Assessment of small molecules as covalent inhibitors of Rac/Cdc42 - PROJECT SUMMARY A major cause of cancer related deaths is due to the metastasis of malignant cells from the primary tumor to other tissues. Aberrant activity of the regulatory Rho family GTPases has been identified as one of the underlying biochemical processes for initiation of this cell migration to occur. In our laboratory we developed the small molecule inhibitor MBQ-167 and in collaboration with the Dharmawardhane laboratory (University of Puerto Rico, Medical Sciences Campus), we showed that it is a dual inhibitor of the activation of the Rho GTPases Rac1 and Cdc42 with IC50 values of 103 nM and 78 nM, respectively. More importantly, we demonstrated that MBQ-167 reduces tumor growth and metastasis in in vivo experiments in mice. As co-founders of the spin- off company MBQ Pharma, we contributed to its pre-clinical development, and combined with its lack of toxicity in different animal models, an IND-application for first-in-human was approved by the FDA for its use in phase 1 clinical trials in metastatic breast cancer patients. We have recently developed novel more potent inhibitors, of which CPV-337 seems especially interesting. It is 3 to 5 times more potent than MBQ-167 both in cytotoxicity to cancer cells and in Rac inhibitory activity. Based on evidence on the mechanism of action of MBQ-167 as an inhibitor of nucleotide binding, as well as molecular docking analysis, we hypothesize that the increased activity of CPV-337 originates via (reversible) covalent binding to the Cys19 residue of Rac. The successful recent FDA approvals of covalent KRAS G12C inhibitors inspired us to develop novel covalent Rac/Cdc42 inhibitors. In the first aim, we will evaluate the binding of CPV-337 to Rac via protein NMR and use the results in the design of novel covalent Rac/Cdc42 inhibitors. In the second aim we elaborate on the knowledge that a major active metabolite of the leukemia drug 6-mercaptopurine is 6-thioguanosine triphosphate (6-thio-GTP). This metabolite has previously been shown to covalently bind to Cys18 of Rac. An immediate precursor of 6-thio-GTP via the ProTide concept, or a precursor with an alternate covalent binding group, could provide novel inhibitors of Rac. Both aims will result in novel small molecules with increased potency and selectivity, better solubility, and improved biopharmaceutical properties, and potentially can be developed into novel anticancer therapies.
