POTENTIATION OF HISTAMINE H1 RECEPTOR CALCIUM RESPONSE BY RATIONALLY DESIGNED GRK2 INHIBITORS

EMILIANA ECHEVERRÍA; MAIA CABRERA; EZEQUIEL JURITZ; FEDERICO MONKZOR; CARLOS DAVIO; CARINA SHAYO; PABLO LORENZANO-MENNA; NATALIA FERNANDEZ

Mar del Plata

Congreso; LXI Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2016

Sociedad Argentina de Investigación Clínica

G protein-coupled receptors (GPCRs) are the largest familyof cell-surface receptors and regulate most biological processes.Because approximately 30% of therapeutic drugs directly targetGPCRs, it is of paramount importance to understand the mecha-nisms that regulate their function. GPCR kinase 2 (GRK2) playsa major role mediating desensitization through phosphorylationdependent and independent mechanisms and has been associatedto the progression of several pathologies. Our aim was to obtainGRK2 inhibitors based on a rational drug design approach. Usingthe crystallographic image of GRK2, 13 compounds were chosenby virtual screening (VS) according to their docking energy andpurchased to the supplier. Considering that histamine H1 receptor(H1R) desensitization depends on GRK2 activity, we evaluated theability of the selected compounds to block H1R desensitization.Histamine-stimulated intracellular calcium release was tested inA549 cells endogenously expressing H1R using Fura2AM dye. 40minutes incubation with 100nM of 3 of the selected compoundspotentiated calcium response. Moreover, the incubation with thesecompounds reverted H1R desensitization induced by 10 minuteshistamine pretreatment. To mitigate the risk of a future failure ofthe compounds due to a potential toxic effect in late phases of drugdevelopment, cytotoxicity was evaluated by trypan blue exclusionon hepatic HEPG2 and hematopoietic derived U937 cells after48hs of treatment. Concentration response assays showed thatthe compounds that displayed cytotoxicity presented a CC50 valuegreater that 10uM. These results indicate that at concentrationsused for calcium assay the compounds display no cytotoxicitythough they inhibited GRK2 mediated H1R desensitization. Basedon these observations, 3 of the candidates obtained by VS arepromissory inhibitors for the treatment of pathologies where GRK2mediated desensitization of GPCRs takes place.