Allelic variation in tumors is a heavily investigated topic in cancer. Some mutants in oncogenic Ras appear more oncogenic than others, leading to an overrepresentation in the frequency of those mutant alleles. However, cancer biologists have not been able to derive the molecular mechanism of allelic variation. I would like to take a molecular approach to addressing this genetic question. K-Ras(G12C) is commonly mutated in 40% of K-Ras-driven non-small cell lung adenocarcinoma tumors, and yet K-Ras(G13C), a supposedly similar mutant, only represents 1% of that tumor population. By designing chemical probes for K-Ras, we would like to elucidate fundamental mechanisms of K-Ras as an oncogenic driver. I would like to investigate the biochemical and structural differences between K-Ras(G12C) and K-Ras(G13C) using chemical probes. I hypothesize that covalent docking is a viable strategy for developing covalent probes for targets previously labeled as chemically intractable such as small GTPases such as Ras. DOCKovalent is a molecular docking software developed at UCSF by the combined efforts of the Shoichet and Taunton groups.
Aim 1 involves testing DOCKovalent on K-Ras(G12C), a clinically relevant oncogene for which our group has amassed chemical and structural data. With the Shoichet lab, I have obtained preliminary data that suggests that DOCKovalent can be used to predict potent and specific small molecule probes for K-Ras(G12C). We will attempt to validate the covalent docking results using structural techniques such as X-ray crystallography and Hydrogen-Deuterium Exchange Mass Spectrometry.
Aim 2 covers the application of covalent docking to the novel and uncharacterized oncogene, K-Ras(G13C), which has no inhibitors to date. Here, I will use a multipronged approach including structure-based linker design, molecular docking screening, and fragment- based disulfide tethering to find new potent inhibitors for K-Ras(G13C). Preliminary data reveals that K- Ras(G13C) is reactive and can be labeled by covalent electrophiles.
Aim 3 involves investigating the mechanism of allelic variation in lung adenocarcinoma for K-Ras(G12C) and K-Ras(G13C). Biochemical investigations of similar mutant alleles of K-Ras have determined that K-Ras mutants differ slightly in their biochemical function and structural conformation. I will use functional assays coupled with X-ray crystallography to investigate if the same is true for K-Ras(G12C) and K-Ras(G13C). I will also use our small molecules to examine if comparable K-Ras(G12C) and K-Ras(G13C) lung adenocarcinoma cell lines have differential sensitivity to inhibition of downstream Ras-driven pathways. Completion of this project will elucidate the differences between these two important oncogenic mutants with the potential for future application to other K-Ras mutants. This project will furthermore validate the DOCKovalent method as a strategy for mutant- specific inhibitor development for small GTPases.
The GTPase Ras has remained an undruggable target for over 30 years, although it is implicated in 30% of all human cancers. Despite this prevalence, the high nucleotide affinity and lack of druggable pockets in Ras have been challenging for design of direct inhibitors until recently. Using DOCKovalent, a covalent docking software, we have identified a new covalent inhibitor of K-Ras(G12C), and we hope to apply this software to develop further mutant-specific inhibitors to study the K-Ras oncogene.
|Nnadi, Chimno I; Jenkins, Meredith L; Gentile, Daniel R et al. (2018) Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange. J Chem Inf Model 58:464-471|