In clinical oncology the new paradigm is the introduction of the molecular targeted therapies exploiting the characteristic genetic alterations of the tumors which is frequently mutation of oncogenes. However, the efficacy of targeted therapies (i.e. anti-EGFR agents) is critically determined by the KRAS mutational status of the tumor, as mutated KRAS leads to resistance to these expensive therapies. Therefore, the mutant oncogenic KRAS in human cancer emerged as arguably the most outstanding issue in clinical oncology that should guide the future of R&D activities.
Nowadays, the successful treatment of cancer remains a great challenge. The molecular targeted therapy promise to lead to clinically useful new procedures. Within this large objective, the present project is associated with oncogenic mutations of KRAS proteins focused on cancer. However, the RAS protein and its KRAS isoform are essential players in signal transduction where some of its point mutations lead to oncogenic changes.
The project involves three universities (BME, Semmelweis University and ELTE) and two innovative companies (KINETO Lab and Fototronic) through the successful cooperation and achieved a promising results. Among these, it should be noted that we developed wild-type and mutant KRAS production and purification of protein constructs at a scale sufficient for the work planned in the project ensuring the production of protein in quantity and functionally active quality. With our protein structure studies we established the binding parameters of several candidate inhibitor molecules and characterized the binding conditions.
We designed promising ligands, the allele-specific KRAS inhibitory effect of which we began to investigate. We found that several new compounds can bind covalently to the G12C KRAS mutant through panel of assays. We have developed high-throughput binding and thiol assays, as well as KRAS functional assay. We have developed in vitro and in vivo model systems for successful toxicological and efficacy studies. With in silico docking simulations, we identified a numerous promising allele-specific inhibitor candidate for KRAS G12C, G12D G12V variants. On this for the rational examination of compounds, we developed a complex screening protocol from in vitro systems to animal models.
The reconstituted purified protein-ligand complexes were subjected to numerous biochemical, biophysical and their investigation using the enzyme kinetic method. Interaction of promising ligand molecules with KRAS mutants analyzed by multidimensional NMR studies and X-ray crystallographic studies.