Three small molecule pan activator families of Ras-related GTPases
The mutated forms of small guanosinetriphosphatases (GTPases) may cause disease either through aberrant activation or through loss of function or diminished acitivity. For example, a dominant negative missense mutation in Rac2 was found to interfere with both Rac1 and Rac2 function and cause impaired resistance to infection, while reduced Cdc42 activity has been found associated with Fanconi anemia. Missense mutations in Rab GTPases or associated regulatory proteins has been associated with decreased normal activity, resulting in immune dysfunction, pigmentation or neurological disorders. Pathogens often evade immune surveillance or degradation by inactivating endosomal Rab5 or Rab7 GTPases. In Bardet Biedl Syndrome, an inherited cystic kidney disease, Rab8 activation is defective impairing exocytosis to cilia. Presently, the only known activation mechanism is via guanine nucleotide-exchange factors (GEFs), which are multidomain proteins that accelerate the exchange of guanosine diphosphate (GDP) by GTP by several orders of magnitude. GEFs catalyze the dissociation of the nucleotide from the GTPase by modifying the nucleotide-binding site such that the nucleotide affinity is decreased, and the GDP is released and replaced with GTP. Results from the current project revealed that the probe compounds ML099 (CID-888706), ML098 (CID-7345532), and ML097 (CID-2160985) function by increasing the affinity of the GTPases for guanine nucleotides, leading to the hypothesis that these activators interact with GTPases bind to an allosteric binding site localized between switch regions I and II. Based on these findings, the project further proposes that in biochemical systems, these novel small activators should prove useful towards: (1) elucidating the mechanism by which GEF regulates GTPase activity; (2) defining protein-protein interactions between GEFs and GTPases; (3) defining the sites of GTPase regulation in crystal structures and (4) platforms for the development of probes that can be used to activate GTPase function.