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 toxicity of OphA, its suitability as a tool compound to further study this K-Ras/CaM associated stemness properties is limited. We have therefore characterized a set of benzazulenones with distant chemical similarity to OphA in a battery of assays. We identified Calmirasone1 which exhibits improved CaM affinity and a significantly lower unspecific toxicity relative to OphA. Furthermore, Calmirasone1 selectively blocked K-Ras membrane organization and inhibited the 3D spheroid growth of K-Ras dependent cancer cell lines.
In the third part of this thesis, I assessed the synergistic potential of targeting CaM and protein phosphatase 2A (PP2A) in Ras-MAPK dependent cancer cell lines. PP2A is a tumor suppressor that catalyze the dephosphorylation of multiple targets in the cell. Using specific CaM inhibitors and PP2A agonists as well as the already clinically approved phenothiazines (PTZs), our results from this study suggests that synergistic targeting of CaM and PP2A improves anti-cancer effects and that PTZs combine CaM inhibitory and PP2A re-activating properties in their cancer killing activity.