# Inverse agonist efficacy of selatogrel blunts constitutive P2Y12 receptor signaling by inducing the inactive receptor conformation

Véronique Pons, Cédric Garcia, Naomi Tidten-Luksch, Aengus Mac Sweeney, Eva Caroff, Céline Galés, Markus A Riederer

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## To cite this version:

Véronique Pons, Cédric Garcia, Naomi Tidten-Luksch, Aengus Mac Sweeney, Eva Caroff, et al. Inverse agonist efficacy of selatogrel blunts constitutive P2Y12 receptor signaling by inducing the inactive receptor conformation. Biochemical Pharmacology, 2022, 206, pp.115291. 10.1016/j.bcp.2022.115291. hal-04845066

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# Inverse agonist efficacy of selatogrel blunts constitutive P2Y12 receptor signaling by inducing the inactive receptor conformation

Véronique Pons<sup>a</sup>, Cédric Garcia<sup>a,b</sup>, Naomi Tidten-Luksch<sup>c</sup>, Aengus Mac Sweeney<sup>d</sup>, Eva Caroff<sup>c</sup>, Céline Galés<sup>a</sup>, Markus A. Riederer<sup>d,*</sup>

## ARTICLE INFO

Keywords:

Thrombosis

P2Y receptor

Inverse agonist

Constitutive activity

Receptor structure

Coronary artery disease

Selatogrel

## ABSTRACT

Selatogrel is a potent inhibitor of adenosine diphosphate (ADP) binding to the P2Y12 receptor, preventing platelet activation. We have previously shown that the P2Y12 receptor constitutively activates Gi- and Go-protein-mediated signaling in human platelets.

Here, we report that selatogrel acts as an inverse agonist of the P2Y12 receptor. Specifically, using bioluminescence resonance energy transfer2 (BRET2) probes, selatogrel, ticagrelor, and elinogrel were shown to stabilize the inactive form of the $G\alpha{i}/o-G\beta{\gamma}$ complex in cells with recombinant expression of the P2Y12 receptor. In dose-response experiments, while selatogrel exhibited a maximal efficacy similar to ticagrelor, selatogrel was approximately 100-fold more potent than ticagrelor. Quantification of relative cyclic adenosine monophosphate (cAMP) levels in cells expressing the cAMP BRET1 sensor (CAMYEL probe) confirmed that selatogrel completely abolished the constitutive activity of the P2Y12 receptor. In agreement, selatogrel increased basal cAMP levels in human platelets, confirming inverse agonism on the endogenous human platelet P2Y12 receptor. In agreement with the biochemical phenotype of inverse agonism efficacy of selatogrel, the 2.8 Angstrom resolution cocrystal structure of selatogrel bound to the P2Y12 receptor confirmed that selatogrel stabilizes the inactive, basal state of the receptor. Selatogrel bound to pocket 1, spanning helix III to VII. Furthermore, the binding mode of selatogrel, suggesting steric overlap with the proposed binding site of ADP and the ADP analog 2-methylthioadenosine diphosphate (2MeSADP), agrees with the functional characterization of selatogrel preventing platelet activation by blocking ADP binding to the P2Y12 receptor.

## 1. Introduction

Platelets play a pivotal role in hemostasis. Upon encountering disrupted surfaces within blood vessels, platelets adhere, become activated and form a hemostatic seal, which prevents blood loss. [1] In this process, adenosine diphosphate (ADP)-mediated P2Y12 receptor activation is essential for the amplification of platelet aggregation. [2,3] In addition, platelets also play a central role in thrombotic complications of atherosclerotic diseases, confirming that under pathological conditions, the platelet system can cause blood vessel occlusion. [4]

The P2Y12 receptor is a member of the G protein-coupled receptor (GPCR) family. Extracellular agonist binding to the GPCR induces conformational changes within the receptor, which trigger dissociation of the intracellular $G\alpha\beta\gamma$ heterotrimer complex and subsequent signaling. [5] Specifically, ADP-induced dissociation of the $G\alpha\beta\gamma$ heterotrimer results in $G\alpha{i2}$ subunit-mediated inhibition of adenylyl cyclase, subsequently lowering the cyclic adenosine monophosphate (cAMP) concentration and consequently increasing platelet activation. [6]

The protein structure of the P2Y12 receptor complexed with an

**Abbreviations:** ADP, adenosine diphosphate; 2MeSADP, 2-methylthioadenosine diphosphate; cAMP, cyclic adenosine monophosphate; BRET, bioluminescence resonance energy transfer; GPCR, G protein-coupled receptor; HTRF, homogeneous time-resolved fluorescence; ACS, Acute coronary syndrome; GFP, Green fluorescent protein; Rluc8, Renilla reniformis luciferase with eight mutations; YFP, Yellow fluorescent protein; Fsk, Forskolin.

* Corresponding author.

E-mail address: markus.riederer@idorsia.com (M.A. Riederer).

<footnote><sup>a</sup> INSERM, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Université de Toulouse, F-31432 Toulouse, France</footnote>

<footnote><sup>b</sup> Laboratoire d'Hématologie, Centre Hospitalier Universitaire de Toulouse, F-31000 Toulouse, France</footnote>

<footnote><sup>c</sup> Idorsia Pharmaceuticals, DD Chemistry, Hegenheimermattweg 91, 4123 Allschwil, Switzerland</footnote>

<footnote><sup>d</sup> Idorsia Pharmaceuticals, DD Biology, Hegenheimermattweg 91, 4123 Allschwil, Switzerland</footnote>
agonist, 2-methylthioadenosine diphosphate (2MeSADP), or an antagonist (6-[4[(benzylsulfonyl)amino]carbonyl]piperidin-1-yl]-5-cyano-2-methylnicotinic acid ethyl ester (AZD1283)) was resolved and provided key insights for the development of orthosteric and allosteric modulators as drug candidates. [7–9] In particular, these studies revealed a very high plasticity of the binding pocket of the P2Y12 receptor characterized by striking conformational changes in the extracellular regions, thus setting the P2Y12 receptor apart from all other known class A G protein coupled receptor (GPCR) structures.

In recent years, advancements in GPCR pharmacology and ligand efficacy have led to major breakthroughs in GPCR drug discovery. In particular, several studies highlighted the physiological relevance of the ligand-independent activation of many GPCRs (i.e., constitutive activity) and the therapeutic potential of inverse agonists in the treatment of diseases.[10–13] In line with this concept, we and others previously reported that the P2Y12 receptor triggered constitutive signaling in human platelets. [14,15] Recently, this P2Y12 receptor constitutive activation started to attract much attention, as it was associated with high thrombotic risk in diabetes patients or correlated with bleeding syndrome when abolished. [15,16]

In patients with type 2 diabetes mellitus, the enhanced expression and constitutive activation of the P2Y12 receptor were shown to contribute to platelet hyperactivity and an increased risk of thrombosis and to hamper the efficacy of antiplatelet drugs. [16,17] Therefore, developing P2Y12 receptor antagonists with potent inverse agonist efficacy, capable of alleviating constitutive P2Y12 receptor activity by switching the receptor from an active to an inactive conformation, should demonstrate attractive therapeutic potential over classical antagonists to prevent basal platelet activation and reduce the pro-thrombotic risk profile of these patients.

Antagonists of the P2Y12 receptor are the foundation of pharmacotherapy in the treatment and prevention of atherothrombosis. To date, reversible (ticagrelor, cangrelor) and covalent irreversible (clopidogrel and prasugrel) antagonists of the P2Y12 receptor have been shown to reduce major adverse cardiovascular events in the management of acute coronary syndrome. [18,19] Ticagrelor, a reversible P2Y12 receptor antagonist with market approval, was demonstrated to significantly reduce the rate of death from vascular causes, myocardial infarction, or stroke, with no increase in the rate of overall major bleeding but with an increase in the rate of non-CABG (non-Coronary Artery Bypass Graft Surgery) related major bleeding. [20] Interestingly, some reversible antagonists have recently been demonstrated to exhibit inverse agonist efficacy at the P2Y12 receptor, thereby specifically counteracting basal receptor signaling. In rat FeCl₃-injury mesenteric arteriole thrombosis and arteriovenous shunt thrombosis models, the inverse agonist AR-C78511 displayed greater antithrombotic effects in diabetic animals than neutral antagonists, again highlighting the therapeutic advantages of inverse agonists over neutral antagonists. [21]

Selatogrel (ACT-246475) is a reversible, highly selective antagonist of the P2Y12 receptor. [22] Selatogrel binds to the P2Y12 receptor with a K_D of 1.5 nM, as determined by binding studies, and in light transmission aggregometry, selatogrel inhibits ADP-induced platelet aggregation with an IC₅₀ value of 14 nM. [23] Nonclinical studies showed selatogrel to have an improved blood-loss profile in comparison to prior P2Y12 receptor antagonists. Specifically, selatogrel was associated with lower blood loss than clopidogrel and ticagrelor at equivalent antithrombotic efficacy in thrombosis/hemostasis models. [22,24] Selatogrel did not interfere with the regulation of vascular tone, in contrast to ticagrelor, and did not exhibit off-target effects interfering with the process of hemostasis as described for clopidogrel and ticagrelor in rat and mouse thrombosis/hemostasis models. [25] Selatogrel has been confirmed to be rapidly absorbed following subcutaneous administration in acute coronary syndrome (ACS) patients, providing fast, potent, and reversible platelet P2Y12 inhibition. [26–28] Further studies of subcutaneously administered selatogrel in clinical scenarios requiring rapid platelet inhibition are currently ongoing. [26,29]

Here, we investigated the pharmacological properties of selatogrel regarding constitutive P2Y12 receptor activity. Using highly sensitive tools based on bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF), we report that selatogrel behaves as an inverse agonist of constitutive P2Y12 receptor signaling both in a recombinant cellular system and in human platelets. In addition, we resolved the crystal structure of the P2Y12 receptor in complex with selatogrel. The observed inactive conformation of the P2Y12 receptor is in agreement with the inverse agonist mode of action of selatogrel.

## 2. Materials and methods

### 2.1. Materials

Selatogrel (ACT-246475) as a hydrochloride salt was provided by Idorsia Pharmaceuticals Ltd. Switzerland. Ticagrelor was extracted from commercially available tablets (Brilique, AstraZeneca, Cambridge, UK). Elinogrel was purchased from Lucerna-Chem (Luzern, Switzerland). Forskolin and IBMX were purchased from Merck (Darmstadt, Germany). Coelenterazines 400a and h were purchased from Intechim (Los Angeles, CA, USA). Plasmids encoding Gα-RLuc8, GFP2-Gγ2, untagged Gβ1, N-terminally Myc-tagged P2Y12 receptor and CAMYEL probe were previously described [15,30].

### 2.2. Cell culture and transfection

HEK293T/17 cells (human embryonic kidney) were purchased from ATCC (American Type Culture Collection) (Manassas, Virginia, USA) and was not cultured after passage 30. HEK293T/17 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) AQmedia (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA), 100 units/mL penicillin and 100 µg/mL streptomycin (Sigma, St. Louis, MO, USA) at 37 °C in a humidified atmosphere containing 5% CO₂. Twenty-four hours after cell splitting, transient transfections were performed using polyethylenimine (PEI 25 K, Polysciences Inc., Warrington, PA, USA).

### 2.3. BRET measurements

For G protein activation, vectors encoding the Gα-RLuc8, GFP2-Gγ2, Gβ1 and Myc-tagged P2Y12 receptors were transiently cotransfected into HEK293T/17 cells as indicated in the figure legends. Forty-eight hours after transfection, the cells were washed and resuspended in phosphate-buffered saline (PBS) containing 0.1% (w/v) glucose at room temperature. Cells were then distributed (80 µg protein/well) into a 96-well microplate (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) and incubated in the presence of the different ligands for 1 min. BRET2, which refers to BRET using green-fluorescent protein2 (GFP2) as an energy acceptor, between RLuc8 and GFP2 was measured after addition of the luciferase substrate coelenterazine 400a (5 µM). BRET2 readings were collected using a modified Infinite F500 (Tecan Group Ltd., Männedorf, Switzerland). The BRET signal (BRET ratio) was calculated by the ratio of emission of GFP2 (510–540 nm) to RLuc8 (370–450 nm). For intracellular cyclic adenosine monophosphate (cAMP) measurements, the cAMP BRET1 sensor (CAMYEL probe) and Myc-tagged P2Y12 receptor were transiently expressed in HEK293T/17 cells as indicated in the figure legends. Forty-eight hours after transfection, cells were resuspended in PBS containing 0.1% (w/v) glucose at room temperature and distributed (80 µg proteins/well) into a 96-well microplate (PerkinElmer Life and Analytical Sciences), stimulated with 10 µM forskolin alone for 10 min and incubated with coelenterazine h (5 µM) two minutes after the addition of forskolin. When indicated, the different ligands (10 µM) were added one minute after the addition of forskolin. BRET1 readings were collected using a modified Infinite F500 (Tecan Group Ltd.). The BRET signal (BRET ratio) was calculated by the
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** P2Y12 receptor constitutively activated Gi/o proteins. **A.** Schematic presentation of the BRET assay system. At basal inactive state (left panel), the preassembled inactive Gαβγ heterotrimeric complex favors the detection of a basal BRET signal due to the close proximity between Gα and Gγ2 protein subunits. This basal BRET will be lower in the presence of a constitutive G protein activation. Thus, an inverse agonist will promote the transition from the active to inactive receptor conformation and trigger the reassembly between the Gα and the Gγ subunits that will be sensed as an increase of the BRET signal. **B.** The Gα-RLuc8 protein probe expression was monitored by luminescence measurement in cells co-expressing the different Gα-RLuc8 together with GFP2-Gγ2 and Gβ1 in the absence (no receptor) or in the presence of P2Y12 receptor. **C.** Basal BRET signal was measured in cells co-expressing the different Gα-RLuc8 together with GFP2-Gγ2 and Gβ1 in the absence (no receptor) or in the presence of P2Y12 receptor. **B–C.** Data represent the mean ± s.e.m of 5 independent experiments and statistical significance between cells expressing P2Y12 receptor or not (no receptor) was assessed using an unpaired *t* test (* P* < 0.05; ** P* < 0.01; ns, not statistically significant).</figcaption></figure>

ratio of the emission of yellow fluorescent protein (YFP) (520–570 nm) to Renilla luciferase (RLuc) (370–480 nm).

## 2.4. Preparation of washed human platelets

Blood sampling from healthy volunteers was performed in line with the principles of the Declaration of Helsinki. Healthy donors were recruited under a protocol approved by the Toulouse Hospital BioResources biobank, declared to the Ministry of Higher Education and Research (DC2016-2804). Blood was processed following hospital guidelines. Specifically, after informed consent was obtained, venous blood was collected and anticoagulated with 0.105 M citrate and centrifuged (180 g, 10 min, room temperature) to obtain platelet-rich plasma (PRP). After two steps of centrifugation and suspension in Tyrode's buffer (140 mM NaCl, 5 mM KCl, 5 mM KH₂PO₄, 1 mM MgSO₄, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 mM glucose and 0.35% (w/v) bovine serum albumin (BSA), pH 6,7),

washed platelets were suspended in the same buffer adjusted to pH 7.4 and containing 1 mM CaCl₂. The final platelet suspension was adjusted to 3.0 x10⁸ platelets/mL and rested for 45 min in the presence of 0.02 U/mL apyrase and 10 µM indomethacin at 37 °C prior to experiments. PGI₂ (0.5 µM) was added before centrifugation steps to avoid platelet activation.

## 2.5. cAMP accumulation assay with human platelets

Quantification of intracellular cAMP was performed using the HTRF cAMP competitive immunoassay (CAMP - Gi kit. Cisbio, Codolet, France) according to the manufacturer's instructions. Briefly, washed platelets were distributed in a 384-well white microplate (Greiner, Kemsmünster, Austria) in the presence or absence of the different ligands (10 µM) and forskolin (5 µM) for 45 min at 37 °C and in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) to prevent phosphodiesterase-mediated cAMP degradation. After the addition of
A

Selatogrel

<figure><img src="image_5.png" /></figure>

Ticagrelor

<figure><img src="image_6.png" /></figure>

Elinogrel

<figure><img src="image_7.png" /></figure>

B - Selatogrel

<figure><img src="image_8.png" /></figure>

C - Ticagrelor

<figure><img src="image_9.png" /></figure>

D - Elinogrel

<figure><img src="image_10.png" /></figure>

(caption on next page)
**Fig. 2.** Seloatogrel, ticagrelor and elinogrel behaved as inverse agonists at P2Y12 receptor, A. Chemical structures of selatogrel, ticagrelor and elinogrel. B-D. Inverse agonist efficacy of compounds was assessed on G protein activation in cells co-expressing the different Gα-RLuc8 together with GFP2-Gγ2 and Gβ1 in the absence (no receptor, gray bars) or in the presence of P2Y12 receptor (orange bars), after stimulation or not for 1 min with 10 µM selatogrel (B), ticagrelor (C) or elinogrel (D). The final antagonist concentration of 10 µM was chosen to guarantee maximal possible effect. Results are expressed as the difference in the BRET signal measured in the presence and the absence of ligand. Data represent the mean ± s.e.m. of 5 independent experiments. Statistical significance of ligand-induced BRET modulation was assessed using a paired *t* test between unstimulated and stimulated cells (# P < 0.05; ## P < 0.01; ### P < 0.001; ns, not statistically significant) or using an unpaired *t* test when comparing cells expressing P2Y12 receptor or not (no receptor) (** P < 0.01; *** P < 0.001; ns, not statistically significant).

cryptate-labeled cAMP (donor) and anti-cAMP-d2 (acceptor) for 1 h, the specific fluorescence resonance energy transfer (FRET) signals were calculated by the fluorescence ratio of the acceptor and donor emission signals (665/620 nm) collected using a modified Infinite F500 (Tecan Group Ltd). Conversion of the HTRF ratio of each sample into cAMP concentrations was performed on the basis of a standard curve to determine the linear dynamic range of the assay.

## 2.6. Quantification of cell surface P2Y12 receptor by ELISA.

Cell surface P2Y12 receptor quantification was performed as previously described [15]. Briefly, HEK293T/17 cells were split into 24-well plates pre-coated with Poly-D-lysine, transiently transfected with pcDNA3.1 (+) empty vector (control) or different amounts of vector encoding N-terminally Myc-tagged P2Y12 receptor. Forty-eight hours post-transfection, cells were fixed (1% paraformaldehyde), saturated (PBS - 1% BSA) and incubated with the primary anti-Myc antibody (Clone 9E10. Santa Cruz Biotechnology. Dallas, Texas, USA) and then with HRP-labeled secondary antibody (Sigma. St. Louis, MO, USA). After washing, cells were incubated for 15 min with HRP substrate: TMB (3,39,5,59-tetramethylbenzidine) (BD Biosciences). The reaction was stopped with HCl 1 N, and the plates were read at 450 nm in a micro-plate reader (Infinite F500. Tecan Group Ltd. Männedorf, Switzerland).

## 2.7. Data and statistical analysis

Results are expressed as the mean values ± standard error of the mean (s.e.m.) or standard deviation (s.d). Statistical analysis was carried out using GraphPad Prism software (GraphPad Software Inc., Chicago, IL, USA). The statistical tests used are indicated in the figure legends. A *P* value < 0.05 was considered significant.

## 2.8. P2Y12 receptor crystallization and data processing

P2Y12 receptor expression and purification were performed as previously described [7] under a contract research service agreement. Briefly, the fusion protein of human P2Y12 receptor and thermo-stabilized apocytochrome B562RIL (P2Y12-BRIL)) was expressed in Sf9 insect cells. Thermostabilized BRIL (PDB code 1M6T) was fused into the third intracellular loop (ICL3) of P2Y12 (T223–R224) with intact N and C termini. The P2Y12-BRIL gene was further modified by introducing the D2947.49 N mutation to improve the yield of purified protein [7]. P2Y12-BRIL was purified in the presence of 2 mM selatogrel using immobilized metal affinity chromatography (IMAC). P2Y12-BRIL crystals were grown in the presence of 2 mM selatogrel using a lipidic cubic phase crystallization method. Diffraction data were collected from 96 crystals at beamline 41-XU of the Spring-8 X-ray source at a wavelength of 1.000 Å with a PILATUS 3 6M detector. The X-ray data were processed, scaled and merged using the software XDS [31] and ZOO [32]. Model building and refinement were carried out with a maximum resolution of 2.78 Å using Coot [33] and Refmac [34].

# 3. Results

## 3.1. Seloatogrel behaved as an inverse agonist of P2Y12 receptor-dependent constitutive Gi/o protein activation in HEK293T cells

We have previously shown that the P2Y12 receptor displays

constitutive activation of the Gi- and Go-protein family. [15] Thus, we assessed the inverse agonism efficacy of selatogrel by using a BRET2-based assay that measures the activation of the G proteins in real time in living HEK293T cells. [15] The BRET2 assay monitors the interaction between the Gα subunit fused to Renilla reniformis luciferase (RLuc8) energy donor (Gα-RLuc8) and the Gγ2 subunit fused to the fluorescent GFP2 energy acceptor (GFP2-Gγ2). In the basal inactive state, the pre-assembled inactive Gαβγ heterotrimeric complex allows the detection of a basal BRET signal reflecting Gα/Gγ2 subunit proximity. Receptor activation promotes dissociation between Gα and Gβγ subunits, thereby increasing the distance between the Gα energy donor and the Gγ2 energy acceptor and leading to a decrease in the BRET signal compared to the basal state (Fig. 1.A). Therefore, these G protein BRET2 probes are also powerful tools to depict the inverse agonism efficacy of ligands [15,35] since an inverse agonist will favor the transition of G proteins from the active to inactive state, leading to an increase in the BRET signal reflecting the reassembly between the Gα and the Gγ subunits.

At the basal state and in the absence of the exogenous P2Y12 receptor (no receptor), we detected a basal BRET signal for the 4 main G protein families (Gi/o, Gs, Gq/11, G12/13), indicative of the Gαβγ inactive complex (Fig. 1. C). Furthermore, at similar Gα probe expression levels (Fig. 1. B), N-terminally Myc-tagged P2Y12 receptor expression triggered a significant decrease in the basal BRET signal for the Gi/o proteins (Fig. 1. C), confirming that under these experimental conditions, the P2Y12 receptor constitutively activated the Gi/o protein family as previously reported. [15] In contrast to what was observed for the Gi/o family, P2Y12 receptor expression did not promote a decrease in the basal BRET signal for the Gq/11, Gs, and G12/13 proteins (Fig. 1. C), indicating a lack of constitutive activity of the P2Y12 receptor on the activation of these G proteins. [15]

Under these conditions, we investigated the pharmacological properties of selatogrel regarding P2Y12 receptor constitutive activation of Gi/o proteins and compared with two other reversible antagonists of the P2Y12 receptor, ticagrelor and elinogrel (chemical structures depicted in Fig. 2. A). The addition of selatogrel, ticagrelor or elinogrel significantly increased the BRET signal for the Gi/o protein family in P2Y12 receptor expressing cells (Fig. 2. B-D, orange bars) compared to control cells (no receptor) (Fig. 2. B-D, gray bars), indicating that all compounds behaved as inverse agonists on P2Y12 receptor-dependent Gi/o protein constitutive activation. Noticeably, elinogrel triggered a different pharmacological profile toward G protein activation compared to selatogrel and ticagrelor, since elinogrel also induced a significant increase in the BRET signal for all the G protein family members, including Gq/11 and Gs, in the absence of receptor expression (no receptor) (Fig. 2. D, gray bars). These results indicated that elinogrel most likely targeted other endogenous GPCRs different from the P2Y12 receptor, suggesting off-target activities of elinogrel.

In conclusion, these data demonstrated that all three reversible P2Y12 receptor antagonists exhibited inverse agonist efficacy at the P2Y12 receptor by inhibiting receptor-dependent constitutive Gi/o protein activation.

## 3.2. Seloatogrel acted as a more potent inverse agonist than ticagrelor

To prevent misinterpretation of data due to the strong off-target activities of elinogrel, subsequent experiments were performed with ticagrelor and selatogrel only. To depict the pharmacological signature of selatogrel compared to ticagrelor, we explored the maximal efficacy
<caption>**Table 1**</caption>

Potencies (EC50) and maximal efficacies (Emax) of ticagrelor and selatogrel on P2Y12 receptor-mediated Gαi2 and GαoB protein activation Data were obtained from experiments shown in Fig. 3D-E and represent the mean of EC50 (M), pEC50 ± s.e.m. and Emax ± s.e.m. The statistical differences between ticagrelor and selatogrel pEC50 and Emax were assessed using a two-way ANOVA followed by a Sidak's multiple comparison test (\*\* P < 0.01; ns, not statistically significant).

<table><thead><tr><th></th><th colspan="3">ticagrelor</th><th colspan="3">selatogrel</th><th colspan="2">selatogrel compared to ticagrelor</th></tr></thead><tbody><tr><th colspan="9" style="text-align: center;">High P2Y12 receptor expression level</th></tr><tr><th></th><th>EC50 (M)</th><th>pEC50 ± s.e.m.</th><th>Emax ± s.e.m.</th><th>EC50 (M)</th><th>pEC50 ± s.e.m.</th><th>Emax ± s.e.m.</th><th>pEC50</th><th>Emax</th></tr><tr><th>Gαi2</th><td>3.662 e-006</td><td>5.436 ± 0.1205</td><td>0.2102 ± 0.01479</td><td>9.511 e-008</td><td>7.022 ± 0.1381</td><td>0.1724 ± 0.01034</td><td>\*\* P&lt;0.01</td><td>ns</td></tr><tr><th>GαoB</th><td>4.008 e-006</td><td>5.397 ± 0.1894</td><td>0.1860 ± 0.01904</td><td>7.481 e-008</td><td>7.126 ± 0.1402</td><td>0.1778 ± 0.009177</td><td>\*\* P&lt;0.01</td><td>ns</td></tr><tr><th></th><th colspan="3">ticagrelor</th><th colspan="3">selatogrel</th><th colspan="2">Selatogrel compared to ticagrelor</th></tr><tr><th colspan="9" style="text-align: center;">Low P2Y12 receptor expression level</th></tr><tr><th></th><th>EC50 (M)</th><th>pEC50 ± s.e.m.</th><th>Emax ± s.e.m.</th><th>EC50 (M)</th><th>pEC50 ± s.e.m.</th><th>Emax ± s.e.m.</th><th>pEC50</th><th>Emax</th></tr><tr><th>Gαi2</th><td>4.333 e-006</td><td>5.363 ± 0.5116</td><td>0.0514 ± 0.01576</td><td>1.213 e-007</td><td>6.916 ± 0.3584</td><td>0.04453 ± 0.00776</td><td>\*\* P&lt;0.01</td><td>ns</td></tr><tr><th>GαoB</th><td>2.646 e-006</td><td>5.577 ± 0.5272</td><td>0.05075 ± 0.01361</td><td>4.992 e-008</td><td>7.302 ± 0.4093</td><td>0.06167 ± 0.006761</td><td>\*\* P&lt;0.01</td><td>ns</td></tr></tbody></table>

(Emax) and the potency (EC50) of both compounds by performing dose-response experiments on Gαi2 and GαoB isoform activity, as the data (presented in Fig. 2. B-C) demonstrated the most prominent effect on these G proteins. To potentially depict some pharmacological differences between the two compounds, we screened high (8 µg receptor-encoding plasmid) and low (0.5 µg receptor-encoding plasmid) expression levels of the P2Y12 receptor. Quantification of cell surface receptor expression by ELISA on non-permeabilized cells confirmed that P2Y12 receptor was significantly detected at the cell surface in both low and high expression level conditions compared to control cells, in the absence of receptor (no receptor, 0 µg receptor-encoding plasmid). And, as expected, P2Y12 receptor was more expressed at the plasma membrane in high than in low expression level conditions (Fig. 3. A). Both receptor expression levels triggered constitutive activation of Gαi2 and GαoB proteins, as demonstrated by the significant decrease in the basal BRET ratio compared to control cells (Fig. 3. B-C)). In the absence of P2Y12 receptor expression (no receptor), neither selatogrel nor ticagrelor promoted significant BRET signal modulation on Gαi2 or GαoB protein (Fig. 3. D-E, no receptor), indicating that neither ligand triggered any P2Y12 receptor-independent response on G protein activation. However, in P2Y12 receptor-expressing cells, selatogrel, much like ticagrelor, increased the BRET signal for the Gαi2 and GαoB proteins, confirming that they both behaved as inverse agonists on P2Y12 receptor-dependent Gi/o protein constitutive activation (Fig. 3. D-E, low and high receptor expression). The BRET signal dose-dependency increased with the receptor expression level, demonstrating that the inverse agonism efficacy of both compounds was strictly dependent on P2Y12 receptor expression. Interestingly, while selatogrel and ticagrelor showed similar maximal efficacy (Emax), regardless of the P2Y12 receptor expression level, they exhibited different potencies, with selatogrel behaving as a more potent inverse agonist than ticagrelor on both the Gαi2 and GαoB proteins (Fig. 3. D-E, and Table 1). Thus, selatogrel was characterized to be approximately 100-fold more potent than ticagrelor in this assay.

### 3.3. Selatogrel displayed inverse agonism efficacy on constitutive adenyl cyclase inhibition both in HEK239T cells and human platelets

We further explored the inverse agonism efficacy of selatogrel by analyzing downstream Gi/o signaling, i.e., inhibition of adenyl cyclase in HEK293T cells. We thus monitored cAMP levels as previously described [15] using the CAMYEL probe [36,37], allowing the dynamic

monitoring of intracellular cAMP. To enable the detection of negative regulation of adenyl cyclase by constitutive activation of Gi/o-coupled P2Y12 receptor, the enzymatic activity of adenyl cyclases was first activated by forskolin (Fsk) to increase cAMP concentration. As expected and previously demonstrated [15], we observed that at similar levels of CAMYEL probe expression (Fig. 4. A), P2Y12 receptor expression significantly decreased Fsk-induced cAMP levels compared to control cells (Fig. 4. B), confirming the constitutive activity of P2Y12 receptor on adenyl cyclase inhibition and in agreement with the constitutive activity detected on the Gαi/o proteins (Fig. 1). [15] Interestingly, selatogrel and ticagrelor did not significantly impact Fsk-induced cAMP production in control cells (no receptor) but completely abolished the receptor constitutive activity in cells expressing the P2Y12 receptor, as shown by the increase in Fsk-induced cAMP production (Fig. 4. C), thereby demonstrating that selatogrel, similar to ticagrelor, displayed inverse agonism efficacy on P2Y12 receptor-dependent adenyl cyclase inhibition in HEK293T cells. Again, and in agreement with experiments on G protein activation (Figs. 2-3), selatogrel exhibited a maximal inverse agonist efficacy similar to ticagrelor. In agreement with data obtained using recombinant HEK293T cells (Fig. 4. C), when we examined the inverse agonism efficacy of selatogrel in human platelets with endogenous expression of the P2Y12 receptor and associated G proteins, we observed that addition of selatogrel (green rhombi) or ticagrelor (blue triangles) potentiated Fsk-induced cAMP concentration (Fig. 4. D), highlighting a reduced Gi/o-mediated constitutive inhibition of adenyl cyclase and thus confirming the constitutive activation of P2Y12 receptor on Gi/o signaling and the inverse agonism efficacy of selatogrel in human platelets.

### 3.4. Crystal structure of P2Y12 receptor in the presence of selatogrel

To further characterize the binding mode of the inverse agonist selatogrel in the P2Y12 receptor, the crystal structure of the P2Y12 receptor in complex with selatogrel was determined. The fusion protein of human P2Y12 receptor with thermostabilized apocytochrome B562RIL (P2Y12-BRIL) was expressed in Sf9 insect cells as previously described. [7] However, in the experiment reported here, the P2Y12-BRIL protein was purified in the presence of 2 mM selatogrel using metal affinity chromatography. The P2Y12-BRIL protein crystals were grown in the presence of 2 mM selatogrel using a lipidic cubic phase crystallization method. X-ray diffraction (XRD) data were collected at beamline 41-XU at the Spring-8 synchrotron facility, allowing the structure of the
P2Y12 receptor-selatogrel to be determined at a resolution of 2.78 Å.
The data processing and structure refinement statistics are shown in
Table 2. The structure was deposited in the Protein Databank (PDB) with
accession code 7pp1.

Compared to the reported structure of AZD1283 bound to P2Y12
(PDB accession code 4ntj) [7], the overall receptor conformation in the

complex with selatogrel was found to be highly similar and therefore
clearly in the antagonized conformation, with only a few amino acid side
chain rearrangements in the ligand binding site. The electron density
allowed the determination of the steric orientation of the selatogrel
molecule in the binding pocket of the P2Y12 receptor. The structure of
selatogrel bound to the P2Y12 receptor strongly suggests that the

A

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>

C - HEK293T cells

<figure><img src="image_13.png" /></figure>

D - Human platelets

<figure><img src="image_14.png" /></figure>

(caption on next page)
**Fig. 4.** Seloatrogel behaved as inverse agonist on P2Y12 receptor-dependent constitutive cAMP inhibition in HEK293T cells and human platelets. **A.** The relative expression levels of CAMYEL sensor was assessed by luminescence measurement in control cells (no receptor) and in P2Y12 receptor expressing cells. Data represent the mean ± s.e.m of 5 independent experiments and statistical significance was assessed using an unpaired *t* test (ns, not statistically significant). **B.** Quantification of relative cAMP levels was assessed in cells expressing the CAMYEL probe alone (no receptor) or together with P2Y12 receptor after treatment or not with forskolin (Fsk, 10 µM) for 10 min. Data represent the difference in the BRET signal measured in the presence and in the absence of forskolin (basal production) and are expressed as the percentage of forskolin treatment in the absence of receptor (no receptor). Data represent the mean ± s.d of 5 independent experiments and statistical comparison was assessed using a Mann-Whitney test (**P** < 0.01). **C.** Quantification of relative cAMP levels was assessed in cells expressing the CAMYEL probe alone (no receptor) or together with P2Y12 receptor. Cells were treated or not with forskolin (Fsk, 10 µM) for 10 min, followed or not by selatogrel or ticagrelor stimulation (10 µM). Data represent the difference in the BRET signal measured in the presence of forskolin alone or with the compounds and in the absence of forskolin (basal production) and are expressed as the percentage of forskolin treatment alone, in the absence of receptor (no receptor, dashed line). Data represent the mean ± s.d of 5 independent experiments. The statistical comparison was assessed using Kruskal-Wallis followed by Dunn's multiple comparison test (*P* < 0.05; ***P* < 0.001; ns, not statistically significant). **D.** Washed human platelets were stimulated with 5 µM forskolin (Fsk) before addition of 10 µM selatogrel (green rhombus) or 10 µM ticagrelor (blue triangle) for 45 min and intracellular cAMP was quantified. Data represent the mean ± s.e.m of 8 healthy donors and are expressed as cAMP concentration. The statistical comparison was assessed using Friedman test followed by Dunn's multiple comparison test (*P* < 0.05; ***P* < 0.001).

<caption>**Table 2**</caption>

Data collection and structure refinement statistics of the P2Y12-BRIL protein crystal with bound selatogrel (ACT-246475).

<table><thead><tr><th></th><th>P2Y12-ACT246475</th></tr></thead><tbody><tr><td><strong>Data collection</strong></td><td></td></tr><tr><td>Space group</td><td>C2</td></tr><tr><td>Cell dimensions</td><td></td></tr><tr><td>α, b, c (Å)</td><td>98.1, 155.3, 47.3</td></tr><tr><td>α, β, γ (°)</td><td>90.0, 111.5, 90.0</td></tr><tr><td>Number of reflections measured</td><td>844,086</td></tr><tr><td>Number of unique reflections</td><td>16,554</td></tr><tr><td>Resolution (Å)</td><td>45.64–2.78 (2.87–2.78)*</td></tr><tr><td>R<sub>merge</sub></td><td>0.3 (0.5)</td></tr><tr><td>Mean I / sI</td><td>18.6 (0.6)</td></tr><tr><td>CC<sub>1/2</sub></td><td>0.99 (0.50)</td></tr><tr><td>Completeness (%)</td><td>98.9 (91.6)</td></tr><tr><td>Redundancy</td><td>50.80 (48.40)</td></tr><tr><td><strong>Refinement</strong></td><td></td></tr><tr><td>Resolution (Å)</td><td>45.68–2.78</td></tr><tr><td>No. reflections</td><td>16,390 (1630)</td></tr><tr><td>R<sub>work</sub> / R<sub>free</sub> (%)</td><td>0.24/0.28</td></tr><tr><td>No. atoms</td><td></td></tr><tr><td>Protein</td><td>2886</td></tr><tr><td>Ligand</td><td>99</td></tr><tr><td>Lipid and other</td><td>372</td></tr><tr><td>Average B factors (Å<sup>2</sup>)</td><td></td></tr><tr><td>Wilson</td><td>83.72</td></tr><tr><td>Protein</td><td>103.77</td></tr><tr><td>Ligand/ion</td><td>116.68</td></tr><tr><td>R.m.s. deviations</td><td></td></tr><tr><td>Bond lengths (Å)</td><td>0.010</td></tr><tr><td>Bond angles (°)</td><td>1.040</td></tr><tr><td>Ramachandran Plot Statistics (%)</td><td></td></tr><tr><td>Favored regions</td><td>98.02</td></tr><tr><td>Allowed regions</td><td>1.98</td></tr><tr><td>Disallowed regions</td><td>0.00</td></tr></tbody></table>

chirality are (S) at the stereocenter in the pyrrolidine group and (R) at the stereocenter in the beta position to the phosphonate (Fig. 5. A). Seloatrogel binds to the same pocket 1 as AZD1283,[7] stretching from helix III to VII (Fig. 5. B). The positioning of two moieties of selatogrel shows highly similar binding compared to AZD1283 as well, e.g., the carbamate moiety of selatogrel binds analogously to the ester group of AZD1283 close to the side chain of Asn 159<sup>4.60</sup>, and the *n*-butyl chain of the carbamate moiety makes hydrophobic contacts to several residues between helices IV and V. Additionally, the phenyl ring of the two molecules both engage in similar edge to face pi stacking with Phe 252<sup>6.51</sup>. The shift of the Tyr 259<sup>6.58</sup> side chain is even more pronounced than in the AZD1283-bound state due to the additional pyrrolidine moiety of selatogrel (Fig. 5. C).

In comparison with the P2Y12 structure bound to an agonist (2MeSADP in 4pxz) [8], it becomes even clearer that selatogrel stabilizes the basal state of the receptor. Similar to the AZD1283 structure, helix VI adopts a completely different orientation in the selatogrel-bound

structure: the extracellular side of helix VI is shifted outward of the ligand binding site by over 8 Å, while on the intracellular side, a shift in the opposite direction of approximately 5 Å is observed. Helix VII is also shifted 6 Å outward on the extracellular side and 5 Å inward on the intracellular side in the selatogrel-bound structure (Fig. 5. D, E, F).

The selatogrel binding site overlaps partially with the site of 2MeSADP: the piperazine moiety of selatogrel overlaps with the binding site of the adenine base in 2MeSADP. The phosphonate binding site of selatogrel is close to but not completely identical to the phosphate binding site of 2MeSADP. While interacting with the same His 187<sup>5.38</sup> and Lys 280<sup>7.35</sup>, the amine position of the lysine is shifted by over 4 Å. One of the 2MeSADP phosphate groups also interacts with Arg 256<sup>6.55</sup>, which is not the case for the selatogrel phosphonate, as Arg 256<sup>6.55</sup> is involved in another hydrogen bond with the carbonyl group of an amide and is also shifted by almost 6 Å (Fig. 5. D, E, F).

As in the related AZD1283 structure, a cholesterol molecule was bound at the interface of helices III and V, stabilizing the receptor-receptor interaction. Weak electron density indicating a partially bound cholesterol between helices I and VII (adjacent to Trp 285<sup>7.40</sup>) was observed, but no cholesterol molecule was included in the model.

In the crystal structure of the P2Y12 receptor bound to selatogrel, cysteine (Cys) 17 and Cys 270<sup>7.25</sup> are involved in a disulfide bond and are located approximately 13 Å from the selatogrel binding site. Cys 175 is disordered, while its partner Cys 97<sup>3.25</sup> is visible in the electron density. Cys 97<sup>3.25</sup> is located approximately 9 Å from the selatogrel binding site. Molecular modeling of the active metabolite of clopidogrel addressing Cys 97<sup>3.25</sup> covalently was analyzed in comparison with the selatogrel-bound structure. For this analysis, two different potential binding modes of clopidogrel were analyzed based on the two pocket model previously reported [7]. One option proposed clopidogrel bound entirely in pocket 2. [9] In this binding mode, no steric clash could be identified with a superimposed selatogrel from the crystal structure. However, we have previously reported that selatogrel prevents clopidogrel binding.[38] Therefore, a second potential binding mode of clopidogrel was also analyzed. In this binding mode, clopidogrel stretches toward pocket 1, potentially addressing His 187<sup>5.38</sup> with its carbonyl group by accepting a hydrogen bond (Fig. 6. A, B).

## 4. Discussion

Biochemical characterization demonstrated selatogrel to be an inverse agonist of the P2Y12 receptor in HEK293T cells recombinantly expressing the P2Y12 receptor and in human platelets, supported by the cocrystal structure of selatogrel bound to the purified P2Y12 receptor, in which the P2Y12 receptor adopts an inactive conformation with selatogrel bound in the previously described "pocket 1".

As a first step, we assessed the inverse agonism efficacy of selatogrel by using a BRET2-based assay to measure the activation of the G proteins in real time in living cells and benchmarked it with ticagrelor and elinogrel. [27] Selatogrel and ticagrelor had no effect on cells without recombinant expression of the P2Y12 receptor. In contrast, elinogrel
<figure><img src="image_15.png" /><figcaption>**Fig. 5.** Protein structure of P2Y12 receptor with selatogrel bound. A. Omit electron density map of selatogrel contoured at two sigma, generated after ten cycles of refinement in the absence of selatogrel. B. X-ray structure of the P2Y12 receptor in complex with selatogrel (green carbons): PDB ID 7pp1. The receptor is shown in dark green ribbons. The numbers of the transmembrane helices are indicated in black roman numbers. Selatogrel is depicted with spheres. C. Superposition of P2Y12 structures with PDB entry IDs 7pp1 (green, selatogrel structure) and 4ntj (yellow, AZD1283 structure), both in the basal state, based on the Cα atoms of the receptors. Both receptors are represented as ribbons, the ligand molecules are represented in balls and sticks, and selected amino acid side chains are represented as sticks. Selatogrel occupies the same binding site as the ligand AZD1283 bound inside the 4ntj structure but pushes Tyr 259<sup>6.58</sup> farther out of the pocket than AZD1283. The edge to face pi stacking interaction between the ligands and the receptor are indicated by a dotted line. D. Superposition of P2Y12 structures 7pp1 (green) and 4pxz (gray) based on the Cα atoms of the receptors. Impact of selatogrel binding on protein structure compared to binding of 2MeSADP. Green helices represent the conformation of the basal state in the presence of bound selatogrel (green carbon atoms). Gray helices represent the active conformation with bound 2MeSADP (gray carbon atoms). Helix numbers are indicated by roman letters. E. Comparison of basal vs active conformation. Active conformation depicted with gray helices from 4pxz (2MeSADP-agonized P2Y12). Basal conformation with bound selatogrel depicted with green helices. Molecular structure of selatogrel presented with green carbon atoms. F. Basal P2Y12 conformation presented as green helices with the molecular structure of selatogrel bound to the P2Y12 receptor depicted with green carbon atoms. Critical amino acid residues are indicated with amino acid numbers and with Ballesteros-Weinstein numbering.</figcaption></figure>

affected Gi, Go, Gq and Gs-protein signaling in HEK293T cells, suggesting off-target activity. Due to the broad effect on multiple subtypes of G-proteins, the identity of involved GPCRs cannot be established. Clinical profiling of elinogrel reported that bleeding requiring medical attention was more common in patients receiving elinogrel. [39] Our data suggest that the off-target activities of elinogrel might have limited the therapeutic window of elinogrel. Therefore, elinogrel was excluded from further profiling in our study.

Subsequent experiments with selatogrel and ticagrelor in cells expressing the P2Y12 receptor demonstrated inverse agonism with the Gαi and Gαo isoforms only, confirming the basal specific signaling of the P2Y12 receptor via these G-protein isoforms. [15] Importantly, selatogrel did not interfere with G-protein signaling in non P2Y12-expressing HEK293T cells, strengthening previous reports describing the absence of off-target activity of selatogrel [23].

In dose-response experiments with the Gαi2 and GαoB isoforms, at comparable effects, selatogrel was approximately 100-fold more potent than ticagrelor. In agreement, direct comparison of selatogrel and

ticagrelor in ADP-induced in vitro platelet aggregation experiments confirmed selatogrel to be approximately 28-fold more potent than ticagrelor (selatogrel IC₅₀ = 14 nM [23], ticagrelor IC₅₀ = 400 nM [40]), suggesting selatogrel to be the most potent inverse agonist of the P2Y12 receptor available thus far.

Activation of Gi/o signaling by ADP translates into decreased cAMP concentrations in human platelets supporting platelet activation. In contrast, our data suggest that the consequence of binding of the inverse agonist selatogrel is twofold: selatogrel prevents ADP binding orthostatically and, in addition, induces an allosteric change from a constitutive preactivated conformation into a quiescent status, consequently preventing the decrease of cAMP and platelet activation. This effect could be particularly important in diabetic patients who are reported to have elevated P2Y12 receptor expression and activation levels and thus enhanced platelet activity. [16] Furthermore, we have previously shown that the addition of selatogrel to preactivated platelets triggered detachment of prebound fluorescein isothiocyanate (FITC)-Fg from the fibrinogen receptor GPIIb-IIIa in vitro. [41] In addition, selatogrel
E Basal vs active conformation grey: 4pxz (2MeSADP-agonised P2Y12), green: selatogrel-antagonized P2Y12

<figure><img src="image_16.png" /></figure>

F 2MeSADP bound to P2Y12 from PDB ID 4pxz

<figure><img src="image_17.png" /><figcaption>Fig. 5. (continued).</figcaption></figure>

induced the dissolution of preformed platelet thrombi in vivo. [41] These observations are in line with what we report herein: selatogrel induces the inactive conformation of the P2Y12 receptor, blunting intracellular signaling, which is essential for the stabilization of the fibrinogen-bound GPIIb-IIIa receptor structure and stabilization of platelet-platelet interactions.

In agreement with the functional consequences of selatogrel binding to the P2Y12 receptor, protein crystallization experiments in the presence of selatogrel resulted in the inactive receptor conformation of the P2Y12 protein. In addition, the binding mode of selatogrel in the P2Y12 receptor protein structure suggested a steric overlap of selatogrel with the hypothetical binding mode of ADP and thus is in line with the biochemical data that demonstrated that selatogrel prevents ADP binding to the P2Y12 receptor. [23]

The binding mode of thienopyridine analogs such as clopidogrel and prasugrel with the P2Y12 receptor is not yet clear. While some studies suggest Cys 97³.²⁵ and Cys 175 to be involved [42,43], other studies suggest Cys 17 and Cys 270⁷.²⁵ to be the target of the active metabolites of thienopyridines. [44] In our crystal structure, as in the structure of AZD1283 bound to the P2Y12 receptor, Cys 175 is not visible. Cys 17 and Cys 270⁷.²⁵ appear to engage in a disulfide bond, reducing the likelihood that they are involved in the binding of thienopyridines. Previous studies have described the disulfide bond between Cys 97³.²⁵ and Cys 175 to be more labile or dynamic in the P2Y12 receptor and thus available for the active metabolites of clopidogrel and prasugrel. [7] In agreement, in our structure, Cys 97³.²⁵ is not involved in a disulfide bond with Cys 175 and thus appears accessible for an interaction with the active metabolites of thienopyridines. Therefore, two potential binding modes of the active metabolite of clopidogrel were modeled and compared to the selatogrel-bound structure of P2Y12. In the first

potential binding mode, clopidogrel would only occupy pocket 2 [9], potentially allowing simultaneous binding with selatogrel. Functional studies using selatogrel have shown in vitro and in vivo that prebound selatogrel prevents the binding of the active metabolites of clopidogrel or prasugrel. [38] Therefore, we consider this binding mode unlikely. In the second potential binding mode (shown in Fig. 6, C), clopidogrel would stretch toward pocket 1 and accept a hydrogen bond from histidine (His) 187⁵.³⁸. This particular hydrogen bond might not be very stable or strong when looking at the covalent complex of clopidogrel-P2Y12. However, given our biochemical data, [38] we hypothesize that the initial contact of clopidogrel to His 187⁵.³⁸ helps to orient the molecule inside the binding pocket with the covalent warhead pointing toward Cys 97³.²⁵ so that ultimately the covalent bond can be formed. When selatogrel is bound prior to clopidogrel, His 187⁵.³⁸ is already involved in interactions with selatogrel and could therefore not orient clopidogrel correctly inside the binding pocket to prepare it for the covalent attack of Cys 97³.²⁵. Alternatively, selatogrel could be a highly effective inducer of the basal state of the P2Y12 receptor, as previously described by Zhang et al. [8] and thus capable of limiting the access of thienopyridines.

We confirmed that selatogrel, similar to the antagonist AZD1283, binds to “pocket one” one of the two distinct pockets in the P2Y12 receptor. However, in molecular docking studies, a binding pocket for the inverse agonist mant-dADP involving tyrosine (Tyr) 105³.³³, glutamic acid (Glu) 188⁵.³⁹, arginine (Arg) 256⁶.⁵⁵, Tyr 259⁶.⁵⁸ and lysine (Lys) 280⁷.³⁵ was described. [45] In a separate study, the amino acids phenylalanine (Phe) 252⁶.⁵¹, Arg 256⁶.⁵⁵, Tyr 259⁶.⁵⁸, leucine (Leu) 276⁷.³¹ and Lys 280⁷.³⁵ were reported to be essential for the binding of AZD1283. (32) The binding pocket of the inverse agonist selatogrel determined in the P2Y12 protein crystals confirmed the residues His
<figure><img src="image_18.png" /><figcaption>B Selatogrel (green) and hypothetical binding of clopidogrel (salmon) to Cys 97³,²⁵</figcaption></figure>

<figure><img src="image_19.png" /></figure>

**Fig. 6.** Binding modes of selatogrel and clopidogrel. **A.** Active P2Y12 conformation presented as gray helices with 2MeSADP bound to P2Y12 from PDB ID 4pxz. Molecular structure of 2MeSADP depicted with gray carbon atoms. Critical amino acid residues are indicated with amino acid numbers and with Ballesteros-Weinstein numbering. **B.** Comparison of the binding mode of selatogrel with the hypothetical binding mode of the clopidogrel active metabolite. Molecular structure of selatogrel depicted with green carbon atoms. Molecular structure of clopidogrel active metabolite depicted with salmon carbon atoms. Dotted lines indicate interactions with relevant amino acids.

187⁵,³⁸, Phe 252⁶,⁵¹, Arg 256⁶,⁵⁵ and Lys 280⁷,³⁵ to be essential for the high affinity interaction between selatogrel and the P2Y12 receptor. In addition, our structure revealed that the induced conformational change with respect to Tyr 259⁶,⁵⁸ is specific for the binding mode of selatogrel.

Study limitations: Although we confirm inverse agonism of selatogrel both, in a recombinant system and using human washed platelets with endogenous P2Y12 receptor expression levels, we cannot exclude that the process of preparation of human washed platelets influences the overall phenotype of the platelets.

In addition, the protein structure of the P2Y12 receptor was generated as a P2Y12-BRIL protein complex. We cannot exclude a potential influence of the presence of the BRIL-protein on the resulting P2Y12-BRIL structure. In addition, protein crystals are frozen in a specific protein conformation and thus potential flexibility within the P2Y12 receptor and thus, alternative conformations might be limited.

In conclusion, selatogrel was confirmed to be an inverse agonist of the P2Y12 receptor in functional assays with P2Y12 receptor-expressing cells and with human platelets. In agreement, crystallization experiments in the presence of selatogrel resulted in the inactive P2Y12 receptor structure. The binding mode of selatogrel within the P2Y12 receptor protein structure is consistent with the competitive antagonism of ADP binding to the P2Y12 receptor in human platelets [23], and the conformational change of Tyr 259⁶,⁵⁸ defines the unique binding mode of selatogrel.

What is known:

* The P2Y12 receptor displays constitutive Gi/o-mediated signaling

* Ticagrelor and cangrelor have been shown to be inverse agonists of the P2Y12 receptor

* Selatogrel is a highly selective, reversible P2Y12 receptor antagonist

What this paper adds:

* Using highly sensitive resonance energy transfer-based probes, selatogrel was demonstrated to act as an inverse agonist of the P2Y12 receptor, approximately 100-fold more potent than ticagrelor

* Inverse agonism by selatogrel specifically blunted basal receptor-dependent Gi/o signaling

* Selatogrel increased basal cAMP levels, confirming inverse agonism in human platelets

* New 2.8 Angstrom crystal structure revealed the binding mode of selatogrel in the inactive conformation of the P2Y12 receptor, agreeing with the inverse agonist efficacy of selatogrel

* The binding mode of selatogrel confirms the antagonism of ADP binding to the P2Y12 receptor, and the conformational change of Tyr 259⁶,⁵⁸ defines the unique binding mode of selatogrel.

CRediT authorship contribution statement

**Véronique Pons:** Conceptualization, Investigation, Methodology, Writing – review & editing.
**Cédric Garcia:** Investigation.
**Naomi Tidten-Luksch:** Investigation, Methodology, Writing – original draft.
**Aengus Mac Sweeney:** Investigation, Methodology, Writing – review & editing.
**Eva Caroff:** Resources.
**Céline Galés:** Conceptualization, Supervision, Methodology.
**Markus A. Riederer:** Conceptualization, Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: C. Pons, C. Gales reports financial support was provided by Idorsia Pharmaceuticals Ltd. Markus A. Riederer reports a relationship with Idorsia Pharmaceuticals Ltd. that includes: employment and equity or stocks. N. Tidten-Luksch, A. Mac Sweeney, E. Caroff reports a relationship with Idorsia Pharmaceuticals Ltd. that includes: employment and equity or stocks. E. Caroff has patent #WO-2009069100-A1 issued to Idorsia Pharmaceuticals Ltd. Markus Riederer has patent #WO-2018167139-A1 pending to Idorsia Pharmaceuticals Ltd.

Data availability

Data will be made available on request.

Acknowledgments

We thank Shunmei Chen of the Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming, Yunnan 650500, China for the purification and crystallization of the P2Y12 fusion protein under a contract research agreement.

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