Protein-Protein Interaction : RhoA


There is an increasing body of evidence supporting a causal role of aberrant activity of small GTPases of the Rho protein family in human diseases including cancers, neuronal disorders, pulmonary and cardiovascular diseases, thus identifying Rho protein signaling cascades as potential targets for new therapeutic strategies. Unfortunately, because of their smooth and globular structure lacking pockets to which small molecules can bind with high affinity, Rho proteins are considered as "undruggable" by traditional drug design approaches.

Rho GTPases (including RhoA, Rac, Cdc42) are critical ubiquitous intracellular signaling molecules that act as binary switches to control many fundamental cellular processes from cytoskeleton organization to gene expression, cell cycle progression, cell motility and contraction. The activity of Rho proteins results from a tightly regulated balance between the GTP-bound, active state and the GDP-bound, inactive state. By catalyzing the exchange from GDP to GTP, Rho guanine exchange factors (GEFs) are major regulators of Rho protein activity that relay a variety of upstream signals from growth factors, hormones, neuromediators, cytokines or other soluble messengers and adhesion molecules. Targeting the interaction of Rho GTPase with its GEFs thus appears as an alternative strategy to block Rho protein signaling and circumvent the low druggability of the GTPase.

The aim of our project is to develop Rho GEF/GTPase interaction-specific small-molecule inhibitors. We will use the mechanistic information of Rho GEF/GTPase interaction to conduct a rational design strategy by structure-based molecular modeling simulations using a wide range of methods to get a comprehensive understanding of RhoGEF site(s) essential for Rho GTPase activation. This strateg will be first applied on the Rho GEF/GTPase pair Arhgef1/RhoA that we have recently identified as a relevant target for the development of new therapies against high blood pressure and atherosclerosis.

Our project is based on the constructive interdisciplinary collaboration of biologists, computational and medicinal chemists from the PIRAMID teams that gather all the skills and expertise required for the success of our strategy. The first step of the project is to identify new ligands targeting Arhgef1/RhoA interaction. Molecular modeling tools of increasing levels of complexity will be used on the GEF/GTPase complexes to (i) refine the x-ray crystallographic data available (ii) identify the key amino acid residues involved in the binding interfaces (iii) provide a detailed description of the structural and energetic parameters of the various partners in interaction (iv) design original ligands targeting the corresponding interfaces. These goals will be reached through the use of successive and complementary molecular modeling approaches. Ligands thus identified will be purchased or synthetized and their ability to inhibit RhoA activity will be assessed by relevant biochemical/biological in vitro assays (cell contraction, cytoskeleton organization, migration, Rac1 and RhoA activity, evaluation of specificity and affinity…). Structure of the best compounds will be examined and modified to optimize their affinity and inhibitory potency. Chemical series will be thus generated and tested. Best candidates will be then assessed in mouse models of hypertension and atherosclerosis. We expected that our project would lead to the discovery of innovative GEF-specific RhoA inhibitors with potential therapeutic applications in hypertension. In addition, our approach may have broad implications for drug discovery efforts targeting Rho GEF/Rho GTPase interactions in other pathophysiological contexts including cancers.