Small-molecule biased formyl peptide receptor agonist compound 17b protects against myocardial ischemia-reperfusion injury in mice

C. Qin1, L. May2, R. Li1, N. Cao1, S. Rosli1, M. Deo1, A. E. Alexander1, D. Horlock1, J. Bourke3, Y. H. Yang4, A. G. Stewart5, D. M. Kaye1, X. Du1, P. Sexton6, A. Christopoulos7, X. Gao1, R. H. Ritchie1. 1Baker IDI Heart and Diabetes Institute, Melbourne, AUSTRALIA, 2Drug Discovery Biology & Dept of Pharmacology, Monash University, Parkville, AUSTRALIA, 3Dept of Pharmacology, Monash University, Clayton, AUSTRALIA, 4Centre of Inflammatory Diseases, Clayton, AUSTRALIA, 5Dept of Pharmacology and Therapeutics, University of Melbourne, Melbourne, AUSTRALIA, 6Drug Discovery Biology & Dept of Pharmacology, Monash University, Melbourne, AUSTRALIA, 7Dept of Pharmacology, Monash University, Melbourne, AUSTRALIA.

Introduction: Formyl peptide receptors (FPR) are integral to inflammation regulation and are thus attractive therapeutic targets for myocardial ischemia-reperfusion (I-R) injury. Dual FPR1/FPR2 agonists potentially offer FPR1-mediated cardiomyocyte preservation together with FPR2 inflammation-limiting actions. Biased G-protein-coupled receptors (GPCR) signalling has revolutionized drug discovery field, especially its capacity to limit receptor-mediated adverse effects. The aim of this study is to test the hypothesis that biased FPR agonists provide superior cardioprotection in vivo.

Method: Firstly, signalling fingerprints (across pERK, pAktT308, pAktS473, Ca2+-mobilization and cAMP inhibition) of the small-molecule agonists Compound 17b and Compound 43 at FPR1 and FPR2 were systemically assessed in CHO-cells stably-expressing human FPR1 and FPR2 and neonatal rat cardiomyocytes. Then, the impact of small-molecule FPR agonists on cardiac injury response both in vitro (cardiomyocytes, cardiofibroblasts) and in vivo (multiple myocardial injury response across 4 different timepoints) post-left anterior descending artery (LAD) occlusion in adult male anesthetized mice (ketamine 80mg/kg, xylazine 20mg/kg and atropine 1.2mg/kg, i.p.).

Results: Signalling fingerprints of FPR agonists revealed for the first time that relative to Compound 43, Compound17b signalling at both human FPRs was significantly biased away, (by 30-fold) from intracellular Ca2+-mobilization (which can trigger inflammation and cardiomyocyte death). Stimulation of ERK1/2-Akt cell survival kinases however remained intact (n=5-6, p<0.05 vs Compound 43). Compound 17b reduced cardia troponin (cTnI) release post stimulated I-R in vitro, in both rat and mouse cardiomyocytes. Interestingly, Compound 17b and Compound 43 exhibited directly contrasting effects on cardiomyocytes Ca2+ mobilization responses, inhibition versus stimulation. Furthermore, using a preclinical mouse model of myocardial I-R injury (LAD ligation), the novel FPR agonist Compound 17b (50mg/kg/day, i.p.) elicited significant cardioprotection when administered prior to reperfusion. Compound17b reduced infarct size from 44±4% to 29±5% (24h), cardiac neutrophil from 3.5±0.4 to 1.9±0.2AU (48h), cardiac apoptosis from 1.5±0.1 to 1.1±0.1AU (apoptotic/non-apoptotic cells, 7 days), and cardiac function (fractional shortening from 25±2 to 36±4%, 28 days), when compared to vehicle-treated I-R mice (n=6-10; p<0.05 vs vehicle-treated I-R mice, one-way ANOVA with Dunnett’s post-hoc test). In contrast, another FPR agonist Compound 43 (50mg/kg/day, i.p.) was devoid of cardioprotection on all endpoints.

Conclusion: These findings demonstrate ligand-selective cardioprotection with dual FPR1/FPR2-agonist Compound 17b. This breakthrough observation is the first to demonstrate GPCR-agonist bias in the context of cardioprotection in vivo, suggesting a new approach for development of small-molecule FPR-pharmacotherapies for treating myocardial I-R injury