Characterization of novel covalent and non-covalent drugs against K-Ras surrogate targets

Mutations in the KRAS gene are associated with approximately 15% of all human cancers. This makes it one of the most frequently mutated oncogenes known. Whilst recent breakthroughs in Ras drug discovery have led to the FDA approval of the first direct and covalent inhibitor of the KRAS-G12C mutant, the majority of KRAS driven cancers are not G12C mutated. Furthermore, recent studies have identified resistance mechanisms against the new inhibitors. Consequently, research into other direct and indirect Ras inhibition strategies, as well as synergistic drug combination efforts are being vigorously pursued. In the first part of this thesis, I describe my contributions to the development and characterization of novel PDE6D inhibitors with activity against KRAS driven cancers. PDE6D is a trafficking chaperone of K-Ras that facilitates its dynamic localization to the plasma membrane. Although some progress had been made in identifying lead drug candidates against this protein, an Arl2 dependent PDE6D cargo ejection mechanism continues to hamper progress. We describe the development of Deltaflexin 1 and Deltaflexin 2, into which we engineered a ‘molecular spring’ to improve resilience to Arl2 ejection of PDE6D cargo. We show that these compounds selectively inhibit K-Ras membrane organization and exhibited K-Ras selective anti-proliferative effects against cancer cell lines from colon and breast tissues whilst blocking the 3D spheroid growth of lung and breast cancer cell lines. In the second part, I describe my main project, the identification of a novel covalent inhibitor of calmodulin (CaM) with anti-cancer activity in K-Ras mutated cancers named Calmirasone1. A relevance of the K-Ras/ CaM interaction for the promotion of cancer cell stemness has been previously suggested. We previously showed that the natural product Ophiobolin A (OphA) blocked K-Ras membrane organization in a CaM dependent manner and cancer cell spheroid formation. However, because of the broad toxicit