Novel insights into mechanisms and selectivity of the heterotrimeric G protein inhibitors BIM-46187 and FR900359

Abstract

Selective silencing of Gα protein subfamilies by small molecules is of great value to explore the contribution of G protein signaling in physiology and disease. It also represents a new opportunity to treat diseases, such as cancer, in which multiple receptors are involved. Consequently, signaling of a large number of receptors could be silenced with a single tool. Only few small cell-permeable molecules acting as Gα-selective inhibitors have been reported to date.<br /> The present thesis classifies BIM-46187, previously reported as pan-G protein inhibitor, as a compound which preferentially silences Gα_q-mediated signaling in a cellular context-dependent manner. In particular, BIM functions as selective Gα_q inhibitor in HEK and CHO cells but silences Gα_q, Gα_i and Gα_s proteins in the human skin cancer cell line MZ7. Cell-context pharmacology might be explained with differences in the relative amount or stoichiometry of signaling components. Gene dosing experiments revealed a correlation between BIM inhibition and Gα_q expression. However, immunoblot detection, which compared expression levels of Gα_q, Gα_s and Gα_i proteins in HEK and MZ7 cells, indicated that different Gα expression levels cannot exclusively account for cell-type-dependent pharmacology of BIM. Investigations concerning the mode of action uncovered an entirely new molecular mechanism: BIM permits GDP exit but precludes GTP entry thereby “freezing” the Gα_q protein in the empty pocket conformation. <br /> The second part of this thesis uncovers the cyclic depsipeptide FR900359 as a suitable tool for selective silencing of Gα_q/11 proteins. A great variety of assays, such as classical second-messenger assays, BRET assays and label-free methods, demonstrate the selectivity of FR900359. Experiments in the commonly used HEK and CHO backgrounds, as well as in the human skin cancer cell line MZ7, reveal its utility in cell-based assays. FR900359 does not compromise agonist binding but interacts with agonist function. Based on radioligand competition experiments, it can be assumed that FR900359 functions as a guanine-nucleotide dissociation inhibitor as it impairs the formation of the high-affinity agonist binding.