Delineation of molecular determinants for FR900359 inhibition of G unlocks inhibition of Gα

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The Gq/11subfamily consists of G, G, G and G proteins of which all but G are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families; G, G and G, but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits G, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359 binding site in G and G We found that the activity of a G mutant with a G-like binding site for YM-254890/FR900359 can be inhibited by FR900359 and a minimum of three mutations are necessary to introduce inhibition in G In all, this suggest that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive G, G and G inhibitors, the mechanism of inhibition between families of G proteins is conserved opening up for targeting by other, novel inhibitor scaffolds. In lack of a selective Gα inhibitor, FR900359 sensitive Gas mutants may prove useful in studies where delicate control over Gα signaling would be of the essence.