ABSTRACT
KRAS is the most frequently mutated oncogene with mutation rates of 95% in pancreatic ductal adenocarcinoma
(PDAC), 45% in colorectal cancer, and 30% in lung adenocarcinomas. The most common K-RAS mutations
occur at codon 12, namely G12D, G12V, G12C, and G12R. K-RAS was considered undruggable due to lack of
well-defined drug-binding pockets. But a recent breakthrough was achieved with covalent inhibitors that form a
bond with K-RAS G12C cysteine. Several of these compounds are in clinical trials, and one was given FastTrack
status by the FDA. Unfortunately, only a small fraction of K-RAS oncogene mutants harbor the G12C mutation,
and G12D, G12V and other mutations do not provide an accessible cysteine nucleophile. In recent work with the
RAS GTPase Ral we showed through high-resolution structures and extensive biochemical studies that covalent
bond formation with Tyr-82 created a well-defined novel binding site located between the Switch II and the Switch
I/II pockets. Additional fragment screening carried out more recently identified a fragment that forms a covalent
bond at K-RAS Switch II Tyr-64 to inhibit activation of the GTPase by the Son-of-Sevenless (SOS) guanine
exchange factor. Here, we hypothesize that covalent bond formation with tyrosine or other amino acids on K-
RAS will inhibit activation or effector binding and block K-RAS signaling in cancer cells. Our preliminary data and
extensive experience in the field puts us in a strong position to accomplish our objectives. In Specific Aim 1, we
employ ligand- and structure-based methods to generate fragment electrophile libraries from large commercial
collections, and we follow a structure-based method to grow hit fragments into neighboring pockets to enhance
their binding affinity and reaction rates. In Specific Aim 2, we will carry out well-established intact protein mass
spectrometry, nucleotide exchange, and effector binding studies to screen fragment libraries for hit compounds,
and to characterize small molecules that emerge from fragment growing strategies. In Specific Aim 3, we will
use X-ray crystallography to solve the structure of hit fragments and derivatives that emerge from fragment
growing efforts. We also carry out cell biological studies to confirm direct engagement of K-RAS, inhibition of K-
RAS signaling, and inhibition of cancer cell proliferation. We expect to identify high quality fragments and small
molecules that form a covalent bond at wild-type and mutant K-RAS oncogenes, inhibit K-RAS wild-type or
oncogene mutant activity in cancer cell lines, and inhibit PDAC and lung adenocarcinoma cancer cell viability.
These compounds will serve as starting points to pursue in lead optimization efforts towards the development of
therapeutic agents for the treatment of K-RAS-driven tumors.