reason for noncompliance (Szabo et al., 2022). The American College of Gastroenterology (ACG) most recent clinical guidelines for the treatment of ALD does not support the use of PTX for severe AH due to a moderate level of evidence that PTX provides survival benefit (Jophlin et al., 2024). More clinical trials are ongoing to test PTX for the treatment of metabolic dysfunction-associated steatohepatitis (MASH) (NCT05284448) and to prevent decompensation in stable cirrhotic patients with prior decompensation (NCT06041932).

Based on strong preclinical evidence that PDE4 inhibition has anti-inflammatory and antifibrotic properties, efficacy of the PDE4 inhibitor, ASP9831, was tested in a proof-of-concept phase 2 clinical trial in biopsy-confirmed patients with MASH and liver fibrosis (Ratziu et al., 2014). The trial did not find any effects of the PDE4 inhibitor on biochemical end points including liver injury markers (ALT, AST, cytokeratin 18), adiponectin and TNFα (Ratziu et al., 2014). The authors
questioned the benefit of PDE4 inhibition as a therapy for MASH due to the failure to attenuate
inflammation and liver injury markers. However, although liver biopsy was performed prior to
patient enrollment, no follow up biopsies were documented. Hence, it is unknown if ASP9831 had
any effect in improving fibrosis stage. Moreover, this study was only of 12 weeks in duration; thus,
there were multiple study limitations. Notably, authors pointed out that the detailed studies
examining the role of each of PDE4 enzymes in the pathogenesis of MASH are lacking. In this
regard, the recent study found that PDE4A, B and D enzymes are expressed in myofibroblasts in
the livers of MASH cirrhosis patients (Elnagdy et al., 2023) implicating the role of these enzymes
in MASH cirrhosis.

In summary, there is ample preclinical and clinical evidence that PDE enzymes are involved in
various liver cells (dys)functions associated with liver pathology. However, more work needs to be
done to characterize the expression patterns and levels of PDEs in a cell-specific manner in the liver
(Figure 13). This will allow us to better understand their role in liver cell pathophysiology for
therapeutic targeting. Moreover, liver diseases are multifactorial and progressive with various
stages of pathology ranging from simple steatosis to inflammation and fibrosis. Animal models do
not recapitulate clinical progression of human liver disease and hence they have limited utility to
test PDE inhibitors for efficacy. Since various selective PDE4 and PDE5 inhibitors have been
approved by FDA for clinical use, perhaps hepatologists will consider them as therapeutic options
for liver diseases, especially for ALD and MASLD.

**2.5 | Neurodevelopmental Disorders**

**2.5.1 | PDE Isoforms, Cognitive impairment and Alzheimer**

Neuronal physiology and pathophysiology

Our memories are what define who we are as a person. Healthy aging, alongside age-related diseases including cognitive decline (ACRD), mild cognitive impairment (MCI), AD, AD-related dementias (ADRD) and Huntington's disease (HD), are characterized by memory deficits and other cognitive impairments (e.g., (Apple et al., 2017; Dean et al., 2017; Ffytche et al., 2017; Kane et al., 2017)). These cognitive domains are known to be regulated by PDEs, and aging and age-related diseases of the brain have been associated with significant disturbances in cyclic nucleotide signaling (c.f., (Kelly, 2018a)). Here we suggest that dysfunction in more than one PDE contributes to age-related pathology and identify those PDE families and isoforms with the greatest therapeutic potential.

As described in detail below, several PDEs demonstrate alterations in expression, localization
and/or activity in the aged brain that can be subverted/exacerbated in the context of ACRD, MCI,
AD, ADRD, and HD. A particularly challenging aspect of this area of research is that these
functional changes are often isoform-specific and vary across brain regions. For example, in the
hippocampus and cortex, aged rodents demonstrate increased high K<sub>m</sub> cAMP-PDE and cGMP-PDE
hydrolytic activity relative to young rodents (Chalimoniuk and Strosznajder, 1998; Stancheva and
Alova, 1991). As such, isoform-selective therapeutics have been much pursued for the treatment of
ARCD, MCI, AD, ADRDs, and HD (Figure 14).

PDE1

During the transition from early to late adulthood, rodent studies show PDE1B mRNA expression
remains unchanged in the hippocampus but decreases in the cerebellum and striatum (Kelly et al.,
2014). In contrast, PDE1C mRNA expression increases in the striatum and PDE1C1 protein—but not PDE1C3—increases in the hippocampus (Kelly et al., 2014). Expression of PDE1A mRNA in these brain regions remains unchanged with age, and no isoform showed age-related changes in cortical expression (Kelly et al., 2014). Functionally, PDE1B is the most explored PDE1 isoform in the context of learning and memory. PDE1B knockout mice performed equivalently to wild-type mice in both the passive avoidance and conditioned avoidance tests (Siuciak et al., 2007b). Adolescent PDE1B knockout and heterozygous mice showed impaired spatial learning and memory in a hidden-platform water maze task relative to wild-type mice (Reed et al., 2002b); however, adult PDE1B knockout mice showed intact spatial learning and memory but impaired reversal learning on the task (Ehrman et al., 2006a). In stark contrast, viral knockdown of only hippocampal PDE1B expression in young adult mice enhanced contextual fear conditioning memory and spatial memory in the Barnes maze (McQuown et al., 2019). Thus, while general knockdown of PDE1B across brain regions impaired memory processes, local deletion in the hippocampus improved memory function. PDE1B is highly expressed in many brain regions other than the hippocampus, including the cortex, striatum, thalamus, and brain stem (Kelly, 2014; Kelly et al., 2014). Thus, an effect of PDE1B deletion in one of these other brain regions may mask any nootropic effect related to deletion within the hippocampus.

That said, the non-selective PDE1 inhibitor, vinpocetine, is sold in over-the-counter supplements (e.g., Cavinton or Intelectol, Richter Gedeon; Cognitex, Life Extension) that claim to improve memory (Baillie et al., 2019a). Indeed, several clinical trials have examined the cognition-enhancing effects of vinpocetine—either alone or in combination with other compounds (e.g., caffeine or Ginko Biloba)—and have generally found improvement in healthy volunteers, individuals with cerebral hypofusion, and possibly aged individuals, but no improvement in AD patients (Balestreri et al., 1987; Caldenhove et al., 2017; Hindmarch et al., 1991; Polich and Gloria, 2001; Richter et al., 2011b; Subhan and Hindmarch, 1985; Szatmari and Whitehouse, 2003; Thal et al., 1989; Valikovics et al., 2012). This is consistent with preclinical rodent models demonstrating therapeutic effects of PDE1 inhibitors in vascular dementia via the PKA-CREB pathway (Zhou et al., 2023), but not with studies showing efficacy of PDE1 inhibitors in rodent models of AD (Shekarian et al., 2020; Shekarian et al., 2023). Reports of side effects associated with vinpocetine have been minimal, and include flushing, rashes, and minor gastrointestinal disturbances (2002).

Intracellular Therapies developed the broad PDE1 inhibitor, lenrispodun, which shows picomolar IC50s for PDE1A, PDE1B and PDE1C in enzymatic assays and >1000-fold selectivity versus its nearest neighbor PDE4 (Li et al., 2016c; Snyder et al., 2016b). Lenrispodun demonstrates cognition-enhancing effects in rodent models of long-term memory and working memory deficits (Li et al., 2016c; Pekcec et al., 2018a; Snyder et al., 2016b). It remains to be determined whether the cognition-enhancing effects of lenrispodun are due to inhibition of PDE1A, PDE1B and/or PDE1C; however, PDE1B may be the most likely candidate given its expression in dopamine D1-expressing neurons (Pekcec et al., 2018a) along with the fact that a PDE1B-selective inhibitor developed by Dart Neuroscience showed similar cognition-enhancing effects (Dyck et al., 2017).

## PDE2

Studies in humans report that PDE2A mRNA levels increase between the prenatal period and childhood and then stabilize into young-middle adulthood in cortical regions, amygdala, and striatum; whereas, hippocampal expression of PDE2A mRNA does not increase beyond prenatal levels until adulthood (Farmer et al., 2020). In stark contrast, studies in rodents report decreased expression of PDE2A in the striatum during the transition from early to late adulthood, but no change in the hippocampus, cortex, or cerebellum (Kelly et al., 2014). PDE2A expression did not change as a function of AD in the hippocampus, cortex, cerebellum, nor striatum (Reyes-Irisarri et al., 2007).
As extensively reviewed elsewhere ((Gomez and Breitenbucher, 2013; Kelly, 2018a; Zhang et al., 2017a); also see (Nakashima et al., 2019; Ruan et al., 2019; Shi et al., 2021a; Yan et al., 2022; Zhou et al., 2021)), PDE2 inhibitors improve many types of memory in young and old rodents as well as in AD rodent models, preventing Aβ-induced cytotoxicity. The ability of PDE2 inhibitors to improve cognition in these models appears to be mediated via its regulation of cGMP signaling since the nootropic effects require signaling via nNOS (Domek-Lopacinska and Strosznajder, 2008) and PKG (Wang et al., 2017a). Takeda Pharmaceuticals initiated Phase I trials with the PDE2 inhibitor, TAK-915, to correlate plasma exposures with central target engagement to inform dose selection for future trials targeting cognitive impairments (Mikami et al., 2017a; Mikami et al., 2017b; Mikami et al., 2017c); however, clinicaltrials.gov does not show any trials registered beyond Phase I (accessed 11/28/2023).

## PDE3

AD upregulates PDE3 expression in cerebral vessels (Maki et al., 2014). In preclinical models, PDE3 inhibitors have prevented or reversed Aβ-induced cytotoxicity, both *in vitro* and in AD mouse models (c.f., (Kelly, 2018a); also see (Yanai et al., 2022; Yanai et al., 2017)). Several prospective and retrospective studies have examined cilostazol as a primary or adjunctive treatment for cognitive deficits associated with AD and schizophrenia (Arai and Takahashi, 2009; Ihara et al., 2014; Sakurai et al., 2013; Shirayama et al., 2011; Taguchi et al., 2013; Tai et al., 2017a; Tai et al., 2017b). As reviewed elsewhere (Heckman et al., 2018a), most of these studies demonstrated positive effects of cilostazol on cognition. The mechanism by which cilostazol elicits improved cognition has yet to be determined empirically. Given there is very little expression of PDE3A or PDE3B in the brain (Kelly et al., 2014; Lakics et al., 2010a), it may be more likely that cognition-enhancing effects of cilostazol are driven by increased cerebral blood flow that comes with chronic—but not acute—dosing (Birk et al., 2004; Kai et al., 2011; Mochizuki et al., 2001). Indeed, recent studies in mice suggest the ability of cilostazol to reverse age-related impairments in hippocampus-dependent memory are related to effects on the blood-brain barrier (Yanai et al., 2017) along with increased cerebral glucose uptake and reduced neuroinflammation (Yanai et al., 2022). Despite its existing FDA approval, the efficacy and safety of cilostazol (Pletal®) is still very much a topic of investigation (c.f., (Baillie et al., 2019a)).

## PDE4

The PDE4 family is arguably the most studied of all the PDE families (c.f., (Baillie et al., 2019a; Kelly et al., 2020)), with PDE4A, PDE4B and PDE4D, but not PDE4C, being expressed in the rodent (Kelly, 2014; Kelly et al., 2014) and human brain (Lakics et al., 2010a). Interestingly, hippocampal PDE4 protein expression appears to decrease from early to late adulthood (Harada et al., 2002; Kato et al., 1998) (but see (Tohda et al., 1996)), yet genetic deletion or broad inhibition of PDE4 rescued many types of Aβ-induced cytotoxicity and age-related decline, including reduced CREB phosphorylation, long-term potentiation deficits, and memory impairments ((Bach et al., 1999; de Lima et al., 2008; Devan et al., 2014; Drott et al., 2010; Kumar and Singh, 2017); c.f., (Baillie et al., 2019b; Kelly, 2018a); see more below). Different studies suggest that individual PDE4 isoforms are differentially affected by disease in a brain region-specific manner (McLachlan et al., 2007; Paes et al., 2021a; Perez-Torres and Mengod, 2003; Sebastiani et al., 2006).

Despite the fact that the broad spectrum PDE4 inhibitor rolipram triggered aging-like impairments in working memory in young adult monkeys (Ramos et al., 2003), more recent studies report nootropic effects of various PDE4 inhibitors in the elderly, patients with schizophrenia, and other human populations (c.f., (Baillie et al., 2019b); see more below). Zembrin is a non-selective PDE4 inhibitor (it also acts as a 5-HT uptake inhibitor) that is not FDA-approved but is a component of a number of herbal supplements claiming calming or mood-stabilizing properties (e.g., Calm,