Discovery of small molecule modulators of Ras superfamily proteins : Studies of MgcRacGAP and Ras

Small guanosine triphosphatases (GTPases) are a family of low molecular weight guanosine diphosphate (GDP)-/guanosine triphosphate (GTP)- binding proteins that act as molecular switches , regulating key cellular processes, such as cell proliferation and differentiation. The activity of small GTPases is controlled by guanine nucleotide exchange factors (GEFs), which facilitate nucleotide exchange from GDP to GTP, and GTPase activating proteins (GAPs), which stimulate intrinsic GTP hydrolysis of the GTPase. Complex, yet unresolved, mechanisms maintain the precise spatiotemporal regulation of these proteins, which is essential for accurate cellular signaling. Accordingly, aberrant function of small GTPases is associated with a large number of human diseases, such as cancer, neurodegenerative diseases and inflammatory disorders. The availability of small molecule modulators of GTPase activity would be highly valuable to improve the understanding of small GTPases of the Ras superfamily and their regulators. However, very few probes of this kind currently exist. To address this gap, I have focused on discovering different small GTPase-modulating probes through different screening approaches. MgcRacGAP is a GAP protein for Rho family small G-proteins and a key regulator of cytokinesis. After almost two decades of research, the detailed functional role, its relevant target GTPase and its GAP activity in cytokinesis are not yet fully resolved. Furthermore, like many other regulators of GTPase activity, its overexpression has been linked to different malignant properties, such as epithelial-to-mesenchymal transition, cell polarity and (invasive) migration, as well as correlated to a poor clinical prognosis in many types of cancers. To be able to investigate the biological role of MgcRacGAP, we first addressed the lack of chemical tools to probe its function by developing a high throughput screening strategy to identify compounds inhibiting its GAP function. The discovery of MgcRacGAP Inhibitor Compound 1 (MINC1), a selective MgcRacGAPRac1 inhibitor, represents the first described selective small molecule inhibitor of a Rho GTPase activating protein and shows that development of inhibitors of small GTPase activating proteins is possible. We utilized MINC1 to study the function of MgcRacGAP in cell division as well as the role in the events that regulate signal transducer and activator of transcription 3 (STAT3) signaling. Notably, we showed that MINC1- mediated inhibition of MgcRacGAP caused impaired mitotic spindle formation during the metaphase, which suggests that the GAP activity of MgcRacGAP plays an important role in mitosis. To address the role of MgcRacGAP in the events that regulate STAT3 phosphorylation and subsequent nuclear translocation, we used both MINC1 treatment and small interference RNA (siRNA)-mediated gene silencing of MgcRacGAP. With these complementary techniques we showed that inhibition of MgcRacGAP triggers STAT3 phosphorylation caused by a Rac1-PAR3-IL6- IL6R-JAK2 mediated autocrine/paracrine mechanism. Due to their key role in different essential cellular processes, non-specific inhibition of GTPases or their regulators is expected to result in significant risk of side effects. Compounds that interfere with specific protein-protein interactions are expected to circumvent this problem, yet most current screening methods fail to detect these. To address this, we have developed a protein-protein interaction inhibitor screening strategy for the oncoprotein Ras and identified ten compounds that inhibited GTP hydrolysis in a concentration dependent manner. Four of these compounds could not be detected with the established method. Of the three compounds that showed efficacy in Ras signaling dependent cell lines, one compound had a direct effect on the activation status of Ras. In summary, this thesis describes i) the discovery of the first RhoGAP inhibitor named MINC1, ii) the application of MINC1 to elucidate the role of MgcRacGAP in the activation and nuclear translocation events of STAT3 and iii) the development and exploration of a new screening strategy to discover Ras protein-protein inhibitors.N.a