Title: Novel allosteric inhibitors of SOS-mediated mutant Ras-driven tumorigenesis
Funder: Cancer Research UK

Abstract: Background K-Ras is the second-most frequently mutated gene in human cancers: some of the most intractable cancers (including pancreatic) show Ras-activating mutation rates of over 90%. Ras is activated by the specific guanine nucleotide exchange factor SOS1, but Ras mutations were presumed able to bypass this normal activation mechanism. We now know, however, that mutant Ras ‘co-opts’ unaltered, wild-type Ras through constitutive activation of SOS via binding to its allosteric Ras site. This implies that inhibiting binding of active Ras to SOS’ allosteric site is a potential therapeutic approach to treatment of mutant Ras-driven cancers. In preliminary screens, we identified small molecule compounds that bind in the allosteric Ras site on SOS and have the potential to inhibit Ras activation via this novel mechanism. Aims Further target validation for this mechanism requires a potent and selective chemical probe, and this is the primary aim of this application. We will use a combination of structural, biophysical, cell biology and medicinal chemistry approaches to: 1. Establish structure-activity relationships around our recently identified small molecule allosteric Ras-site ligands, using structure-guided medicinal chemistry approaches to improve affinity of these compounds to yield potent, cell-permeable chemical leads (sub-1 µM in biochemical assay); 2. Progress and hybridise the fragment hits that we will identify in our follow-up XChem fragment screen at Diamond Light Source; 3. Assess the potency of our compounds in inhibition of Ras-driven signalling in wildtype and mutant Ras cancer cell lines. Methods We will use an X-ray cocktail soaking approach to screen the generic XChem and Leeds ‘diversity’ fragment sets at Diamond Light Source. We will then use our established secondary screens (NMR, SPR and ITC; MANT-GDP exchange assays) together with our Newcastle collaborators’ expertise in computational and medicinal chemistry, to drive series hybridisation and potency optimisation against SOS-mediated Ras nucleotide exchange. We will follow this by measuring phenotypic responses in wild-type and mutant Ras-dependent cell lines. How the results of this research will be used Although the literature evidence supporting the importance of constitutive SOS activation by mutant K-Ras in hyperactivation of wild-type Ras alleles and consequent cellular transformation is powerful, confirmation is lacking that interruption of this mechanism with a small-molecule inhibitor of allosteric Ras-site binding will achieve therapeutically worthwhile cellular or tissue-level responses. We will furnish evidence bolstering this mechanism as a promising avenue for development of new therapeutics for mutant Ras-driven cancers, including currently intractable pancreatic disease.

Lay abstract: Background K-Ras is the second-most frequently mutated gene in human cancers: some of the most intractable cancers (including pancreatic) show Ras-activating mutation rates of over 90%. Ras is activated by the specific guanine nucleotide exchange factor SOS1, but Ras mutations were presumed able to bypass this normal activation mechanism. We now know, however, that mutant Ras ‘co-opts’ unaltered, wild-type Ras through constitutive activation of SOS via binding to its allosteric Ras site. This implies that inhibiting binding of active Ras to SOS’ allosteric site is a potential therapeutic approach to treatment of mutant Ras-driven cancers. In preliminary screens, we identified small molecule compounds that bind in the allosteric Ras site on SOS and have the potential to inhibit Ras activation via this novel mechanism. Aims Further target validation for this mechanism requires a potent and selective chemical probe, and this is the primary aim of this application. We will use a combination of structural, biophysical, cell biology and medicinal chemistry approaches to: 1. Establish structure-activity relationships around our recently identified small molecule allosteric Ras-site ligands, using structure-guided medicinal chemistry approaches to improve affinity of these compounds to yield potent, cell-permeable chemical leads (sub-1 µM in biochemical assay); 2. Progress and hybridise the fragment hits that we will identify in our follow-up XChem fragment screen at Diamond Light Source; 3. Assess the potency of our compounds in inhibition of Ras-driven signalling in wildtype and mutant Ras cancer cell lines. Methods We will use an X-ray cocktail soaking approach to screen the generic XChem and Leeds ‘diversity’ fragment sets at Diamond Light Source. We will then use our established secondary screens (NMR, SPR and ITC; MANT-GDP exchange assays) together with our Newcastle collaborators’ expertise in computational and medicinal chemistry, to drive series hybridisation and potency optimisation against SOS-mediated Ras nucleotide exchange. We will follow this by measuring phenotypic responses in wild-type and mutant Ras-dependent cell lines. How the results of this research will be used Although the literature evidence supporting the importance of constitutive SOS activation by mutant K-Ras in hyperactivation of wild-type Ras alleles and consequent cellular transformation is powerful, confirmation is lacking that interruption of this mechanism with a small-molecule inhibitor of allosteric Ras-site binding will achieve therapeutically worthwhile cellular or tissue-level responses. We will furnish evidence bolstering this mechanism as a promising avenue for development of new therapeutics for mutant Ras-driven cancers, including currently intractable pancreatic disease.

Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS