Doctor's Best; Mood, Procera; Nutri-calm, Nature's Sunshine) (Terburg et al., 2013). Roflumilast has also been tested for its ability to improve cognition and information processing in healthy humans, (Heckman et al., 2018b; Van Duinen et al., 2018).

The cognition-enhancing effects of roflumilast described above are consistent with a press release from Dart Neuroscience claiming that 45mg of their PDE4 inhibitor, HT-0712, the lowest dose tested, improved long-term memory for word-lists in elderly subjects experiencing cognitive decline (see (Baillie et al., 2019a)). Like Dart Neuroscience, Tetra Therapeutics appears to be pursuing an indication related to cognitive functioning for their PDE4D negative allosteric modulator zatolmilast. These effects in humans are again consistent with preclinical studies showing zatolmilast improved a number of behaviors in a mouse model of Fragile-X Syndrome and antagonized the amnestic effects of scopolamine in mice (Gurney et al., 2017; Zhang et al., 2018a). Preclinical cognition-enhancing effects of GSK's PDE4 inhibitor, GSK 356278 were similarly reported (Rutter et al., 2014) as were the ability of several PDE4 inhibitors to ameliorate memory deficits and pathology in dementia-related rodent models (Cong et al., 2023; Feng et al., 2019; Gomaa et al., 2023; Hasan et al., 2022; Liang et al., 2020; Nazir et al., 2021; Virk et al., 2021; Wang et al., 2020; Xia et al., 2022).

From a therapeutic perspective, then, it would be preferable to only target those isoforms in relevant brain regions. As described below, select PDE4A and PDE4D splice variants are the most likely to play a role in molecular mechanisms of memory, because numerous studies have reported that genetically manipulating all/select PDE4B isoforms alter synaptic plasticity but largely have no effect on learning and memory (Campbell et al., 2017; Rutten et al., 2011; Siuciak et al., 2008a; Zhang et al., 2008a).

PDE4A. Many studies have analyzed the effects of genetically manipulating PDE4A on memory. PDE4A knockout mice exhibit normal object recognition memory and spatial water maze memory yet improved passive avoidance memory relative to wild-type mice (Hansen et al., 2014). The selective effect on passive avoidance memory may be related to the aversive nature of the stimuli employed in passive avoidance given the fact that PDE4A deletion produces anxiogenic-like phenotypes on the elevated-plus maze, light-dark transition, and novelty-suppressed feeding tests (Hansen et al., 2014). This is highly interesting given that negatively-valanced memories are thought to trigger a stronger encoding, storing, and reactivation of sensory detail in controls (Hansen et al., 2014) and patients with AD (Maria and Juan, 2017). As extensively reviewed elsewhere (Baillie et al., 2019a), each PDE(4) isoform is uniquely anchored by protein-binding partners through its unique *N*-terminal domain, which leads to regulation of different nanodomains of cAMP. When full-length PDE4A5 that includes its unique *N*-terminal targeting domain is virally overexpressed in hippocampal excitatory neurons, forskolin-induced hippocampal late long-term potentiation (LTP) is impaired and hippocampus-dependent long-term, but not short-term, memory for object location and contextual fear conditioning are attenuated (Havekes et al., 2016a). In contrast, hippocampal delivery of a catalytically dead PDE4A5 that displaces endogenous PDE4A5 (i.e., a dominant negative approach) rescued localized cAMP signaling deficits and hippocampus-dependent memory impairments that were caused by sleep deprivation (Havekes et al., 2016a; Havekes et al., 2016b; Vecsey et al., 2009). Interestingly, overexpression of PDE4A5 in the hippocampus did not alter anxiety-related behaviors in this study, suggesting either that the PDE4A isoforms regulating anxiety may differ from those regulating memory function or that PDE4A5 expression outside of the hippocampus (e.g., in the amygdala or prefrontal cortex) regulates anxiety-related behaviors (Kelly et al., 2020). Importantly, overexpression of PDE4A1, which targets different hippocampal nanodomains, leaves memory undisturbed (Havekes et al., 2016a). As such, compounds or biologicals that target the unique *N*-terminal domain of each individual
PDE4A isoform may be necessary for a beneficial effect in the context of cognitive deficits (as described in (Baillie et al., 2019a)).

**PDE4D.** Expression of PDE4D has been genetically and pharmacologically manipulated to examine its role in hippocampus-dependent memory and plasticity. Genetic deletion of PDE4D strengthened recent long-term memory in the radial arm maze, hidden platform water maze, and object recognition tests while increasing levels of cell proliferation and phosphorylation of CREB in the mouse hippocampus (Li et al., 2011). That said, weaker recent long-term memory for contextual fear conditioning was also observed in the global PDE4D knockout mouse (Rutten et al., 2008). It is quite possible that this particular memory impairment may reflect the loss of PDE4D outside the hippocampus, particularly from the amygdala, because selective knockdown of *PDE4D* in the hippocampus alone improved recent long-term memory for contextual fear conditioning while increasing the number of training-induced stubby spines in CA1 (Baumgartel et al., 2018). Further, *PDE4D* knockout mice require less tetanic or theta burst stimulation to induce long-term potentiation relative to wild-type mice, although the maximum strength of LTP obtained in *PDE4D* knockout mice matches that of wild-type mice (Rutten et al., 2008). Further, PDE4D expression is downregulated via gene methylation in aging rats undergoing a moderate-intensity intermittent training program that attenuated ARCD of spatial learning and memory while improving synaptic structure of the hippocampus (Zhang et al., 2023b). Similarly, PDE4D miRNA infused selectively into prefrontal cortex reversed Aβ1-42-induced cognitive impairment (Shi et al., 2021b). That said, PDE4D was reported to be upregulated in the prefrontal cortex of aged rats in a manner that positively correlated with working memory and inversely correlated with Tau phosphorylation (Leslie et al., 2020). Thus, the role for PDE4D in regulating memory appears to be brain region and memory type specific.

It is likely the long forms of PDE4D, specifically, are negative regulators of hippocampus-dependent memories. Infusion of miRNAs within the dentate gyrus of the hippocampus that target PDE4D4 and PDE4D5 strengthened recent long-term memory in the radial arm maze, hidden water maze, and object recognition tests (Li et al., 2011); however, infusion of miRNAs targeting PDE4D1/2 or PDE4D3 did not. PDE4D4 and PDE4D5 miRNAs also rescued Aβ-42-induced memory deficits in the hidden platform water maze and object recognition tasks (Zhang et al., 2014). Knockdown of *PDE4D* long forms also increased phosphorylation of CREB in the hippocampus (Li et al., 2011), a reduction of which is associated with aging (Kelly, 2018a). When long forms of PDE4D were knocked down in the prefrontal cortex of mice, novel object recognition and spatial memory were similarly improved, as was phosphorylation of CREB and pyramidal neuron dendritic branching/length (Wang et al., 2013). Further, knockdown of PDE4D long forms in the prefrontal cortex rescued memory impairments in mice undergoing chronic unpredictable stress (Wang et al., 2015b). Thus, PDE4D4 and PDE4D5, both within and outside of the hippocampus, play critical roles in constraining neuroplasticity and memory formation. Together, these data suggest that PDE4A5, PDE4D4, and PDE4D5 may be the key PDE4 splice variants to target in the treatment of memory deficits.

## PDE5

PDE5 is probably best known as a drug target for erectile dysfunction (c.f., (Baillie et al., 2019a); however, several studies have pointed to a potential role in regulating brain function. In rodent cerebellum, PDE5A mRNA expression increases between early to late adulthood (Kelly et al., 2014). In animal models, inhibitors of PDE5A have provided protection against age-related decline (Devan et al., 2014; Domek-Lopacinska and Strosznajder, 2008; Orejana et al., 2012; Palmeri et al., 2013). Still, a number of clinical trials have tested the effects of the PDE5 inhibitors tadalafil, sildenafil and vardenafil on various measures of cognition in healthy volunteers, patients with schizophrenia, or elderly patients with cerebral small vessel disease and have largely found no
effects (Goff et al., 2009; Grass et al., 2001; Pauls et al., 2023; Reneerkens et al., 2013a; Reneerkens et al., 2013b; Schultheiss et al., 2001). In contrast, the temporal cortex of patients with AD show a 5x increase in PDE5A expression relative to controls (Ugarte et al., 2015). Further, PDE5A inhibitors rescue memory deficits, synaptic dysfunction, Tau hyperphosphorylation, Aβ burden, and cytotoxicity in AD mouse models (Acquarone et al., 2019; Cuadrado-Tejedor et al., 2011; Fiorito et al., 2013; Garcia-Barroso et al., 2013; Huang et al., 2020b; Justo et al., 2023; Kang et al., 2022; Puzzo et al., 2014; Puzzo et al., 2009; Tabrizian et al., 2021; Zhang et al., 2013; Zhu et al., 2019a) in a PKG-dependent manner (Zhang et al., 2013).

## PDE7

Expression of PDE7A mRNA decreases in rodent cortex from early to late adulthood (Kelly et al., 2014). This age-related reduction in PDE7A mRNA is particularly interesting given that a SNP in PDE7A has been genetically associated in humans with age-related cognitive decline (Andrews et al., 2016; De Jager et al., 2012) and cognitive dysfunction related to brain tumors (Correa et al., 2019). In contrast PDE7A mRNA is decreased in CA2 of the hippocampus in patients with AD relative to controls (Perez-Torres et al., 2003). Thus, it may be surprising that PDE7 inhibitors have positive effects in models of diseases where cognition, neuroprotection, neuroinflammation, and/or motor function are impaired (Banerjee et al., 2012; Garcia et al., 2014b; Jankowska et al., 2017a; Lipina et al., 2013; Mestre et al., 2015; Morales-Garcia et al., 2015a; Morales-Garcia et al., 2015b; Morales-Garcia et al., 2017; Morales-Garcia et al., 2014; Perez-Gonzalez et al., 2013; Redondo et al., 2012), including models of AD (Bartolome et al., 2018; Perez-Gonzalez et al., 2013).

## PDE8

PDE8A3 and PDE8A4/5 protein expression increase in the rodent hippocampus from early to late adulthood (Hegde et al., 2016; Kelly et al., 2014), whereas PDE8A1 protein levels do not change in this brain region (Kelly et al., 2014). PDE8A mRNA levels also increase across the lifespan in the rodent striatum (Kelly et al., 2014). In contrast, PDE8B mRNA is increased in hippocampal CA2 of patients with AD (Perez-Torres et al., 2003) and *in vitro* in response to an accumulation of carboxy-terminal amyloid precursor protein fragments (Kametani and Haga, 2015). This AD-related increase in PDE8B expression may contribute to cognitive deficits associated with the disease since PDE8B inactivation in rodents strengthens recent long-term memory for hippocampus-dependent memories (Tsai et al., 2012). Further, recently characterized PDE8 inhibitors demonstrated therapeutic effects in mouse models of vascular dementia (Huang et al., 2020c; Wu et al., 2022). Together, these results suggest PDE8B inhibitors may be a therapeutic approach for cognitive decline; however, this potential may be limited by anxiogenic side effects (Tsai et al., 2012).

## PDE9

Multiple PDE9A isoforms also exhibit age-related changes in expression in a brain region-specific manner (Patel et al., 2018). In particular, PDE9A isoforms decreased during early postnatal development in the cerebellum and hippocampus of rodents (Patel et al., 2018). Importantly, PDE9A mRNA also decreases during early life in the human hippocampus (Patel et al., 2018). This age-related decrease in hippocampal PDE9A mRNA may reflect a healthy adaptive process, because hippocampal PDE9A mRNA is synergistically elevated in the hippocampus of individuals with a history of traumatic brain injury plus dementia relative to controls, even though patients with only traumatic brain injury or dementia showed no change relative to controls (Patel et al., 2018). The lack of change in PDE9A expression in dementia-only patients may help explain why the PDE9 inhibitors PF-04447943 and BI-409306 failed to improve either cognition or dementia-related behavioral disturbances in patients with AD in Phase II clinical trials (Frolich et al., 2019; Schwam et al., 2014). These clinical failures stood in the face of preclinical studies showing that PDE9A
inhibitors rescue cytotoxicity, plasticity impairments, and memory deficits in AD rodent models (Kroker et al., 2014; Li et al., 2016a; Rosenbrock et al., 2019). Further, the development of novel PDE9 inhibitors continues to be pursued for neurodegeneration (Ribaudo et al., 2021; Swetha et al., 2022; Zhang et al., 2020b).

Not only do expression levels of PDE9A isoforms change with age in a brain region-specific manner, but so does their subcellular localization (Patel et al., 2018). For example, across early development PDE9A6/13 and PDE9X-120 shift from the membrane to the nucleus in the prefrontal cortex and cerebellum but not the striatum or hippocampus (Patel et al., 2018). As discussed elsewhere (Salpietro et al., 2018), the issue of subcellular localization may also have contributed to the aforementioned clinical failures of PF-04447943 and BI-409306 for AD. That is, PDE9A is enriched in the nucleus and membrane (Patel et al., 2018) and, thus, is not in a position to directly regulate the cytosolic pools of cGMP that appear to be dysregulated in AD (Baltrons et al., 2002; Baltrons et al., 2004; Bonkale et al., 1995).

## PDE10

Prenatally, PDE10A mRNA is widely expressed throughout human cortical regions, hippocampus, amygdala, and striatum; however, PDE10A levels dramatically drop by childhood in all regions except in the striatum (Farmer et al., 2020). In the adult human brain, then, PDE10A is predominantly expressed in striatal medium spiny neurons (Farmer et al., 2020; Geerts et al., 2017), and has been largely studied in the context of corticostriatal disorders such as HD and schizophrenia (c.f., (Baillie et al., 2019a)). In adult rodents, PDE10A mRNA is also predominantly expressed in the striatum; however, it is also found at very low levels in the cortex, cerebellum, and hippocampus (Farmer et al., 2020; Kelly et al., 2014). Even so, PDE10A deletion or inhibition in rodents has largely proven ineffective, if not harmful, for learning and memory (Sano et al., 2008; Schmidt et al., 2008; Siuciak et al., 2006a; Siuciak et al., 2006c; Siuciak et al., 2008b). PDE10A is widely reported to be downregulated in the striatum of patients with HD, with the extent of PDE10A loss corresponding to the number of CAG repeats within the Huntington gene (Ahmad et al., 2014; Fazio et al., 2020; Hebb et al., 2004; Russell et al., 2014; Russell et al., 2016; Wilson et al., 2016). PDE10A mutations linked to hyperkinetic movement disorders that phenocopy many features of HD reduce PDE10A expression due to irregular subcellular trafficking that leads to increased PDE10A degradation in the cytosol (Tejeda et al., 2020). Experimentation using highly-specific, PDE10A positron emission tomography tracers shows PDE10A expression continues to decline over years, suggesting the enzyme could be a useful biomarker for assessing the initial diagnosis and subsequent progression of HD (Russell et al., 2016). This downregulation of PDE10A in the HD brain may reflect a compensatory mechanism aimed at increasing cAMP/cGMP signaling, which is known to be reduced in patients samples (e.g., (Gines et al., 2003)). Indeed, HD mouse models also show reduced striatal PDE10A expression (Beaumont et al., 2016; Hebb et al., 2004; Hu et al., 2004; Leuti et al., 2013; Miller et al., 2014), yet PDE10 inhibitors rescue behavioral, neurodegenerative, and electrophysiological deficits (Beaumont et al., 2016; Giampa et al., 2010; Giampa et al., 2009; Giralt et al., 2013; Harada et al., 2017). That said, Pfizer's PF-02545920 failed to improve symptoms in patients with HD and, therefore, further development for was terminated (c.f., (Baillie et al., 2019a)). Omeros and Palobiofarma similarly explored HD as an indication for their PDE10 inhibitors; however, those efforts are suspended or have been terminated (c.f., (Baillie et al., 2019a)).

## PDE11A

In the rodent brain, PDE11A is quite unique in that it is the only PDE whose mRNA expression emanates predominantly (if not solely) from the hippocampus (Kelly et al., 2014). Age-related increases in PDE11A mRNA and PDE11A4 protein expression have been reported in the mouse,
rat, and human hippocampus (Kelly et al., 2014; Pilarzyk et al., 2022). Interestingly, these age-
related increases in protein expression are driven, at least in part, by phosphorylation of Ser¹¹⁷ and
Ser¹²⁴ in the PDE11A4 N-terminal regulatory domain, which also triggers the protein to ectopically
accumulate within filamentous structures termed ghost axons (Pilarzyk et al., 2023; Pilarzyk et al.,
2022). These age-related increases in PDE11A4 expression are likely a direct contributor to the
age-related increases in hippocampal PDE hydrolytic activity and decreases in CREB function
described above (Kelly et al., 2010; Pilarzyk et al., 2022; Smith et al., 2021) as well as age-related
decreases in expression of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor that
occur post-synaptically in the prefrontal cortex (McQuail et al., 2021; Pilarzyk et al., 2019). These
age-related increases in hippocampal PDE11A4 protein expression may also contribute to age-
related changes in microglia activation and cytokine expression in the hippocampus (Pathak et al.,
2017; Pilarzyk et al., 2021; Porcher et al., 2021).

Genetic deletion of PDE11A in mice leads to a transient amnesia for social memories that ultimately produces stronger, remote long-term social memories in young adult mice and prevents age-related cognitive decline of remote long-term social memories in old mice (Pilarzyk et al., 2019; Pilarzyk et al., 2022). This transient amnesia correlates with changes in the overall activation levels and functional connectivity of frontal cortical regions and hippocampal/parahippocampal regions and reduced expression of the glutamate receptor NR1 subunit in the prefrontal cortex (Pilarzyk et al., 2019). Further, viral restoration of PDE11A4 selectively to ventral CA1 of adult *Pde11a* KO adult mice was sufficient to reverse the memory phenotypes caused by the deletion, suggesting the nootropic effect of the deletion was due to the acute loss of PDE11A4 signaling in the adult brain as opposed to an effect on development (Pilarzyk et al., 2019; Pilarzyk et al., 2022). Based on these findings, potent and selective PDE11 inhibitors are currently being developed for treating age-related cognitive decline (Mahmood et al., 2023).

Disrupting PDE homodimerization may also prove to be an effective way to target PDE11A4 function in a subcellular domain-specific manner (GAF-B domain) to rescue cognitive deficits (Pathak et al., 2017). Interestingly, age-related increases in ventral hippocampal PDE11A4 protein are localized to the membrane (Pilarzyk et al., 2022), which suggested the isolated GAF-B domain might prove quite beneficial in the context of age-related cognitive decline (Pilarzyk et al., 2022). Indeed, viral expression of the isolated GAF-B domain in CA1 of mouse hippocampus was able to reduce PDE11A4 protein expression in a compartment-specific manner, reverse age-related cognitive decline of remote long-term social associative memory, and improve social recognition memory in old mice, albeit at the expense of being unable to access recent long-term social memories (Pilarzyk et al., 2023).

Challenges and outlooks

As discussed elsewhere (Baillie et al., 2019a), this is clearly an exciting time in the PDE field but
there is much work that remains to be done. For therapeutics to be efficiently developed, we need
to have a more thorough understanding of exactly where cyclic nucleotide signaling is disrupted in
a given disease, and in which tissue, cell types, and subcellular compartments. We then need to
target a PDE in a defined locale, with the understanding that subcellular compartmentalization of a
given PDE may vary depending on species, age, tissue type, or disease status (Ahmad et al., 2009;
Al-Tawashi and Gehring, 2013; Houslay, 2010; Houslay and Baillie, 2005; Huston et al., 2006;
Nagel et al., 2006; Patel et al., 2018; Penmatsa et al., 2010; Perera et al., 2015; Richter et al., 2008).
This consideration is equally important in the evaluation of potential efficacy and potential side
effects. To maximize potential efficacy while minimizing potential side effects, one would target a
PDE that is enriched, if not exclusively expressed, in the tissue of interest and that controls the same
pool of cyclic nucleotide that is altered by the disease. At the same time, efforts to unravel the
intramolecular signals responsible for trafficking each PDE also need to continue to inform more
sophisticated therapeutic approaches that can preferentially target a given PDE in a given subcellular compartment. Along these same lines, we need to grow our understanding of how to stimulate PDE activity and how to target the PDE catalytic activity of dual-specificity PDEs in a functionally-selective manner (i.e., target only its cAMP- or cGMP-hydrolytic activity, see (Kelly, 2015) for further discussion). Perhaps by increasing the specificity of our approach, we can retain efficacy while mitigating the numerous side effects described above that have plagued PDE inhibitors to date.

PDE Isoforms, Dopamine Signaling, and Disease: Implications for Treatment

There is a high degree of overlap between expression of PDE isoforms and dopamine signaling pathways that mediate motor movement and motivated behaviors. Moreover, the use of new genetic models and novel pharmacological inhibitors has shown that many of the prominent brain PDEs directly impact cyclic nucleotide-dependent dopamine signaling pathways in a region-specific and cell type-specific manner. Certain PDE isoforms, then, represent targets for novel pharmaceutical approaches to a wide array of neuropsychiatric and neurodegenerative diseases that affect dopamine neurons and their target cells, including schizophrenia, Parkinson's disease (PD), HD, and depression. Though the role of PDE isoforms in brain and behavioral disease is the subject of other contributions to this review, we review briefly, here, the potential utility of PDE inhibitors for the treatment of the dopamine-related neurodegenerative disease, PD.

*Parkinson's disease and Symptomatic Treatment:* PD is characterized as a progressive degeneration of dopamine (DA) containing neurons in the brain. It is most often characterized by motor deficits, notably bradykinesia, limb rigidity and resting tremor. Dopamine depletion - most notable as the degeneration of nigrostriatal dopamine neurons - is considered the primary cause for the loss of volitional movement in PD. This effect may contribute to the associated non-motor symptoms of the disease, including cognitive dysfunction, loss of affect, and depression (Bernheimer et al., 1973; Hornykiewicz, 1966) though non-dopaminergic systems are also clearly involved (Braak et al., 2003; Jellinger, 1991). Mutations in specific genes, including *LRRK2*, synuclein, *PINK1*, are linked to familial forms of PD (Biskup et al., 2008) but may also play important roles in the many cases (i.e., >95 % of patients) of the disease for which the biological cause is unknown. There is no cure for PD. Rather, the disease is currently addressed with symptomatic treatments that temporarily restore motor function, including replacement therapies like L-dihydroxyphenylalanine (L-DOPA or levodopa), the immediate precursor for DA synthesis (Jankovic and Aguilar, 2008). Unfortunately, long-term L-DOPA therapy becomes ineffective in treating motor disabilities with chronic use.

The use of adjunctive or alternate first-line therapies which delay the introduction of L-DOPA therapy or reduce the required dose of L-DOPA can positively affect the course of the disease and the appearance of motor side effects (Schrag and Quinn, 2000). Dopamine receptor agonists (e.g., pramipexole, ropinirole), may slow disease progression (Olanow, 2009) and have become part of the arsenal for the treatment of early-stage disease, perhaps with fewer drug-induced dyskinesias (Rascol et al., 2000). Interestingly, *in vitro* preclinical studies show that dopamine receptor agonists may exert anti-apoptotic effects directly on dopamine neurons (e.g., via autoreceptors) (Olanow, 2009); furthermore, dopamine receptor agonists may act at post-synaptic sites (downstream of actions on dopamine neurons) to normalize dopamine activity within the basal ganglia motor system and slow disease progression. The strategy of normalizing motor symptoms to slow disease progression by intervening at points distant from the affected dopamine neurons is the basis for several novel therapeutic approaches.

At present, the only medication recognized by the FDA for efficacy in the treatment of LIDs is amantadine (Metman et al., 1999), a mixed-action drug that produces a modest attenuation of LIDs
in some patients via molecular mechanisms which are unclear, but which likely involve NMDA
receptor blockade and dopamine agonist activities (Jankovic and Aguilar, 2008). Based on these
data, a major avenue for development of new PD therapies is the discovery of stand-alone or
adjunctive therapies that will increase “on” time, enable replacement of L-DOPA or lower
maintenance doses of L-DOPA, and prolong the useful lifetime of PD therapy, delaying or
preventing the appearance of motor fluctuations, including LIDs.

**PDEs as Novel Targets for PD Therapy:** PDEs are exciting and novel targets for development of new therapies to treat the symptoms (motor and non-motor symptoms), motor side effects, and possibly to modify disease progression in PD. The interest in these enzymes stems, in part, from their abundant expression in brain regions that sustain loss of motor function in PD (e.g., basal ganglia) or regions that subsume important roles in cognition (e.g., the prefrontal and dorsolateral cortex and hippocampus) – a prominent non-motor symptomatic deficit in PD (Lakics et al., 2010b). In addition, the ability of PDEs to control levels of the second messengers, cAMP and cGMP, that are the key signaling molecules in the actions of dopamine on motor and cognitive function (Greengard et al., 1999), is another appealing functional property. While it is unclear whether modulation of cAMP or cGMP might be differentially beneficial in addressing symptoms and progression in PD, we will here focus on four PDE families with possible benefit for PD – two which are cAMP-preferring in their actions (PDE4 and PDE7A/B), and two which hydrolyze both cAMP and cGMP (PDE10A and PDE1).

*Inhibitors of cAMP-Preferring PDE4 Enzymes in PD.* PDE4 enzymes have been proposed as drug targets of interest for PD as family members, including PDE4B are abundantly expressed in nigrostriatal dopamine terminals and in striatal medium spiny neurons (MSNs), in proximity to presynaptic and postsynaptic dopamine signaling machinery (Yamashita et al., 1997). Pharmacological inhibition of PDE4 with rolipram increases dopamine synthesis in cultured mesencephalic dopamine neurons (Yamashita et al., 1997). This effect is consistent with the ability of PDE4 inhibition to increase cAMP levels in these neurons, leading to phosphorylation of the dopamine synthetic enzymes, tyrosine hydroxylase (TH) at a site (Ser⁴⁰) that catalyzes dopamine synthesis. These data are supported by *in vivo* studies demonstrating increases in TH phosphorylation and dopamine turnover in the striatum in response to rolipram (Nishi et al., 2008). PDE4 inhibitors also exhibit anti-depressant and pro-cognitive effects in a variety of animal models (Barad et al., 1998; Bolger et al., 1994; Bourtchouladze et al., 1998; D'Sa et al., 2005; Takahashi et al., 1999b; Zhang et al., 2009) that would address non-motor symptoms of PD that are poorly responsive to L-DOPA pharmacotherapy (Chaudhuri and Schapira, 2009). The ability of PDE4 inhibitors to drive dopamine synthesis and release and provide non-motor support, suggests that they might be useful in addressing early-stage PD.

The positive pharmacological effects of PDE4 inhibitors, however, are complicated by postsynaptic
effects that mimic the actions of dopamine D₂-receptor antagonists. D₂-receptor blocking drugs,
such as the antipsychotic medication, haloperidol, produce motor disturbances in animals that
resemble extrapyramidal motor symptoms and tardive dyskinesia (Klawans and Weiner, 1974).
Dopamine receptor antagonists can interfere with the restoration of motor activity by L-DOPA in
animal models of PD (Boyce et al., 1990; Grondin et al., 1999). The PDE4 inhibitor, rolipram,
preferentially increases DARPP-32 phosphorylation at Thr³⁴ in striatopallidal neurons (Nishi et al.,
2008), which are predominantly controlled by dopamine D₂ receptors. Thus, PDE4 inhibitors
produce an effect in this subset of striatal neurons characteristic of dopamine D₂-receptor
antagonists such as haloperidol, which is shared with PDE10A inhibitors (see below), such as
papaverine (Siuciak et al., 2006b). Overall, inhibition of PDE4 enzymes modestly reduces motor
activity in normal mice and rats and potentiates the catalepsy produced by neuroleptic drugs (Kanes
et al., 2007; Siuciak et al., 2007a). Thus, despite the favorable potential effects of PDE4 inhibitors
for non-motor symptoms of PD, including treatment of cognitive deficits and depression (Zhang, 2009), it is unlikely that the motor effects of pan-PDE4 inhibitors would be tolerated in PD patients.

Perhaps the greatest limitation to the development of PDE4 inhibitors for PD, and for CNS-based disorders in general, are the associated gastrointestinal and emetic side effects. The emetic response to PDE4 inhibitors has been attributed to the inhibition of the PDE4D isoform in the brain (Mori et al., 2010) Robichaud et al., 2002); indeed, PDE4D expression is enriched in the area postrema, a region controlling the emetic response. Emesis limits the tolerability of PDE4 inhibitors, thus, stalling their development for brain disorders. Peripherally restricted inhibitors of PDE4 isoforms, including roflumilast and apremilast, have been successfully developed and approved by the FDA for treatment of peripheral inflammatory disorders such as COPD; roflumilast has also been investigated for CNS disorders (Prickaerts et al., 2017), albeit with a narrow therapeutic window. Efforts continue toward the design of PDE4 inhibitors that minimize PDE4D-related safety concerns. These efforts have focused on the design of compounds (e.g., zatolmilast) that work as negative allosteric inhibitors of the PDE4D enzyme and, thereby, lack full emetic potential (Burgin et al., 2010a). To this end, Tetra Therapeutics has advanced a PDE4D inhibitor, zatolmilast, into phase III clinical development for the treatment of Fragile X syndrome (NCT05163808).

*Anti-inflammatory and neuroprotective potential of cAMP-preferring PDE4 and PDE7 inhibitors.*

Another area of drug development focus has been the design of PDE4 inhibitors that target, selectively, the PDE4B isoform, which is proposed not regulate the emetic response (Fox 3rd et al., 2014). PDE4B-preferring inhibitors have been discovered and tested pre-clinically for CNS activity (Pearse and Hughes, 2016) and for safety in assays thought to predict emetic potential in humans. Inhibitors (e.g., ABI-4) of the brain enriched PDE4B isoform exert strong anti-inflammatory and neuroprotective actions in cell-based assays. For example, the release of TNFα from LPS-stimulated human PBMCs and murine primary microglia was suppressed by ABI-4 in a concentration-dependent manner (Hedde et al., 2017). Similarly, ABI-4 suppressed the brain and plasma levels of pro-inflammatory cytokines, including IL1β and IL-6 (though not TNFα) in mice *in vivo* (Hedde et al., 2017). Aging has been associated with enhanced systemic inflammation (Franceschi et al., 2007); subchronic administration of AB4-1 in aged mice significantly reduced brain levels of TNFα and IL1β. Furthermore, lower levels of plasma TNFα were observed in mice genetically lacking the PDE4B isoform (Hedde et al., 2017). Despite the promising preclinical effects of more selective PDE4B inhibitors, like ABI-4 in inflammation and aging models, a suitable PDE4 inhibitor is yet to be evaluated clinical for the treatment of motor and/or non-motor symptoms of PD.

The anti-inflammatory effects of PDE4B-preferring compounds are particularly interesting with regard to PD as it has become increasingly evident that neuroinflammation and immune system dysfunction are likely to be causative or exacerbating factors in the symptomatology and progression of PD (Tansey et al., 2022). As discussed above, PDE4 inhibitors, including PDE4B-preferring molecules, are reported to have potent anti-inflammatory actions which might protect neurons in models of PD and other neurodegenerative diseases. This property of PDE enzymes will be discussed below in reference to other PDE families, including those for PDE7, PDE10A and PDE1.

*Inhibitors of cAMP-Preferring PDE7A/B Isoforms in PD.* The PDE7 family has also been proposed as a potential target for PD therapy due, in part, to high expression in striatal neurons and the potent anti-inflammatory/neuroprotective effects of inhibitors. To date, only a few studies have been published on this cAMP-preferring PDE. PDE7 enzymes (predominantly the PDE7B isoform) are abundantly expressed in the brain. PDE7B mRNA levels are high in rat dentate gyrus, striatum and olfactory tubercle (Reyes-Irisarri et al., 2005). PDE7B mRNA is localized to striatal MSNs and its translational regulation under control of the dopamine D₁-receptor confirmed (Sasaki et al., 2004). More recently, double *in situ* hybridization analysis has shown that the PDE7B signal also localizes
to dopamine D₂-receptor-containing striatal neurons (De Gortari and Mengod, 2010), further supporting the potential significance of this PDE as a target for the development of therapies for PD. Expression of PDE7B in the hippocampus further connects this isoform with brain circuitry underling cognitive dysfunction in PD. A link between familial PD genes and PDE7B is supported by a recent study in mice overexpressing mutant A53T-alpha (α) synuclein (Kurz et al., 2010). Mutant α-synuclein, which was associated with reduced levels of several indices of striatal dopamine signaling, was found to negatively regulate striatal gene expression, including the gene encoding PDE7B. High-affinity inhibitors of this PDE (e.g., OMS182401) are being investigated for motor benefit in animal models of PD (see http://www.michaeljfox.org/).

PDE7 inhibitors, like PDE4B inhibitors, elicit strong anti-inflammatory effects and may be responsible for protective effects on dopamine neurons (Chen and Yan, 2021; Garcia et al., 2014a; Zorn and Baillie, 2023). Inhibition of PDE7B or silencing of the gene encoding PDE7B dampens expression of inflammatory cytokines (like TNFα) in rodent neurons treated with 6-OHDA (a dopamine-depleting neurotoxin) or the inflammogen, LPS (Chen et al., 2021). The neuroprotective effects of PDE7 inhibitors are accompanied by increases in tissue levels of cAMP suggesting that they protect dopamine neurons via pathways involving cAMP (Sasaki et al., 2004). The PDE7A isoform, furthermore, is highly expressed in human proinflammatory and immune cells (Smith et al., 2004a) providing a broader role for this PDE in the regulation of brain and systemic inflammation.

*Inhibitors of the Dual cAMP/cGMP PDE10A Enzyme in PD.* A high level of basic research and drug development interest has focused on PDE10A, one of the dual cAMP/cGMP hydrolyzing PDE families. Initial interest in PDE10A inhibitors was as a novel target for the treatment of schizophrenia (Schmidt et al., 2009). PDE10A inhibitors, including the tool compound, papaverine, mimic the effects of established antipsychotic medications possessing dopamine D₂-receptor antagonist activity in a variety of behavioral models (Siuciak et al., 2006b; Siuciak et al., 2007a). Deletion of the gene encoding *PDE10A* mimicked the behavioral actions of antipsychotic medications in mice (Siuciak et al., 2006b). These studies, however, noted that pharmacological inhibition of PDE10A or deletion of the *PDE10A* gene consistently elicited disruptions in motor function, including reduced spontaneous locomotor activity and increases in response latency in sensorimotor tests (Siuciak et al., 2006b). Clinical investigations of PDE10A inhibitors established a lack of efficacy in the treatment of psychosis (Menniti et al., 2021; Walling et al., 2019).

Despite concerns that dopamine D₂-receptor antagonist-like properties of PDE10A inhibitors could further compromise motor activity, mild dopamine D₂ antagonist activity provided by these agents might be of value in the management of LIDs. Dopamine receptor sensitization that results from the loss of striatal dopamine innervation and the effects of dopamine replacement therapy likely contribute significantly to the development of LIDs (Nutt, 1990). Thus, mild dopamine D₂-receptor antagonist-like activity that would normalize dopamine receptor responses to L-DOPA might delay the onset or lessen the severity of motor responses to replacement therapy. Dopamine D₂-receptor antagonists effectively suppress the appearance of specific behaviors in animals that are analogous to human dyskinesias, including axial, limb, and orolingual movements, without significantly comprising L-DOPA effects on spontaneous motor activity (Monville et al., 2005; Taylor et al., 2005). Antagonists of specific receptors within the D₂ family, like the D₃-type dopamine receptor, have recently been shown to suppress LIDs, supporting the idea that molecules with D₂-receptor antagonist-like activity may be useful anti-dyskinetic agents (Visanji et al., 2009). Clinically, several studies support the efficacy of atypical antipsychotic drugs, such as clozapine and aripiprazole, for the control of LIDs. The positive effects of these drugs may be due to their complex pharmacology, which includes activity at several different receptors. Thus, the interesting dopamine signaling effects of both PDE4 and PDE10A inhibitors may warrant their consideration for
neurological indications such as LIDs. For example, abnormal orolingual movements induced by chronic neuroleptic drug treatment, a model for dyskinetic behaviors seen after L-DOPA, is attenuated by rolipram treatment (Sasaki et al., 1995).

*Inhibitors of the Dual cAMP/cGMP PDE1 Enzyme in PD.* PDE1 enzyme also represent an interesting target for addressing the motor symptoms of PD. The concept is supported by the enrichment of the PDE1B isoform in basal ganglia (Polli and Kincaid, 1994), and the demonstrated actions of pan-PDE1 inhibitors in enhancing cAMP-dependent actions of dopamine at the biochemical and behavioral level (Pekcec et al., 2018b; Snyder et al., 2016a). PDE1B was originally recognized as an attractive candidate for dopamine-related indications such as PD based on its striatal enrichment and close association with brain regions receiving heaving dopaminergic innervation (Polli and Kincaid, 1994; Yan et al., 1994). Further, *PDE1B* gene knockout amplifies dopamine signaling via D₁-receptor pathways and motor activity stimulated by low-level dopamine agonist administration (Ehrman et al., 2006b; Reed et al., 2002a; Siuciak et al., 2007a). The data support the idea that PDE1B inhibition enhances dopamine signaling in a stimulus-bound manner, as deletion of the *PDE1B* gene in mice resulted in no significant change in basal protein phosphorylation and negligible changes in basal locomotor activity. This quality is consistent with the unique regulatory properties of the PDE1 family of enzymes. As all three PDE1 family members are stimulated by Ca²⁺/CaM (the only one of 11 PDE families with this property), their activity is likely controlled by neuronal activity. This property confers an “on demand” quality to PDE1 activity that would be anticipated to provide phasic amplification of dopamine signaling contingent upon stimulation of MSNs by endogenous factors. The “on demand” activity of PDE1B may be a superior attribute as a drug target compared with dopamine agonists which tonically activate dopamine receptors (Wennogle et al., 2017). The tonic activation of receptors by agonists is one factor which results in dopamine receptor changes that contribute to the development of motor fluctuations (Olanow, 2009). Establishing PDE1B as a therapeutic target for PD will need to be evaluated with potent and selective inhibitors.

Over the past decade, several potent and selective PDE1 inhibitors have been discovered and reported to have activity on motor features of PD and/or on behavioral dimensions such as cognition, which are prominent non-motor features compromised in PD and poorly treated by current PD medications. The first fully characterized, orally active, brain permeant and selective inhibitor of PDE1 was Lenrispodun. Lenrispodun has been reported, primarily, to enhance memory performance in rodents using the novel object recognition test (NOR) (Snyder et al., 2016a). Pekcec and colleagues further demonstrated the ability of the compound to elevate brain levels of cAMP and cGMP and facilitate dopamine D₁-receptor and PKA-dependent neural transmission in prefrontal cortical brain slices (Pekcec et al., 2018b). Behaviorally, these investiagtors showed that the compound acted like a dopamine D₁-receptor agonist to reverse MK-801-induced cognitive deficits in a continuous alternation task. Interestingly, the compound preferentially improved the cognitive performance of “low performing” rats in the 5-CSRTT attentional assay, while having little effect on “high performing” rats. These results fit nicely with, what is referred to, as an “inverted U” curve noted with cognitive responses of animals to treated with dopamine D₁-receptor agonists. Animals typically show improved cognitive performance in response to dopamine D₁-receptor agonists only at optimal levels of dopamine D₁-receptor agonism; lower and higher levels of activity are associated with poorer cognitive performance (Goldman-Rakic et al., 2000).

Most recently, a novel inhibitor of PDE1 has been disclosed by Sumitomo Dianippon Pharma Co., Ltd, which has efficacy in reducing the expression of dyskinetic behavior in MPTP-lesioned primates receiving long-term treatment with L-DOPA (Enomoto et al., 2021). To date, it is unclear if this PDE1 inhibitor in being advanced into clinical testing for PD or any other indications.
It is noteworthy, that like inhibitors of PDE4, PDE7 and PDE10A, PDE1 inhibitors also possess potent anti-inflammatory activity in cell-based and *in vivo* inflammation models. Early on, the PDE1B isoform was found to be expressed in immune cells. Bender and Beavo demonstrated enhanced expression of PDE1B levels in monocytes when stimulated to differentiate into macrophages (Bender and Beavo, 2006). Another study found that the pan PDE1 inhibitor, Lenrispodun, suppressed the release of the pro-inflammatory cytokine, TNFα, from microglia-like BV2 cells in response to LPS treatment (O'Brien et al., 2020). Lenrispodun also inhibited expression of proinflammatory genes in LPS-stimulated BV2 cells, including IL1β and CCL2. Functionally, these gene expression changes were correlated with inhibition of BV2 migration toward the chemoattractant, ADP, in a Boyden chamber assay, implying that the compound was capable of dampening recruitment of microglia to sites of inflammation (O'Brien et al., 2020). Transcriptome analysis using RNAseq showed that most genes regulated by Lenrispodun in LPS-treated cells were distinct from those regulated by rolipram, a pan-inhibitor of PDE4; PDE4 is the nearest cross-reactive PDE family to Lenrispodun (Li et al., 2016b; Snyder et al., 2016a). These data support the idea that inhibitors of the PDE1 family of enzymes appear able to distinctly regulate unique gene networks separate from those controlled by a well-characterized pan-PDE4 inhibitor (O'Brien et al., 2020). Whether PDE7 and PDE10A inhibitors also control networks of genes distinct from PDE1 and other PDE families has yet to be clarified. Taken together, the prominent anti-inflammatory effects of PDE1 inhibitors argues for possible neuroprotective effects in PD in addition to possible motor-based symptomatic effects.

*cGMP and corticostriatal correlates of dyskinesia: possible role for PDE inhibitors.* The therapeutic potential of PDE inhibitors and, in particular, inhibitors of cGMP hydrolysis catalysed by dual-specificity PDEs, in PD is highlighted by recent research on the molecular basis of dyskinesia. These studies have identified a dysfunction in striatal cGMP signaling as an electrophysiological correlate of LIDs in animals. Work by Calabresi and colleagues has identified a deficit in long-term depression (LTD) of striatal responses to high frequency stimulation (HFS) of corticostriatal slices in dopamine-depleted animals displaying dyskinesia after chronic L-DOPA treatment (Picconi et al., 2003). Rats depleted of striatal dopamine with the neurotoxin 6-OHDA received repeated daily doses of L-DOPA that resulted in a subset of animals developing LIDs and another subset of rats that remained non-dyskinetic under similar treatment conditions. Corticostriatal slices from dyskinetic rats were distinguishable from those from non-dyskinetic animals based on the loss of LTD responses. Thus, the absence of LTD provides an electrophysiological correlate for dyskinesia (Picconi et al., 2003). Rats with established LIDs expressed lower striatal levels of cAMP and cGMP compared with normal animals. Treatment of these animals with agents such as zaprinast, an inhibitor of cGMP preferring PDEs, partially restored striatal cyclic nucleotides and attenuated dyskinesias (Giorgi et al., 2008). Zaprinast, and other cGMP-elevating agents, further induced LTD responses in corticostriatal slices (Calabresi et al., 1999; Picconi et al., 2011). Together, these data indicate that LIDs may be associated with abnormal corticostriatal plasticity that results from deficits in cGMP levels. Thus, PDE inhibitors that elevate striatal cGMP levels and signaling are potentially beneficial for attenuating LIDs. These data support a possible therapeutic role for inhibitors of several striatal-enriched PDEs in this indication including PDE1B.
Several generations of research in the biology of cancer have led to the definition of a number of
properties that collectively define the neoplastic state, commonly referred to as the Hallmarks of
Cancer (Hanahan and Weinberg, 2000; 2011). The original definition of these hallmarks included
six biological capabilities acquired during the multistep development of human cancer. These
included: (1) sustaining proliferative signaling, (2) evading growth suppressors, (3) resisting cell
death, (4) enabling replicative immortality, (5) inducing angiogenesis, and (6) activating invasion
and metastasis. Essential for the acquisition of these hallmarks is the concept of genome instability,
which contributes to the mutations required for the development of neoplasia, and inflammation,
which promotes several hallmark functions. To these six hallmarks, two more were subsequently
added: (7) reprogramming of energy metabolism and (8) evading immune destruction. Study of
cyclic nucleotide signaling in cancer provide an opportunity to observe these hallmarks in action.
Alterations in cAMP and cGMP signaling can profoundly contribute to the neoplastic state.
Modulation of cAMP and, especially, cGMP signaling can profoundly attenuate and modulate the
cancer phenotype. Collectively, these advances in cyclic nucleotide signaling in neoplasia have the
potential to lead to important advances in the prevention, diagnosis, and treatment of several
important human cancers.

**2.6.1 | Effects in neoplastic cells**

cAMP PDE: a role for driver mutations

The concept of driver mutations is essential to understanding the functional, pathogenic and clinical
role of cAMP and cGMP signaling in cancer. Driver mutations are defined as germline or somatic
mutations in DNA that play an essential role in generating the transformed phenotype (Bailey et
al., 2018; Gorelick et al., 2020; Greenman et al., 2007; Sack et al., 2018; Sanchez-Vega et al., 2018).
Driver mutations can therefore be defined as follows: (1) they materially affect the expression or
structure of the RNA/protein encoded by the mutated gene(s), leading to alterations in its
physiological function(s), producing a growth advantage; (2) they localize to specific "hot spots"
within the gene product essential to its cellular function, such as its enzymatic activity or regulation;
and (3) they are present in a substantial proportion of clinical specimens obtained from a specific
cancer type. "Passenger" mutations, in contrast, differ from driver mutations in that they do not
play a clear role in cancer formation. Passenger mutations typically (1) do not change the
physiological or biochemical functions of the gene product; (2) do not concentrate in "hot spots";
and (3) are found in only a small proportion of clinical specimens obtained from a specific cancer
type.

Using the strict criteria for cancer driver mutations, as defined above, we can identify cancer-
associated driver mutations in 11 different genes encoding members of cAMP-signaling pathways,
and which are involved in at least 9 different cancers (Table 1) (see (Ahmed et al., 2022; Bolger,
2022) for recent reviews). In the PDE space, germline and tumor-associated driver mutations in
PDE8B and PDE11A are the best characterized driver mutations, as described in detail previously
(Bolger, 2022). The PDE8B mutations have been identified in patients with adrenal hyperplasia,
adenomas and carcinomas and have been shown to attenuate PDE8 enzymatic activity. Other PDE
germline mutations may also predispose to adrenal tumors (Bolger, 2022).

As thoroughly reviewed elsewhere (Kelly, 2015; Kelly, 2018b), a large number of PDE11A
intronic, synonymous (i.e., non-coding), missense (i.e., nonsynonymous coding), and nonsense
mutations (i.e., truncating) have been associated with a variety of endocrine-related tumors. The
majority of these variants are common, but rare mutations have also been reported in a small number
of patients with various tumor types (Kelly, 2018b). The tumor-associated PDE11A mutations
largely produce a loss-of-function phenotype either by reducing expression, catalytic activity, or
proper localization of the enzyme; however, it is important to note that the effects of the mutations
are often cell-type specific (Kelly, 2015) (Kelly, 2018b), (Faucz et al., 2011; Horvath et al., 2006; Horvath et al., 2009; Libe et al., 2008; Libe et al., 2011; Pathak et al., 2015; Peverelli et al., 2009; Vezzosi et al., 2012). Tumors of endocrine tissues have also found to be associated with reduced PDE11A expression of non-mutational genetic causes, such as transcriptional and epigenetic regulation (Boikos et al., 2008; Mirabello et al., 2012)). Together, these studies suggest that a loss of PDE11A function more likely is a risk modifier than an inducer of tumors (Kelly, 2015), which is supported by PDE11A KO mouse studies that show no increased presence of tumors (Kelly et al., 2010).

### cGMP PDE: a Role of PDE5 and PDE10 in cell cycle regulation and apoptosis

Highly potent and selective inhibitors of PDE5 (e.g., sildenafil) and PDE10 (e.g., Pf2545920) have been reported to induce cell cycle arrest and apoptosis of cancer cell lines grown *in vitro* at concentrations that activate cGMP/PKG signaling (Lee et al., 2016; Li et al., 2015b; Mei et al., 2015). As depicted in Figure 15, PKG activation results in the suppression of both β-catenin transcriptional activity and RAS/MAPK signaling. The mechanism for suppressing β-catenin transcriptional activity appears to result from PKG mediated phosphorylation of β-catenin on residues known to induce ubiquitination and proteasomal degradation resulting in reduced nuclear levels needed to activate Tcf/Lef transcription (Lee et al., 2016; Lee et al., 2021). The disruption of MAPK signaling by PKG activation may be attributed to disrupting RAS membrane localization and activation (Cho et al., 2016) or by interfering with receptor tyrosine kinase activity (Tao et al., 2012). The ability of activated PKG to block oncogenic β-catenin and RAS signaling simultaneously is consistent with reports that PDE10 inhibitors can suppress both signaling pathways in lung and ovarian cancer cell lines (Borneman et al., 2022; Zhu et al., 2017a). This possibility is supported by the broad anticancer activity of PDE10 inhibitors and is significant given that mutations in APC/β-catenin and RAS/MAPK pathway components drive most human cancers.

### 2.6.2 | Preclinical and clinical studies: cGMP PDE inhibition and anticancer activity

Based on effect of PDE subtypes on tumor biology, (pre)clinical research has been conducted, which was hitherto limited to cGMP PDEs. We will now discuss the history and the gathered evidence for the application of cGMP PDEs in cancer treatment, and pinpoint cross talk with other pivotal signaling pathways.

#### Early evidence for cGMP-PDE involvement: studies of exisulind and NSAIDs

The association between cGMP-degrading PDEs and the inhibition of cancer cell proliferation and survival was first reported from studies of sulindac sulfone, a metabolite of the nonsteroidal anti-inflammatory drug (NSAID), sulindac (Thompson et al., 2000). Sulindac sulfone (exisulind) was in clinical trials for the treatment and prevention of precancerous colon adenomas, but its mechanism of action was unknown. Exisulind and several analogs were found to inhibit the proliferation and induce apoptosis of colorectal cancer (CRC) and bladder cancer cell lines at concentrations that caused a sustained elevation of cGMP and activation of PKG that was not found with PDE5-selective inhibitors or other PDE inhibitors (Piazza et al., 2001; Thompson et al., 2000). Nonetheless, PDE5 was initially suspected as a likely target because it was the predominant cGMP-PDE degrading isoenzyme expressed in CRC and bladder cancer cell lines, and exisulind did not affect cAMP levels. Despite modest potency and no apparent PDE isoenzyme selectivity, exisulind significantly suppressed tumor formation in multiple, chemical-induced rodent models of colon, bladder, breast, lung, and prostate tumorigenesis, suggesting that the well-established cancer chemo preventive activity of NSAIDs may involve a COX-independent mechanism of action (Malkinson et al., 1998; Narayanan et al., 2007; Piazza et al., 1997; Piazza et al., 2001; Reddy et al., 1999; Thompson et al., 1997). Exisulind showed promising efficacy in clinical trials of patients with familial adenomatous polyposis (FAP) or sporadic adenomas and selectively induced apoptosis of
colonocytes in precancerous lesions without affecting colonocytes in the adjacent normal mucosa (Arber et al., 2006; Stoner et al., 1999). However, exisulind was not approved by the FDA because of liver toxicity that was likely attributed to low potency and lack of PDE isoenzyme selectivity.

The NSAID sulindac (Clinoril®) also inhibits adenoma formation in FAP patients (Giardiello et al., 1993) and has broad cancer chemopreventive activity in experimental models of tumorigenesis. However, the long-term use of sulindac and other NSAIDs is not FDA-approved for long-term use because of potentially fatal toxicities resulting from COX-1 and COX-2 inhibition. While the antineoplastic activity of sulindac and other NSAIDs is commonly attributed to COX-2 inhibition and suppression of prostaglandin synthesis (Wang and Dubois, 2010), numerous investigators have concluded that both COX-dependent and COX-independent mechanisms are involved (Figure 15) (Gurpinar et al., 2014).

Initial evidence suggesting that the anticancer activity of NSAIDs is mediated by an off-target mechanism involving cGMP PDE inhibition is based on experiments showing that the rank-order potency of a chemically diverse group of NSAIDs, including the COX-2 selective inhibitor, celecoxib, to inhibit *in vitro* growth of HT-29 CRC cells correlated with the inhibition of cGMP PDE but not of COX-2 (Tinsley et al., 2010). In addition, concentrations of NSAIDs required to inhibit cell growth far exceeded those required to block COX-1 or COX-2. These data, along with results of other experiments showing that cancer cell lines, which do not express COX-2, are sensitive to NSAIDs, and the inability of prostaglandins to rescue cell growth inhibition by NSAIDs, support a COX-independent mechanism for the cancer chemopreventive activity of sulindac and possibly other NSAIDs and COX-2 inhibitors. However, the contribution of COX-2-derived prostaglandins should not be excluded given their broad biological activity that can have an impact on multiple oncogenic pathways (Gurpinar et al., 2014; Wang and DuBois, 2006).

*Preclinical research on PDE5 and PDE10 inhibition as possible anti-cancer therapies*

Publications reporting that inhibitors of cGMP/PKG signaling, including NO donors, guanylyl cyclase activators, cell permeable cGMP analogs, and certain cGMP PDE inhibitors, inhibit cancer cell proliferation and induce apoptosis suggest that PDE5 is essential for cancer cell proliferation and survival (Tinsley et al., 2009; Tinsley et al., 2010). Multiple investigations have supported this possibility by reporting that PDE5 is overexpressed in colon, bladder, breast, and lung cancers compared with non-involved adjacent tissue (Bisegna et al., 2020; Chan et al., 2002; Iwasaki et al., 2021; Mei et al., 2015; Piazza et al., 2001; Pusztai et al., 2003b; Tinsley et al., 2009; Tinsley et al., 2010; Tinsley et al., 2023; Whitehead et al., 2003b). Consistent with a role of PDE5 in regulating cancer cell growth, PDE5 knockdown by siRNA selectively inhibited the growth of colon and breast cancer cell lines expressing high PDE5 levels compared with normal colonocytes or mammary epithelial cells with low PDE5 expression (Tinsley et al., 2011; Tinsley et al., 2009). In addition, gene silencing of *PDE5* in the highly aggressive human breast cancer cell line, MDA-MB-231, resulted in decreased cell motility and formation of lung metastases (Marino et al., 2014). Conversely, PDE5 overexpression in MCF-7 breast cancer cells led to increased motility and invasion (Catalano et al., 2016). Sildenafil and vardenafil were also reported to inhibit proliferation and induce caspase-dependent apoptosis in B-cell chronic lymphatic leukemia, which suggested a role of cGMP in regulating hematological malignancies (Sarfati et al., 2003). Finally, a sulindac derivative, sulindac benzylamide, with PDE5 selectivity that did not inhibit COX-1 or COX-2, potently inhibited CRC cell growth (Whitt et al., 2012).

The finding that sulindac sulfide, a metabolite of the NSAID, sulindac, attenuated CRC cell growth at concentrations that blocked PDE5 and PDE10 activity, inspired a drug discovery campaign to identify cGMP PDE inhibitors based on the same chemotype that did not target COX-1 and COX-2. This resulted in the synthesis of a large library of compounds sharing the indene scaffold of
sulindac. Screening this library against recombinant COX, to confirm a lack of effect on prostaglandin synthesis, and PDE5 and PDE10, led to the discovery of several lead compounds, which displayed appreciably greater potency as inhibitors of cancer cell growth at concentrations that blocked PDE5 and/or PDE10. Chemical optimization of these indene-based inhibitors to improve their drug-like properties holds promise for further development of this novel anti-cancer therapy (Borneman et al., 2022; Lee et al., 2021; Li et al., 2015a; Li et al., 2013; Piazza et al., 2009; Tinsley et al., 2023; Whitt et al., 2012).

Given that known PDE5 inhibitors lacked sufficient binding affinity to arrest the proliferation of cancer cell lines *in vitro*, attention turned towards PDE10 because selective inhibitors (e.g., PF2545920) were found to attenuate cancer cell growth with low micromolar potency. PDE10 is overexpressed in colon and lung adenocarcinomas relative to non-involved adjacent tissue (Li et al., 2015b; Zhu et al., 2017a) and interventions that reduced PDE10 activity (selective inhibitors, siRNA-mediated knockdown) arrested the proliferative response. Conversely, transfection of plasmid DNA encoding PDE10 to normal or precancerous colonocytes stimulated proliferation (Li et al., 2015b). Conversely, PDE10 inhibitors had a negligible impact on the proliferation of cells (colonocytes, airway epithelia) in which PDE10 levels were low or undetectable.

Further experiments showed that PDE10 is essential for CRC cell proliferation and survival by suppressing β-catenin transcriptional activity (Li et al., 2015a). The concentration range of a potent and selective PDE10 inhibitor (PF2545920) needed to inhibit CRC cell growth matched the concentration required for activation of cGMP/PKG signaling and suppression of β-catenin transcriptional activity and Wnt-induced translocation of β-catenin to the nucleus. Interestingly, PDE10 was found to co-localize with the oncogenic form of β-catenin in dysplastic colon regions from APC<sup>min</sup> mice (i.e., mice containing a multiple intestinal neoplasia [min] allele of the adenomatous polyposis coli [APC] locus, which encodes a nonsense mutation at codon 850 (Lee et al., 2021)). These observations were significant since CRC is mostly driven by mutations in the APC/β-catenin axis. Oral administration of PF2545920 did not have antitumor activity in subcutaneous CRC mouse tumor models, but peri-tumoral injection to bypass liver metabolism suppressed growth in subcutaneous mouse tumor models (unpublished). Following these observations, a novel orally bioavailable PDE10 inhibitor derived from sulindac (ADT-061) was reported to potently and selectively inhibit CRC cell growth *in vitro* and suppress colon tumorigenesis in the APC<sup>min</sup> mouse model (Lee et al., 2021).

Two other research groups independently found a link between poor prognosis in patients with non-small cell lung cancer (NSCLC) and PDE10A expression. In one of those studies, an activating mutation in the *PDE10A* gene as a driver of NSCLC was identified by using a combination of structural biology and genomic data (Shen et al., 2017). Fusco and colleagues observed a negative correlation between *PDE10A* mRNA and protein levels and overall and recurrence-free survival in NSCLC patients by comparing genomic data from high-risk and low-risk individuals (Fusco et al., 2018). The role of PDE10 in lung cancer was supported by studies showing that *PDE10* mRNA and protein were overexpressed in lung cancer cell lines compared to normal airway epithelial cells and lung tumors relative to normal lung tissue. Further, PDE10 inhibitors and gene knockdown of *PDE10* selectively inhibited lung cancer cell growth by blocking both β-catenin transcriptional activity and MAPK signalling (Zhu et al., 2017a). Another study extended those observations by reporting that PDE10 inhibitors, including ADT-061 (aka MCI-030) inhibited ovarian cancer cell growth by suppressing both β-catenin transcriptional activity and MAPK signaling (Borneman et al., 2022).

*Translational and clinical studies with PDE5 inhibitors*
A role of PDE5 during early stages of colon tumorigenesis was shown by Browning and colleagues who reported that sildenafil can suppress adenoma formation in mouse models of inflammation-induced CRC (Islam et al., 2017; Sharman et al., 2018). Similar cancer chemopreventive activity was reported with the guanylyl cyclase C agonist, Plecanatide (Chang et al., 2017). These findings supported the testing of PDE5 inhibitors and guanylyl cyclase C activators for CRC cancer chemoprevention in clinical trials, which was feasible given that such drugs are FDA-approved for chronic indications (e.g., erectile dysfunction and constipation) and generally well tolerated.

Case studies have reported that the long-term use of PDE5 inhibitors can reduce the risk of death or developing metastasis in male patients diagnosed with CRC, as well as lowering the risk of developing CRC in men with benign colon neoplasia (Huang et al., 2019a; 2020a). While PDE5 inhibitors are FDA-approved for erectile dysfunction and pulmonary hypertension, their use for cancer prevention remains experimental and could have undesirable side effects (e.g., thrombocytopenia) from long-term use. Recent studies by Browning and colleagues describing a novel PDE5 inhibitor (malonyl-sildenafil) that inhibits the proliferation of colon epithelium in mice following oral administration without systemic absorption, holds promise for CRC chemo prevention (Lee et al., 2023). In addition, existing drugs that activate guanylyl cyclase C and are approved for constipation are being studied for CRC chemoprevention, which also act locally in the colon (Rappaport and Waldman, 2020). The potential benefits of combining exisulind or sildenafil with chemotherapeutic drugs has also been studied in experimental models and in clinical trials as summarized in *Table 2* but have not resulted in significant benefits for patients with advanced stage malignancies.

## Dual PDE5 and PDE10 inhibition

Research has showed additive, antiproliferative activity following simultaneous blockade of PDE5 and PDE10 with small molecule inhibitors (MY5445 and papaverine, respectively) or hybrid PDE5/PDE10 inhibitors (e.g., ADT-094). Similar data were obtained by silencing, simultaneously, PDE5 and PDE10, suggesting that the expression of these two enzyme families in neoplastic cells may cooperate to maintain low intracellular cGMP levels and, thus, provide proliferative and survival advantages to neoplastic cells during tumorigenesis (Li et al., 2015a).

While the concentration range required for PDE5 and PDE10 inhibitors to suppress cancer cell growth is similar to those needed to inhibit cGMP hydrolysis in cell lysates and activate cGMP/PKG signaling in intact cells (Zhu et al., 2017a), appreciably lower concentrations are needed to inhibit the isolated enzymes. This suggests that the effect of such inhibitors in cells is not isoenzyme selective and that the high concentrations non-selectively inhibit both isoenzymes. The co-expression of PDE5 and PDE10 in cancer cells may explain the discrepancy between cellular and biochemical experiments involving recombinant enzymes whereby the expression of one isoenzyme in cancer cells may compensate for the effects of an inhibitor that is specific for the other isoenzyme, resulting in the need for higher concentrations to inhibit both PDE5 and PDE10. In support of this hypothesis, experiments using dual PDE5/10 inhibitors or dual genetic knockdown of PDE5 and PDE10 resulted in greater growth suppression than inhibition of either isozyme alone (Li et al., 2015b). Further, in the case of PDE5 inhibitors, sildenafil is a known substrate for ATP-binding efflux transporters, which may also account for the apparent discrepancy between potencies involving experiments in cells vs. isolated enzymes (Ding et al., 2011).

## Neoplastic effects of PDE5 and PDE10 inhibition: conclusions

Mutations in β-catenin and RAS and their pathway components (e.g., APC, RAF) account for most human cancers. These oncoproteins have been extensively studied but considered challenging or “undruggable” cancer targets, given that the cellular pathways they support are essential for the proliferation and survival of both cancer and normal cells. For example, Wnt-driven activation of
β-catenin-dependent transcription is well known to be crucial for maintaining the survival of normal stem cells, and growth factor activation of RAS-driven/ MAPK/AKT signaling is needed for normal cell turnover or in response to injury. The observation that PDE10 is overexpressed in certain cancers, and essential for cancer cell growth and that PDE10 inhibitors can suppress both β-catenin and RAS signaling suggests an unrecognized strategy to kill cancer cells selectively. Although conventional PDE10 inhibitors were developed for CNS conditions and may not be able to achieve adequate systemic levels for anticancer activity in experimental mouse tumor models, novel PDE10 inhibitors that can achieve systemic levels needed to kill cancer cells selectively warrant further investigation for treating cancers harboring mutations in β-catenin or RAS or pathway components. There is potential for safety given that PDE10 has low expression in most peripheral tissues with no known physiological function. Lastly, evidence that PDE5 and PDE10 inhibitors can activate mechanisms of antitumor immunity suggests potential benefits in combination with immunotherapy (see Figure 15).

## 2.7 | The role of PDEs in immune regulation

### 2.7.1 | General background

In previous chapters the important contribution of inflammation in the role of PDE function in pathophysiology is accented ubiquitously. The immune response and its mediators are involved in the pathogenesis of various diseases, and we here highlighted tissue remodeling effects in internal organs, fibrosis and extracellular matrix formation, and neurodegeneration. Alterations in the immune environment of tissues have categorically become a part of the standard variables to be measured in studies on the pathobiology of diseases. With respect to PDEs, comprehensive knowledge has especially been built up in the framework of tumor progression which is discussed in depth. In this chapter, the involvement of cyclic nucleotide signaling in regulation of cells of the innate and adaptive immune system is summarized. This will start with a comprehensive review of the role in tumor microenvironment. Thereafter, the relevance of each individual immune cell effect is illustrated with typical examples in cancer and internal organ disease to facilitate the extrapolation to the expertise of the readership. Due to its comprehensiveness this latter part uses a two-layer mode to transfer the knowledge, the first layer being the cell type, and as a second layer the role of the PDE subtype.

### 2.7.2 PDEs in the tumor immune microenvironment

#### General aspects

It has become increasingly clear that the effectiveness of many anticancer drugs relies, in part, on the host immune system. Mounting evidence indicates that the immune composition of the tumor microenvironment (TME) can profoundly influence tumor response to treatments. For example, immunologically “hot” tumors, which show signs of inflammation and are infiltrated with T lymphocytes, tend to respond well to immune checkpoint inhibition (ICI) therapy; In contrast, “cold” tumors, characterized by lack of T cell infiltration, are resistant or refractory to immunotherapy (Galon and Bruni, 2019). In terms of T cells, their functional status is an important determinant of the host antitumor immunity. It has been well-established that chronic antigenic stimulations which often occurs during chronic viral infections and cancer development, can lead to functional exhaustion in CD8+ T cells, characterized by gradual loss of the ability to proliferate, persist and produce inflammatory cytokines (Schietinger et al., 2016; Wherry and Kurachi, 2015). Exhausted CD8+ T cells are phenotypically and functionally heterogenous, consisting progenitor, transitory and terminally exhausted cells, each characterized by distinct transcriptional and epigenetic signatures as well as varied responsiveness to anti-PD-1 ICI therapy (Beltra et al., 2020; Miller et al., 2019; Sade-Feldman et al., 2018; Siddiqui et al., 2019). The TME is often enriched in regulatory T cells ($T_{reg}$), which are a subset of CD4+ T cells known to suppress antitumor immunity.
Besides T lymphocytes, myeloid cells in the TME are also known to impact tumor progression and response to therapies. Extensive studies have demonstrated that a subset of aberrantly developed immature myeloid cells, termed myeloid-derived suppressor cells (MDSCs), promote tumor growth, metastasis and immune evasion, presenting a major hindrance to the effectiveness of various types of cancer treatments.

It has been shown that β-catenin signaling in tumor cells can prevent dendritic cell (DC) recruitment, resulting in T cell exclusion in the TME and tumor resistance to checkpoint immunotherapy (Luke et al., 2019; Spranger et al., 2015; Spranger et al., 2017). Not only does tumor-intrinsic β-catenin signaling promote tumor immune evasion (Spranger and Gajewski, 2015) but β-catenin activation in DCs also contributes to immune tolerance (Suryawanshi and Manicassamy, 2015). Tumors can induce the activation of β-catenin in DCs in the draining lymph nodes, rendering them tolerogenic, which induces Treg cells to suppress antitumor activity.

## The role of PDE in inflammation and TEM

The fact that PDE11A loss-of-function is associated with an increased risk of various tumors may be related to its likely role in regulating inflammation. Interestingly, PDE11A expression can be induced by stress and immune activation signals in cells that do not basally express the enzyme (Bazhin et al., 2010; Witwicka et al., 2007; Zhu et al., 2019b). Further, reduced PDE11A4 expression in the brain correlates with increased expression of the proinflammatory cytokine, IL-6, increased cytokine release, and increased microglial activation (Pathak et al., 2016; Pilarzyk et al., 2021). Conversely, increased PDE activity may also be relevant. As PDE inhibition emerges as an attractive therapeutic strategy for cancer treatment, there is growing interest in understanding whether and how PDE5 and PDE10 inhibitors impact the various immune components in the TME. Mechanistic explanations of the role of PDE5 on tumor biology can be found in from their effects on various immune cells. With respect to MDSC, it was first reported that PDE5 inhibition by sildenafil or tadalafil led to enhanced intratumoral T cell infiltration and activation, along with improved tumor growth control in multiple mouse transplant tumor models (Serafini et al., 2006). These beneficial effects were lost in immune-deficient mice, indicating that the antitumor effect of PDE5 inhibitors was immune-mediated. Mechanistically, the restoration of antitumor immunity in tumor-bearing mice was due to abrogation of MDSC-mediated immune suppression as PDE5 inhibitors down-regulated arginase 1 and NO synthase-2, the main mediators of MDSC immunosuppressive activity. A subsequent study using a spontaneous mouse melanoma model confirmed that PDE5 inhibition by sildenafil resulted in reduced MDSC accumulation and immunosuppressive function, accompanied by restoration of CD8+ T cell effector activity and improvement in mouse survival (Meyer et al., 2011).

In further explanation of the role of PDE5, it is conceivable that suppressing β-catenin signaling, either in tumor cells or DCs, with PDE5 inhibitors can overcome some of the major immunosuppressive mechanisms (MDSCs, tolerogenic DCs and Treg cells) in the TME, thereby improving tumor response to immunotherapy. The observed beneficial effects of PDE5 inhibitors in mouse tumor models and clinical studies, including reduced Treg presence and increased tumor infiltration of activated CD8+ T cells, may be driven by fully activated DCs as the result of β-catenin suppression secondary to PDE5 inhibition. The mechanisms linking PDE inhibitor-induced β-catenin suppression to DC and T cell recruitment/activation in the TME requires elucidation.

Evidence for a role of immune modulation by PDE5 in tumor progression has also been found in humans. Clinical trials were conducted to evaluate whether PDE5 inhibition can revert tumor-induced immunosuppression and promote antitumor immunity in patients with head and neck squamous cell carcinoma, metastatic melanoma, or multiple myeloma (Califano et al., 2015; Hassel et al., 2017; Weed et al., 2015). These trials indicate that tadalafil administration correlated with
reduced MDSC accumulation and/or suppressive function and improved T cell activation, with one
trial reporting additional reduction of Treg cells. However, future research should examine whether
PDE5 inhibition also affects MDSC induction or recruitment because a reduction in MDSC
accumulation was observed in some but not all published studies. Moreover, the exact function and
expression kinetics of PDE5 in MDSCs and Treg cells warrant further investigation.

The immunological impact of PDE10 inhibition has remained largely unexplored. It is reasonable to speculate that PDE10 inhibitors mirror PDE5 inhibitors in exerting immunomodulatory effects because their mechanisms of action overlap in terms of activation of cGMP/PKG signaling and suppression of β-catenin transcriptional activity. It is worth noting, that some of the newly developed PDE10 inhibitors suppress oncogenic RAS signaling (Borneman et al., 2022; Zhu et al., 2017b), raising the possibility that they may improve tumor immunogenicity. Aberrant RAS activation occurs in about 20% of all malignancies, with high incidences found in pancreatic cancer (90%), colorectal cancer (50%) and lung cancer (30%) (Bos, 1989). Oncogenic RAS signaling is known to promote immune suppression (Weijzen et al., 1999). It has been reported that KRAS mutations induce Treg cells (Cheng et al., 2019; Zdanov et al., 2016), upregulate PD-L1 in cancer cells (Chen et al., 2017; Coelho et al., 2017; Sumimoto et al., 2016), but downregulate MHC class I molecules (Atkins et al., 2004; El-Jawhari et al., 2014). In addition, RAS activation promotes tumor production of G-CSF and GM-CSF, which induce and expand MDSCs to facilitate tumor progression, metastasis, and immune suppression (Phan et al., 2013; Pylayeva-Gupta et al., 2012). With the advances in developing mutant-specific inhibitors targeting KRAS G12C (Ostrem et al., 2013), RAS is no longer considered an undruggable target (Molina-Arcas et al., 2021). It is reassuring that in pre-clinical studies, many of these newly developed KRAS inhibitors significantly improved antitumor immunity by reducing MDSCs, enhancing antigen-presentation and CD8+ T cell priming. These data validate RAS inhibition as a potent immune-potentiating strategy in addition to its direct tumoricidal effect (Briere et al., 2021; Canon et al., 2019; Kemp et al., 2023; Mugarza et al., 2022; Zhang et al., 2022). Likewise, the emergence of novel PDE10 inhibitors that are capable of suppressing oncogenic RAS and β-catenin activities have the potential to reverse an immunosuppressive TME, a feature that awaits to be exploited to drive durable therapeutic outcomes.

2.7.3 | The role of PDEs in cells of the innate and adaptive immune systems

Cyclic nucleotide signaling in the regulation of immune response has been on the map for a long
time. Previous publications described that cAMP plays a critical role as a second messenger (Rall
and Sutherland, 1958; Sutherland and Rall, 1958) and has been shown to be a key regulator of
activation and function of cells of the innate (Schafer et al., 2019; Schafer et al., 2014) and adaptive
immune system (Amarandi et al., 2016; Bourne et al., 1974; Wang et al., 1978). Numerous anti-
inflammatory drugs successfully target molecules of the cAMP signaling pathway including several
FDA-approved medications (Baillie et al., 2019b; Blokland et al., 2019; De Souza et al., 2012;
Dong et al., 2016; Jarnagin et al., 2016; Milara et al., 2012; Rabe, 2011; Schafer et al., 2019; Schafer
et al., 2014; Schett et al., 2010; Sriram and Insel, 2018; Tenor et al., 2011; Victoni et al., 2014;
Wittmann and Helliwell, 2013). As described in Chapter 1, cAMP is degraded by cyclic nucleotide
PDEs (Conti and Beavo, 2007; Lerner and Epstein, 2006), which constitute a group of enzymes
known to hydrolyze cAMP and cGMP and, hence, maintain spatial and temporal control over its
activity. Cells of the innate and adaptive immune system play a critical role in inflammation and its
modulation (Medzhitov, 2021; Meizlish et al., 2021). Function and regulation of different
subpopulations of T cells, B cells and natural killer (NK) cells, as well as myeloid cells, such as
neutrophils, monocytes, macrophages, and DCs involve activation of the cAMP pathway.
Additionally, interactions between leukocytes and endothelial cells are critical during the formation
of inflammatory lesions and can be regulated by cAMP/cGMP signaling. As a general rule, cAMP
levels in cells of both the innate and adaptive immune systems are correlated with their inflammatory activities (Bourne et al., 1974; Mosenden and Tasken, 2011; Rueda et al., 2016; Schafer et al., 2019; Schafer et al., 2014; Vang et al., 2013; Wang et al., 1978). While PDEs have been recognized as potential drug targets for anti-inflammatory drugs, developing specific PDE inhibitors has faced challenges, primarily due to side effects; nevertheless, progress has been made with the approval and clinical use of selected PDE4 inhibitors for treating major inflammatory diseases (vide supra). This chapter will discuss the role of PDEs in cells of the innate and adaptive immune system (Table 3).

### 2.7.4 | Role of PDEs in specific immune cell types

**Dendritic Cells:** DCs are heterogenous and are commonly classified into subtypes including plasmacytoid DC (pDC), myeloid or conventional DC1 (cDC1) and myeloid or conventional DC2 (cDC2), as well as tissue-specific DCs such as Langerhans cells (Collin and Bigley, 2018). Early studies indicated that during differentiation of DCs, PDE4 activity decreased, while activities of PDE1 and PDE3 increased. Of note, rolipram, at PDE4-selective concentrations, blocked LPS-induced TNFα release by ~37%. In contrast, the PDE3 inhibitor, motapizone, only marginally influenced TNFα synthesis, but a synergistic inhibitory effect was noted in combination with rolipram (Gantner et al., 1999). In addition, the PDE4 inhibitor, roflumilast, has been shown to be a potent immunomodulator of DC cell function (Hatzelmann and Schudt, 2001). In mice, the cAMP-PKA-CREB signaling pathway orchestrates many functional aspects of cDC2s (Lee et al., 2020; Schafer et al., 2014). Notably, PDE4B is highly expressed in mouse DCs (Chinn et al., 2022). Using mice deficient in Gαs, PDE4B was shown to be a key regulator of cellular cAMP concentrations in DCs and played a role in DC-mediated T helper cell type 2 (Th2) dependent immunopathology (Chinn et al., 2022; Jin et al., 2010). PDE4 inhibition in DCs has also been shown to reduce their ability to induce T helper cell type 1 (Th1) cells from naïve T cells in vitro, an effect that was largely attributed to an effect on PDE4A (Heystek et al., 2003).

**Granulocytes:** The immunosuppressive effect of the PDE4 inhibitor, roflumilast, on a wide range of neutrophil functions in vivo and in vitro is well documented (Chang et al., 2024; Cortijo Gimeno and Morcillo Sanchez, 2010; Hatzelmann et al., 2010; Hatzelmann and Schudt, 2001; Jones et al., 2005; Koga et al., 2016; Lin et al., 2023a; Nials et al., 2011; Sanz et al., 2007; Tsai et al., 2023; Wollin et al., 2005). Several studies demonstrated that PDE4 inhibitors suppress oxidative stress and chemotaxis in neutrophils (Chang et al., 2024; Cortijo Gimeno and Morcillo Sanchez, 2010; Hatzelmann et al., 2010; Hatzelmann and Schudt, 2001; Jones et al., 2005; Koga et al., 2016; Lin et al., 2023a; Nials et al., 2011; Sanz et al., 2007; Tsai et al., 2023; Wollin et al., 2005). Additionally, PDE10A was shown to regulate neutrophil infiltration in a mouse model of lung inflammation (Hsu et al., 2021). PDE inhibitors (ciclamilast, piclamilast, IBMX) have also been shown effectively suppress pulmonary eosinophilia in rodent models of allergic airway disease (Lee et al., 2020; Zheng et al., 2019).

#### Monocytes and macrophages

**Monocyte differentiation:** Changes in cyclic nucleotide levels have been shown to have a profound impact on the phenotypical differentiation of monocytes (Gantner et al., 1997a; Hertz and Beavo, 2011; Schudt et al., 1995; Tenor et al., 2011). Importantly, during in vitro differentiation of human blood-derived monocytes, the PDE profile undergoes significant remodeling which parallels that in human alveolar macrophages (Schudt et al., 1995; Tenor et al., 1995a). Major changes in PDE1, PDE3 and PDE4 activities are seen, while PDE4 activity, the major PDE isotype of peripheral blood monocytes, rapidly declines under in vitro culture (Gantner et al., 1997a). Subsequent investigations have delineated the role and function of various PDEs in monocytes and macrophages and those finding are described in more detail below.
**PDE1:** Bender and colleagues reported the selective up-regulation of PDE1B2 during monocyte-to-macrophage differentiation (Bender et al., 2005). Recently, an inhibitor of Ca²⁺/CaM-dependent PDE1 was shown to suppress LPS-induced expression of genes encoding pro-inflammatory cytokines and motility in rodent microglial cells (O'Brien et al., 2020; Zhou et al., 2023).

**PDE4:** The expression and function of specific PDE4 isoforms in monocytes and macrophages are differentiation dependent (Schafer et al., 2014; Shepherd et al., 2004). In gene knockout studies in mice, Conti and colleagues demonstrated in vivo and ex vivo the selective regulation of the LPS-Toll-like receptor signaling pathway in macrophages by Pde4b but not Pde4a or Pde4d (Jin et al., 2005).

**PDE10:** Recent reports indicate a role of PDE10A in lung inflammation mediated by macrophages. Treatment of murine macrophages with LPS in vitro induces a sustained expression of PDE10A, in contrast to a more transient induction of PDE4B. Similarly, LPS-induced cytokine and chemokine production were differentially affected by selective inhibition of PDE10 versus PDE4. These results were supported by in vivo experiments performed in Pde10a-deficient mice, or in mice treated with a PDE10 selective inhibitor (Hsu et al., 2021).

**Natural Killer cells:** As with other cells of the immune system, raising the levels of cAMP suppresses the activity of NK cells (Shepherd et al., 2004). Studies have focused on the regulation of NK cells by a broad variety of PDEs and selective isoforms including PDE3 and PDE4 (Chen and Yan, 2021; Schafer et al., 2010; Walker and Rotondo, 2004; Whalen and Crews, 2000). The PDE4 inhibitor, apremilast, has been shown to suppress the pro-inflammatory activity of most cells of the innate and adaptive immune system, including TNFα production by NK cells in a model of psoriasis (Schafer et al., 2010). Notably, PGE₂, mediated NK cell suppression in a tumor environment can be reversed through external exposure to IL-15 which acts, in part, by upregulating the expression of PDE4A (Chen and Yan, 2021). Similarly, the broad spectrum PDE inhibitor, IBMX, suppresses interferon (IFN) γ synthesis by NK cells (Walker and Rotondo, 2004).

**T cells:** PDEs in human lymphocytes, particularly T cells, have proven to be very effective therapeutic targets for treating inflammation, with three PDE4-selective inhibitors now approved and in the clinic for treatment of several diseases, including COPD, psoriasis, psoriatic arthritis, atopic dermatitis, and seborrheic dermatitis. There is evidence that PDEs other than PDE4 may also be effective therapeutic targets for treating certain inflammatory conditions, and in the following sections we review what is known about PDEs that are expressed in T cells, and how they might be useful as targets for treating inflammation.

**PDE1:** The Ca²⁺/CaM-dependent PDE1 gene family all hydrolyze cGMP with Kms in the low micromolar range, but differ in their affinities for cAMP (1 µM, 7-24 µM and 50-100 µM for PDE1C, PDE1B, and PDE1A, respectively (Lerner and Epstein, 2006)). Early analyses of quiescent human peripheral blood lymphocytes (HPBL) showed little or no expression of PDE1 (Epstein and Hachisu, 1984; Epstein et al., 1987; Giembycz, 1996; Tenor et al., 1995b). It was also shown in early studies that cAMP PDE activity was highly elevated in murine (Hait and Weiss, 1976) and human (Epstein et al., 1977) transformed lymphocytes associated with hematological malignancies, and PDE activity was greatly induced in HPBL following activation with mitogenic agents such as phytohemagglutinin (Epstein et al., 1980). Subsequently, PDE1 activity was shown to be expressed in a human B lymphoblastoid cell line (Epstein et al., 1987), and further analyses showed that PDE1B1 was present in both T and B lymphoblastoid cell lines and induced in HPBL following mitogenic activation (Jiang et al., 1996; Jiang et al., 1998; Kanda and Watanabe, 2001). The full open reading frame of the PDE1B1 cDNA was cloned from a human lymphoblastoid cell line and antisense oligodeoxynucleotides designed to inhibit the expression of PDE1B1 induced apoptosis in these cells (Jiang et al., 1996), but not in quiescent HPBL (Epstein, 1998).
The recent development of potent, selective inhibitors of PDE1 have now facilitated the testing of PDE1 as a therapeutic target for combating inflammation in a number of experimental systems. As noted in the section on monocytes and macrophages, the PDE1 inhibitors, lenrispodun (PDE1B IC₅₀ = 58 pM) (O'Brien et al., 2020) and compounds 4a and 5f (PDE1C IC₅₀S = 2.5 nM and 4.5nM, respectively) in Zhou and colleagues suppressed brain neuroinflammation by preventing microglia migration as well as blocking nuclear factor-κB-mediated release of inflammatory mediators and activation of the cAMP/CREB axis (Zhou et al., 2023). Lenrispodun also reduced inflammatory cytokine levels and ameliorated vascular function and inflammatory responses in an Ercc1Δ/- murine model of aging (Golshiri et al., 2021a). Naringenin, a polyphenolic flavonoid isolated from citrus fruit, was shown to bind to CaM and inhibit CaM-stimulated PDE1 activity; it also inhibited LPS-induced inflammatory cytokine release from the SK-OV-3 and K562 human cell lines, that were immortalized from patients with ovarian serous cystadenocarcinoma and chronic lymphocytic leukemia, respectively (Afshari et al., 2023). Early studies had shown that nimodipine, a 1,4-dihydropyridine calcium channel antagonist, was capable of directly inhibiting PDE1 at low micromolar concentrations (Epstein et al., 1982). Based on this observation, structural modifications of nimodipine were made that increased potency and selectivity for PDE1. One of those compound, 2g, synthesized by the Wu laboratory (PDE1C IC₅₀ = 10 nM), exhibited anti-inflammatory properties by reducing the expression of TGFβ and attenuating fibrosis in a bleomycin-induced lung fibrosis rat model (Huang et al., 2022). Another potent PDE1 inhibitor, a quinoline-2 (1H)-one derivative, compound 10c (PDE1C IC₅₀ = 15 nM), inhibited the LPS-induced release of inflammatory cytokines from the murine macrophage cell line RAW264.7 and exhibited appreciable clinical improvement in a dextran sodium sulfate-induced mouse model of inflammatory bowel disease (Zhang et al., 2023a; Zhang et al., 2023b). Hence PDE1 may prove to be an effective target for treating inflammation.

**PDE2:** Human T cells express little or no PDE2 (Giembycz, 1996; Tenor et al., 1995a). In contrast, in murine thymocytes PDE2 represents as much as 80% of the total hydrolytic activity when activated by cGMP (Michie et al., 1996). A recent study investigated the expression of PDE2A and PDE3B in murine conventional (T<sub>con</sub>) and T<sub>reg</sub> cells at rest and after activation with anti-CD3/CD28 and cGMP-elevating natriuretic peptides on cAMP levels and on early activation markers, CD25 and CD69 (Kurelic et al., 2021). In that study, engagement of the T-cell receptor (TCR) led to the selective upregulation of PDE2A protein expression in T<sub>con</sub> cells, whereas no change in expression was detected in T<sub>reg</sub> cells. In contrast, TCR engagement significantly increased PDE3B levels in both resting and activated T<sub>con</sub> subsets but not in T<sub>reg</sub> cells. Thus, it seems that an elevation of cGMP led to an increase in cAMP levels in non-activated T<sub>con</sub> cells, presumably through inhibition of PDE3B (which would be more predominant in the non-activated state) and this led to a decrease in cAMP levels in activated T<sub>con</sub> cells, where PDE2A, the cGMP-activated PDE, is induced. When assessing if this cGMP/cAMP crosstalk following activation of T<sub>con</sub> cells might have functional effects, it was found that cGMP elevation by atrial natriuretic peptide enhanced T<sub>con</sub> activation as indicated by enhanced expression of the early activation markers CD25 and CD69. Furthermore, this effect was blocked by a PDE2A selective inhibitor indicating that this was due to a cGMP-mediated reduction in cAMP levels secondarily to the activation of PDE2A. Hence, at least in mice, PDE2A may play a functional role in T cell activation.

**PDE3:** An early investigation of PDE activity in HPBL supernatants identified a single, prominent form of cAMP PDE based on DEAE anion-exchange chromatography, isoelectric focusing and glycerol gradient analysis, enzymes kinetics and inhibitor sensitivity (Epstein and Hachisu, 1984). Subsequently, a study of whole homogenates of purified human T lymphocytes showed two separable high affinity cAMP PDEs on HPLC columns, with one peak characteristic of PDE4 being purely cytosolic, and the other peak characteristic of PDE3 localized exclusively in the particulate fraction (Robicsek et al., 1991; Robicsek et al., 1989). Other studies with purified human T cells
confirmed the presence of PDE3 in the particulate fractions and showed PDE3 activity to be represented exclusively by PDE3B with no evidence of PDE3A (Ekholm et al., 1997; Giembycz, 1996; Sheth et al., 1997; Tenor et al., 1995a). In contrast to T cells, purified human B cells express no mRNA for PDE3B and only very marginal expression of PDE3A mRNA (Gantner et al., 1998). The expression of PDE3B in T<sub>reg</sub> cells is considerably reduced compared to that in T<sub>con</sub> cells and it appears that the low catalytic activity of PDE3B is critical for the regulation of T<sub>reg</sub> cell-specific gene expression (Gavin et al., 2007). Functionally, PDE4 inhibitors suppressed PHA- and anti-CD3-induced proliferation of purified human CD4<sup>+</sup> and CD8<sup>+</sup> T-lymphocytes and the release of IL-2 and IFNγ, whereas inhibitors of PDE3 did not. However, although inactive by themselves, PDE3 inhibitors potentiated the inhibitory activity of PDE4 inhibitors on these processes (Giembycz, 1996). In a similar vein, inhibition of PDE3B augmented PDE4 inhibitor-induced apoptosis in a subset of patients with chronic lymphocytic leukemia who were resistant to PDE4 inhibition alone (Moon et al., 2002).

**PDE4:** Early studies showed PDE4 to be the predominant isoenzyme in the cytosolic fraction of human lymphocytes (Epstein and Hachisu, 1984) with *PDE4A*, *4B*, and *4D*, but not *4C*, contributing, presumably, to the overall hydrolytic activity (Gantner et al., 1997c; Giembycz, 1996; Jiang et al., 1998; Peter et al., 2007). PDE4 has long been known to play a key role in regulating T cell activation and functions (Abrahamsen et al., 2004; Arp et al., 2003; Asirvatham et al., 2004; Barnette et al., 1998; Bielekova et al., 2000; Bjorgo and Tasken, 2006; Claveau et al., 2004; Ekholm et al., 1997; Erdogan and Houslay, 1997a; Gantner et al., 1997b; Gantner et al., 1997c; Hatzelmann and Schudt, 2001; Jimenez et al., 2004; Jin et al., 1998; Jin et al., 2010; Kanda and Watanabe, 2001; Michie et al., 1996; Michie et al., 1998; Peter et al., 2007; Sommer et al., 1997). A key mechanism appears to be the modulation of signal transduction through the TCR by signaling through PGE<sub>2</sub> via EP2 and EP4 receptors, adenosine via A2a and A2b receptors that yields cAMP (Figure 16). Activation of the TCR leads to cAMP production localized in lipid rafts, activation of PKA and, subsequently, the inhibition of the TCR signal through a PKa-Csk inhibitory pathway scaffolded by Ezrin-EBP50-PAG/Cbp anchoring complex (Abrahamsen et al., 2004; Bjorgo and Tasken, 2006; Wehbi and Tasken, 2016). However, engagement of the co-stimulatory receptor, CD28, leads to the recruitment of β-arrestin and PDE4 to lipid rafts and a decrease in the local cAMP pool and of PKA activity (Figure 17). PDE4 inhibitors downregulate the TCR signal by increasing the local cAMP concentration and PKA activity, which counteract the CD28-induced recruitment of PDE4. Thus, localized activities of cAMP, PKA, and PDE4 regulate the upstream TCR signal necessary for T cell activation and the subsequent initiation of effector functions (Schafer et al., 2014; Wehbi and Tasken, 2016). As for downstream effects of cAMP signaling, inhibition of PDE4 in CD4<sup>+</sup> T cells by broad-spectrum and selective inhibitors leads to suppression of effector functions, including cell proliferation in response to TCR signals and co-stimulation, as well as cytokine production and cell motility (Bielekova et al., 2000; Ekholm et al., 1997; Gantner et al., 1997c; Hatzelmann and Schudt, 2001; Jimenez et al., 2001; Pette et al., 1999; Sommer et al., 1997). In a series of in vivo experiments using gene knockout mice, it was shown that cytokine production by Th2 cells is dependent on PDE4B expression while Th1 cells were apparently unaffected (Jin et al., 2010).

**PDE7:** PDE7, primarily PDE7A, is also expressed in human lymphocytes, albeit to a lesser extent than PDE3 and PDE4; PDE7A1 is primarily cytosolic whereas PDE7A2 mainly associates with a particulate fraction (Bender and Beavo, 2006; Bloom and Beavo, 1996; Giembycz, 1996). Subsequent studies showed that PDE1B, PDE7A, and PDE8A were induced following activation of human lymphocytes (Glavas et al., 2001; Jiang et al., 1998; Kanda and Watanabe, 2001). Moreover, a critical requirement of PDE7A induction for full T cell activation has been reported (Guo et al., 2009; Li et al., 1999). While the potential of PDE7 as a therapeutic target to treat inflammation has been investigated in several laboratories, it still remains controversial (Szczypka,
2020; Zorn and Baillie, 2023). In this respect, an antisense oligonucleotides approach has implicated PDE7A in T lymphocyte activation (Li et al., 1999). In contrast, T cells from Pde7a-deficient mice were activated normally by anti-CD3/CD28 (Yang et al., 2003). Similarly, PDE7 inhibitors did not impair CD3/CD28-dependent activation of human CD4⁺ T-lymphocytes (Nueda et al., 2006). It has been suggested that PDE7 may be a target for treating inflammation in conjunction with inhibition of PDE4. Thus, the PDE7 inhibitor, BRL 50481, enhanced the inhibitory effect of rolipram on lymphocyte proliferation and cytokine release (Smith et al., 2004a). Additionally, T-2585, a potent PDE4 inhibitor (IC₅₀ = 0.013 nM), which also inhibits PDE7 with an IC₅₀ = 1.7 µM, inhibited proliferation and cytokine release from T cells under conditions in which the highly selective PDE4 inhibitor, piclamilast had no effect (Nakata et al., 2002). Similarly, the PDE inhibitor, ASB16165, which inhibits PDE7A with an IC₅₀ = 15 nM and PDE4 with an IC₅₀ = 2.1 µM, also inhibited anti-CD3/CD28-stimulated T cell proliferation and cytokine release (Kadoshima-Yamaoka et al., 2009c). Additionally, in an in vivo mouse model of smoke-induced lung inflammation, combined antisense inhibition of the expression of PDEs 4B, 4D and 7A produced a much greater anti-inflammatory effect than use of the PDE4-selective inhibitor, roflumilast, alone(Fortin et al., 2009). Given that inhibition of PDE7 can often enhance the effects of a PDE4 inhibitor, medicinal chemistry efforts were initiated to produce compounds that can selectively and potently inhibit both of the cAMP PDEs although, at the time of writing, a definitive role for PDE7 as a target for mitigating inflammation remains to be established (Huang et al., 2023).

**PDE8:** Few PDEs are currently the targets of FDA-approved drugs and there is a significant knowledge gap about the potential therapeutic role of other PDE isoforms particularly for the treatment of inflammatory diseases. The cAMP-specific PDEs, PDE8A and PDE8B, have been the subject of numerous studies (Basole et al., 2017; Brown et al., 2013; Chen et al., 2009b; Demirbas et al., 2013; DeNinno et al., 2011; Dong et al., 2015; Dong et al., 2006; Dong et al., 2010; Fisher et al., 1998; Glavas et al., 2001; Hayashi et al., 1998; Johnstone et al., 2017; Kelly, 2018b; Kobayashi et al., 2003; Maurice, 2013; Shimizu-Albergine et al., 2016; Soderling et al., 1998; Tsai and Beavo, 2012; Vang et al., 2016; Vang et al., 2010). PDE8A and PDE8B are expressed widely across human tissues (Wang et al., 2008a) and have been implicated in testosterone and corticosteroid production (Demirbas et al., 2013; Tsai et al., 2010), myocyte contraction (Patrucco et al., 2010), lymphocyte adhesion and chemotaxis (Basole et al., 2017; Dong et al., 2015; Dong et al., 2006; Vang et al., 2010; Vang et al., 2013), memory and coordination (Tsai et al., 2012), human airway smooth muscle relaxation (Johnstone et al., 2017), immune protection against intracellular pathogens (Blanco et al., 2017), brain disorders associated with inflammation (Chimienti et al., 2019) and systemic lupus erythematosus (Orlowski et al., 2008). In addition, a recent study identified SNPs in the *PDE8B* locus that were associated with a susceptibility to Sjögren's Syndrome (Taylor et al., 2017).

T cell activation induces PDE8A1 (Glavas et al., 2001), a splice variant that has an affinity for cAMP that is up to 100 times higher than PDE4 isoforms (Bender and Beavo, 2006; Fisher et al., 1998; Gamanuma et al., 2003; Hayashi et al., 1998; Soderling et al., 1998). This property of the PDE8 family suggests that they may regulate changes of baseline cAMP gradients around cell signaling complexes. The availability of PDE8 inhibitors and disruptors has greatly enhance the ability of scientists to interrogate the function of PDE8 in vitro and in vivo. It has been shown that PDE8A regulates motility of lymphocytes and breast cancer cells, including adhesion to endothelial cells under physiological shear stress and chemotaxis (Basole et al., 2017; Dong et al., 2015; Dong et al., 2006; Vang et al., 2010; Vang et al., 2013). These functional effects seem to be uniquely controlled by PDE8 and are distinct from PDE4-regulated outcomes (Vang et al., 2016). The therapeutic activity of biologicals and compounds interacting with molecular targets on pathogenic T cells has been demonstrated in vitro and in vivo (Brocke et al., 1999; Healy and Antel, 2016; Steinman, 2005; Yednock et al., 1992). Current observations reveal PDE8 to be one of those targets
for blocking T<sub>eff</sub> cell motility and, potentially, inflammation (Basole et al., 2017; Dong et al., 2015; Dong et al., 2006; Vang et al., 2016; Vang et al., 2010; Vang et al., 2013). The possible role of PDE8 as an anti-inflammatory target has been examined in vivo. Thus, in experimental autoimmune encephalomyelitis (EAE) induced by immunization with a myelin oligodendrocyte glycoprotein peptide, a model of multiple sclerosis, the PDE8 inhibitor, PF 04957325, suppressed clinical signs of EAE, inflammatory lesion formation and accumulation of Th1 and Th17 effector T cells in the CNS (Basole et al., 2022; Brocke et al., 1999). Collectively, these data demonstrate the efficacy of pharmacologically targeting PDE8 as a treatment of autoimmune inflammation by reducing the inflammatory lesion load.

**PDE9:** PDE9A1 and a novel splice variant, PDE9A5, have been detected in human T cells (Wang et al., 2003b). PDE9A5 was localized to the cytosolic whereas PDE9A1 was expressed exclusively in the nucleus. The function of PDE9 in T cells and whether it has a regulatory role in controlling inflammation is unknown.

**Regulatory T Cells:** It is well established that T-effector (T<sub>eff</sub>) cells and T<sub>reg</sub> cells express high and relatively low levels of PDEs, respectively. The low abundance of PDEs in T<sub>reg</sub> cells and high level of cAMP have been linked to the mechanism by which this T-cell subset suppresses the function of T<sub>eff</sub> cells through the direct cell-to-cell transfer of cAMP (Bopp et al., 2007; Bopp et al., 2009; Vaeth et al., 2011). Mechanistically, the transcription factor, forkhead box P3 (Foxp3), expressed in T<sub>reg</sub> cells has been shown to selectively repress genes, including those encoding PDEs, leading to elevated levels of intracellular cAMP (Bopp et al., 2007; Bopp et al., 2009; Vaeth et al., 2011). Remarkably, T<sub>reg</sub> cell subsets in mice show significantly lower expression of *Pde1a*, *Pde1b*, *Pde2a*, *Pde3b*, *Pde4b*, *Pde5a*, *Pde7a* and *Pde8a* compared to naive T<sub>eff</sub> cell subsets (Vang et al., 2013). Consistent with these findings, Foxp3 represses *Pde3b*, and that reducing *Pde3b* expression by genetic means permits normal T<sub>reg</sub> cell homoeostasis and T<sub>reg</sub> cell-specific gene expression (Gavin et al., 2007). In contrast, T<sub>reg</sub> and T<sub>eff</sub> cells express comparable levels of *Pde4b3*, *Pde4d* and *Pde7a* (Vang et al., 2013). It has also been reported that microRNA-mediated repression of *Pde3b* critically regulates peripheral immune tolerance (Anandagoda et al., 2019). While the regulation of selected PDE isoforms including *Pde8* through Foxp3 in T<sub>reg</sub> cells is well established, the exact role of PDE isoforms regulating T<sub>reg</sub> cell function remains to be elucidated.

**B cells:** Studies conducted in the 1990s revealed that *PDE3A*, *PDE4A*, *PDE4B*, *PDE4D* and *PDE7A* were the predominant isoenzymes in human isolated CD4⁺ and CD8⁺ T lymphocytes (Giembycz, 1996; Tenor et al., 1995a). Human isolated CD19⁺ B lymphocytes express a similar complement of PDE mRNA transcripts but, in contrast to T cells, PDE3 activity is marginal. Indeed, *PDE3B* was absent by PCR analysis and only a weak signal for *PDE3A* mRNA was detected. No evidence for PDE1, PDE2 and PDE5 activity was found in these purified B cells (Gantner et al., 1998). It has been reported that the expression of *PDE7B* mRNA and protein in B-chronic lymphocytic leukemia (B-CLL) cells is 23-fold higher than in normal human B cells and the most abundant PDE transcript expressed (Zhang et al., 2008b). Inasmuch as PDE7 inhibitors induce apoptosis of B-CLL cells, it has been suggested that PDE7B may be a therapeutic target for treatment of CLL (Zhang et al., 2008b). A role for PDE7B as a therapeutic target for treating inflammation has not been reported.
# CHAPTER 3: GENERAL PERSPECTIVES AND FUTURE DIRECTIONS

Over the last 50+ years (1972-date), it has become clear that cyclic nucleotide PDEs represent a large superfamily of enzymes that can be exploited to therapeutic advantage with selective inhibitors (Figure 1). Many diseases of the internal organs and the immune system appear to be associated with aberrant cyclic nucleotide signaling or can be effectively treated with interventions that elevate either cAMP or cGMP. This has resulted in the approval by the FDA of 31 PDE inhibitors for the treatment a variety of conditions that relate to cardiovascular, respiratory and male sexual health. PDE inhibitors have also found their way to dermatological application, which has been reviewed elsewhere (Milakovic and Gooderham, 2021; Napolitano et al., 2018; Sidbury et al., 2023). Other inhibitors are undergoing preclinical evaluation or have entered clinical trials for the treatment of cancers, fibrosis and several neurological disorders. Today, our extensive knowledge of the diversity and properties of the 11 PDE families suggest that therapeutic utility could be improved by developing allosteric PDE inhibitors or interventions that modify protein/protein interactions at specific PDE signalosomes of interest. Resonating throughout this entire review is the awareness that the specificity of nanodomain-level measurements and PDE subtype or isoform inhibition requires improvement. In addition, the development of specific PDE stimulators is an area of interest that needs to grow.

## 3.1 | Toward drug specificity

PDE subtype-specific functions within the myocardial cell is a typical example of the importance of development of more specific inhibitors and more precise measurement of nanodomain processes. The cellular mechanisms controlled by each PDE family or subtype umistakenly point to the need to target specific PDE isoforms in circumscribed microdomains of myocardial cells, rather than using a pan-PDE approach to produce global changes in cyclic nucleotide concentrations. At the background of this knowledge the measurement of global tissue, circulating or excreted cyclic nucleotide concentrations seems to lose its meaningfulness. Even PDE-isoform-selective modulators can be ineffective when the isoform is expressed in different cell types or compartments, particularly when those compartments serve opposing functions, for instance when it concerns the relocation of a PDE subtype or isoform as a consequence of the pathophysiological status of the cell (Chapter 2.1 and 2.2). The biology of PDE1A, B and C in smooth muscle cells can serve as a typical example.

On this ground the search for more selective ways to inhibit and activate specific isoforms and spatially restricted pools of PDEs will continue. The lessons learned during the rapid evolution of PROTACS from proof-of-concept to clinical trials heralds a new opportunity for PDE4 modulation (Li and Crews, 2022). Differential targeting of PROTACS to a subset of proteins with similar structures, or a subpopulation of a single target, can be achieved by leveraging small divergencies in protein conformations, oligomerization, cellular location, and activation state (Tao et al., 2022a). These advances are all germane to PDEs, whose structure between members of the same family can be similar and the cellular location of individual “pools” of single PDE isoforms influence the function of that “pool”. The event-driven pharmacology of PROTACS is also a good fit for PDE modulation as targeted degradation occurs at lower concentrations than conventional pharmaceuticals and is much less likely to induce side effects due to less reliance on high target occupancy (Li and Crews, 2022). The synergy between what is known about PDEs and PROTAC development has started to build and may be an important aspect for PDE pharmaceuticals in the future.

Independent of the chemical identity of the drug, beneficial effects might stem from isoform-specific PDE inhibition or activation (for example, by modifying PDE expression, targeting GAF domains or, regulatory domains or by post-translational modifications) as well as alteration of PDE
location (Baillie et al., 2019a). The development of compartment-specific, PDE-isoenzyme-selective modulators (e.g., by using cell-permeable peptide disrupters to displace the PDE isozyme from its signalling complex) (Blair and Baillie, 2019) could help to achieve this goal. In addition, the complex crosstalk between cAMP and cGMP intracellular signalling, and the presence of various PDE isoforms in different compartments, could require multi-drug therapy to fine-tune the cyclic nucleotide level in specific subcellular compartments. For example, activating PDE4D near RYR2 or PDE4B or PDE2A near LTCCs while, at the same time, inhibiting PDE3A or PDE5 near phospholamban, could restore normal compartmentalization of cAMP and cGMP during hypertrophy and prevent the transition towards HF.

## 3.2 | Targeting specific driver mutations

An alternative approach to enhancing PDE specificity involves the concept of driver mutations, particularly relevant in oncology. Having that said, one of the most important incentives to define driver mutations in cancer is that they are powerful predictors of response to therapeutic agents that target that mutation or its associated cellular actions. For example, somatic mutations in codon V600 of the *BRAF* proto-oncogene predict responsiveness to BRAF inhibitors in numerous human cancers, and mutations in each of *EGFR*, *ALK* and *ROS1* in lung cancer predict responsiveness to inhibitors specifically targeting the protein tyrosine kinases encoded by each of these genes. The impressive therapeutic responses to these drugs in these mutationally-defined cancers provides strong support for the role of the associated driver mutations in pathobiology of these cancers. This concept provides impetus for the development of drugs targeting the *PDE8B* and/or *PDE11A* driver mutations in adrenal, testis and other cAMP-signalling cancers. Because these are loss-of-function mutations, such therapeutics would need to activate residual PDE activity in these cancers, potentially by increasing expression of the non-mutated (wild-type) *PDE8B* or *PDE11A* allele present in the germline state, by augmenting the functions of other PDE families in these cells, or by lowering cAMP levels by other means, thereby compensating for the loss of PDE8 or PDE11 action. Although there are no commercially available drugs that augment the enzymatic action of these PDEs, there is a tool compound that allosterically stimulates PDE11A4 activity (Jager et al., 2012).

Further attempts to develop allosteric modulators for therapeutic implicate PDE4 (Baillie et al., 2019a; Omar et al., 2019). One area of interest is in targeting PDE4D7 in prostate cancer (Figure 18) (Gretarsdottir et al., 2003; Wang et al., 2003a). Prostate cancer is characterized by its responsiveness to steroid hormones, such as dihydrotestosterone (Huggins and Hodges, 1941). These hormones are agonists at the androgen receptor, which is a ligand-dependent transcription factor that regulates the expression of numerous genes. The androgen receptor is phosphorylated and, thereby, activated by several kinases, including PKA (Cox et al., 2000; Dagar et al., 2019; Desiniotis et al., 2010; Kvissel et al., 2007; Merkle and Hoffmann, 2011; Moen et al., 2017; Nazareth and Weigel, 1996; Sarwar et al., 2014; Waldkirch et al., 2010). There is abundant evidence for active, adenylyl cyclase-cAMP signaling in prostate cancer cells. These include those driven by the β₂-adrenoceptor and receptors for vasointestinal peptide and pituitary adenylate cyclase-activating peptide (Flacke et al., 2013; Gutierrez-Canas et al., 2003; Zhang et al., 2011a). Numerous PDE isoforms have been detected in normal and neoplastic prostate tissue (Uckert et al., 2001; Uckert et al., 2006) and prostatic smooth muscle cells (Kedia et al., 2012). However, in contrast to the extensive and growing list of pharmacologically targetable mutations in many human cancers, there has been no documentation to date of mutations in cAMP signaling in prostate cancer. Early reports of somatic mutations or SNPs in *PDE4B*, *PDE6C*, *PDE7B* and *PDE10A* in prostate cancer (de Alexandre et al., 2015) have not been reproduced by other groups (Abida et al., 2019; Quigley et al., 2018; Wedge et al., 2018), and their significance as driver mutations has yet to be demonstrated. However, a number of studies have shown PDE4D7 is down-regulated in late-stage,
aggressive, androgen-independent prostate cancer (Henderson et al., 2014), and that loss of
PDE4D7 promotes androgen-independence and alterations in DNA transcription, replication, and
repair (Gulliver et al., 2023a). Alterations in PDE4D7 mRNA and/or protein abundance in prostate
cancer patient specimens may have prognostic value (Alves de Inda et al., 2018; Bottcher et al.,
2016; Bottcher et al., 2015; van Strijp et al., 2019; van Strijp et al., 2018) and they have been
proposed to represent novel biomarkers to help classify the risk of disease progression (Gulliver et
al., 2022; Henderson et al., 2019). These observations have provided the impetus to develop
therapies that specifically target PDE4D7 in prostate and possibly other cancers. Since major
portions of PDE4D7 are also seen in other “long” PDE4D isoforms, including the
regulatory/dimerization UCR1 and UCR2 domains, as well as the catalytic site (Figure 18), such
therapies would probably not target these regions of the protein but, instead, be directed to its unique
N-terminus.

**3.3 | In conclusion**

The importance of prioritizing PDE selectivity to enhance clinical effectiveness, while concurrently amplifying selectivity to minimize adverse drug reactions, constitutes a pivotal aspect in the developmental trajectory of PDEs. This narrative traces back to the inception of PDE research, marked by the serendipitous discovery of the beneficial effects of caffeine, epitomized by asthmatic Henry Hyde Slater's experience in 1886. The field has expanded dramatically over the last 4 decades and moved on to form a vast, multidisciplinary research landscape that occupies a growing part of the pharmacopoeia. The versatility and nanodomain functions of PDEs makes one aware of the intricacy of cellular signaling, and of the possibility that hitherto we might only have scratched the surface of all potential the research field offers for improvement of pharmacotherapy.

The research also forces scientists to look over the edges of current technical possibilities and to
move to the magnification of increasingly smaller details, leading to growth of drug development
possibilities, not only through allosteric binding of PDEs but also through the modulation of binding
partners or perhaps even the correction of gene expression that is affected by driver mutations. The
increased importance of bringing more detail in the research also entails the question what is the
proper model for each isoform, per cell type, and even nanodomain. Are cell culture experiments
representative, or are even rodents or larger animals apt to predict which PDE is a drug target in
humans? And what about the emerging field of organoid research? These considerations will shape
the success of drug discovery. It is expected that the list of possible clinical indications and of
compounds will be growing the coming decades. In other words: PDE research will remain an
exciting field to follow, from molecular biology all the way to the clinic.
**Acknowledgements**

The authors acknowledge Dr. Fabrizio Facchinetti, Chiesi Farmaceutici S.p.A., Parma, Italy for providing information on the tanmilast clinical development programme. MZ acknowledges funding by the British Heart Foundation (RG/17/6/32944, RE/18/3/34214) and the Oxford NIHR Biomedical Research Centre. RC acknowledges the grant R01EY033403 to RHC from the National Eye Institute of the National Institutes of Health. SMJ acknowledges funding by the ZonMw and Dutch research council (NWO), (ZonMw01142042330003). MAG is supported by grants awarded by the Canadian Institutes for Health Research (PJT 152904, PJT 190230) and the Natural Sciences and Engineering Research Council (Discovery Grant, RGPIN-2018-04312). RF is supported by grants from the Leducq Foundation for Cardiovascular Research (19CVD02) and ANR-23-CE14-0027-01. MS received support by the Deutsche Forschungsgemeinschaft (IRTG1874/DIAMICON-SP2), a Novartis unrestricted grant 50199468 and a Alzheimer Nederland grant WE.03-2019-05. The work of SB has been supported by the National Multiple Sclerosis Society. DAK received support by the National Institutes of Health R35-HL135827 and R35-HL166565. GAP received support by the NIH/NCI R01CA254197, R01CA238514. LG received funding from National Institute on Alcohol Abuse and Alcoholism (R01AA029798). GSB is supported by grant from the British Heart Foundation PG/17/26/32881CAMS. MPK acknowledges grants by R01AG061200 and R01067836. KT received support by the Regional Health Authority for South Eastern Norway, grant no. 2021082 and Norwegian Cancer Society grant no. 215850. PME thanks the Smart Family Foundation and Lea's Leukemia Research Foundation Inc. for their continued support of his work and dedicates his portion of this review in memory of his sweet, beautiful, loving daughter, Serena Rae Epstein (March 22, 1984 – July 1, 2011) whose spirit continues to inspire him in everything he does.

**Data availability statement**

N/A.

**Authorship contribution**

All authors contributed equally to drafting and reviewing the manuscript.

**Conflict of interest**

SB is a member of the Scientific Advisory Board of MindImmune, Inc. DAK is on the advisory board of Cardurion Pharmaceuticals that is testing PDE9 inhibitors In humans with heart failure. GAP and ABK are co-founders of ADT Pharmaceuticals Inc. and consultants. GLS, Ph.D. is a full-time employee of Intra-Cellular Therapies Inc and holds equity in the company. GSB is founder and scientific advisor of Disruptyx Therapeutics. CH and PK are employees of Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach an der Riss, Germany. A.J.M.R receives funding from Health-Holland/Erasmus MC/Intracellular Therapies TKI grant # EMCLSH23035 for research on PDE1 in vascular aging.
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