Targeting the Ras superfamily via their regulatory GEF complexes

The Ras superfamily of small GTPases are guanine nucleotide-dependent molecular switches which are involved in numerous signalling pathways in the cell. Mutations or dysregulation of these proteins are associated with a variety of diseases, and so the small GTPases are important targets in drug discovery. Extensive medicinal chemistry efforts have largely been unsuccessful due to the difficulty in developing inhibitors to compete with GDP or GTP in an analogous manner to the ATP-competitive inhibitors of kinases. The activation of the small GTPases is regulated by guanine nucleotide exchange factors (GEFs). This thesis describes the efforts to identify small molecules which could be developed into novel inhibitors of the small GTPases by targeting their regulatory GEF complexes.
The second chapter details a kinetic analysis of the GEF-catalysed activation cycle to convert an inactive GDP-bound GTPase into its active GTP-bound form. The modelling of a competitive inhibitor of a GEF/GTPase complex within this cycle supported the hypothesis that a GEF/GTPase inhibitor could slow the rate of nucleotide exchange in a manner not achieved by an archetypal competitive inhibitor which binds to the GTPase alone.
The following chapter presents the cloning, protein production, and crystallisation efforts of over 50 proteins to develop a pipeline within the Structural Genetics Consortium (SGC). The chapter reports the determination of three novel crystal structures, including the GEF Kalirin and its small GTPase Rac1, which was chosen as a therapeutic target for compound screening due to its role in neurodegenerative diseases and suitability for X-ray crystallography ligand screening.
Chapter 4 describes the high-throughput X-ray crystallography fragment screening conducted on Kalirin/Rac1 and the subsequent identification of an isoxazole-urea hit fragment within the canonical binding site. Computational docking of purchased analogues and the synthesis of bioisosteres is also reported, as well as the development of a biochemical fluorescence-based assay to detect compounds which inhibit the nucleotide exchange reaction.
Electrophilic covalent fragments were also found to bind to the guanine nucleotide pocket of Kalirin/Rac1. Determination of their co-crystal structures, and subsequent ongoing efforts to synthesise novel and merged fragments are detailed in Chapter 5, including a lead compound which demonstrated potency in the biochemical assay.
The final chapter delineates a virtual screening effort to discover new scaffolds with similar pharmacophores which bind within the GDP/GTP binding pocket of Kalirin/Rac1 and the consequent synthesis of non-covalent and covalent analogues arising from the identification of a potent non-covalent fragment.
Overall, this thesis proposes several compounds as suitable vectors for the development of probes or therapeutics for Kalirin/Rac1, as well as providing the groundwork for the expression, structural determination, and compound screening of other members of the Ras superfamily. Future work arising from this thesis, including the development of lead compounds, establishment of cellular assays, and the progression of other GEF/GTPase complexes is also considered.

Keywords: XChem
Chemical biology
Medicinal chemistry
Ras
Advisor: Brennan, P
Von Delft, F