P7: Designing engineered proteins to foster development of unnatural natural ligands: A chemogenetic proof-of-concept study

Currently existing chemogenetic platforms comprise kinases, non-kinase enzymes, G protein-coupled receptors and ion channels. In this project, the Kostenis group wishes to enable non-invasive chemical control over heterotrimeric αβγ G proteins of the G12/13 family. G12/13 proteins are one of the four major families of large G proteins and among all, the least studied subgroup.

Strategies to investigate their biological roles so far comprise genetic knockout and knockdown approaches, but assays for quantification of G12/13-mediated signaling are scarce and chemical tools to selectively interfere with the G12/13 subgroup, and that can be added at will to act quickly are lacking. Kostenis wants to overcome this deficit by establishing G12/13 signaling assays along with engineered variants of Gα12 and Gα13 that retain wildtype biological activity, yet are sensitive towards inhibition by FR900359 (FR). FR is a cyclic depsipeptide, a “chemical”, that shuts down Gq signaling in a highly selective manner, and is accessible via P4.

The group previously succeeded in transplanting FR’s mechanism of action to G16 proteins which are naturally not FR-regulated. Here they plan to place synthetic Gα12 and Gα13 variants under FR control as biopolymeric tools using Gq null systems or cells engineered to express catalytically active but FR-resistant Gαq variants.

This is anticipated to lead to the analysis of inhibitor-target interactions enabling prediction of structural modifications for conversion of FR from a Gq-selective to a G12- or G13-selective inhibitor, and assisting P9 in eludicating the biological relevance of GPR84-G12/13 interactions. At the very least, the G12/13 assay platform will complement the orthogonal functional assays established in P9 to characterize the developed drug conjugates.