Preclinical pharmacology study of rationally designed GRK2 inhibitors

EMILIANA ECHEVERRÍA; MAIA CABRERA; PABLO LORENZANO-MENNA; SONIA RIPOLL; FEDERICO MONCZOR; NATALIA FERNANDEZ; LUCAS FABIÁN; CARINA SHAYO

Mar del Plata

Congreso; LXIII Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica; 2018

Sociedad Argentina de Investigación Clínica

GPCR kinase 2 (GRK2) plays a major role in GPCRs desensitization and has been extensively validated as an effective target for heart failure (HF) treatment. Its overexpression is associated to disease progression due to the lack of cardiac BAR responsiveness. Accordingly, we have previously obtained four compounds (C2, C3, C4, C5) that exert significant in vitro GRK2 inhibitory activity, postulating them as suitable candidates for in vivo testing. However, there are several risks inherent in preclinical drug discovery that might lead to drugs attrition in late stages. Therefore, the objective of this work was to identify potential early failures of our hits before reaching in vivo phases.To achieve this, we evaluated their ADMET (Absorption-Distribution-Metabolism-Excretion-Toxicity) properties. As a lipophilicity descriptor, experimental logP was obtained by RP-HPLC. Hits cytotoxicity was assessed in U937 and HepG2 cells by trypan blue exclusion test after 48hs treatment. Even though all compounds exhibited an appropriate lipophilicity, with logP values ranging from 1 to 3, compounds C3 and C5 stood out as they did not affect cellular viability, while C2 presented an EC50=10,5µM and C4 an EC50=17,2µM.Moreover, GRK2 desensitizes GPCRs that couple to different G-proteins. Since compounds that specifically potentiate cAMP could be of interest for HF treatment, we compared their ability to increase responsiveness of GPCRs that couple to different G-proteins. Initial cell-based screening assays proved that the hits increased cAMP response of H2R (histamine type 2 receptor). Nonetheless we observed that compound C5 also increased histamine-stimulated intracellular calcium release in A549 cell line endogenously expressing H1R (histamine type 1 receptor), revealing an undesirable promiscuous behavior.In conclusion, we applied strategies to mitigate the risks of drug attrition in late phases of clinical trials, increasing the confidence in our candidate compound C3 for proceeding to HF animal models research.