Author ORCID Identifier


Date of Graduation


Document Type

Dissertation (PhD)

Program Affiliation

Cell and Regulatory Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Alemayehu A. Gorfe

Committee Member

John F. Hancock

Committee Member

John A. Putkey

Committee Member

Shane R. Cunha

Committee Member

Jeffrey T. Chang


RAS, one of the most well characterized membrane-associated small GTPases, is a notorious oncogene with >15% of all tumors harboring RAS mutations. When RAS is mutated it becomes constitutively active sending cell growth, survival and proliferation into overdrive, which subsequently leads to cancer. Although, RAS has been aggressively targeted with drug design efforts for more than 30 years an FDA approved direct inhibitor has not yet been developed. There are three isoforms of RAS in cells; HRAS, NRAS and KRAS. We focused on KRAS since it is the most frequently mutated isoform in cancer. To identify novel non-covalent small molecules that bind to mutant KRAS, we conducted a high-throughput virtual screen of drug-like compounds against a previously characterized allosteric pocket on a molecular dynamics simulation-derived KRASG12D structure. The in silico predicted hits were then validated with a battery of cell-based and biophysical assays. Specifically, the hits effects on KRAS signaling, binding affinities for KRAS and mechanisms of activity were evaluated. We found two key hit compounds (i) a pyrazolopyrimidine based molecule compound 11 and (ii) a indazole based molecule compound M1. Compound 11 exhibited a monotonous dose-dependent inhibition of KRAS signaling, however compound M1 demonstrated a biphasic dose-depended effect. With the potential to elucidate the structure-activity relationships between these molecules and their unique structures we tested both in cell-based and vi biophysical assays. We found that compound 11 binds to KRAS with nanomolar affinity and completely abolishes CRaf binding in vitro, subsequently leading to a significant reduction in RAS dependent CRaf/ERK activation and son of sevenless (SOS) mediated nucleotide exchange. Moreover, treatment at low micromolar concentrations of compound 11 reduced cell proliferation in six cancer cell lines. Further, compound M1, binds to KRAS in NMR-based studies and both reduced and enhanced signaling in cell-based assays as indicated by western blotting. We attributed this to M1 enhancing and disrupting nucleotide exchange through different mechanisms. Additionally, we noted that M1 showed remarkable selectivity towards inhibiting proliferation of MiaPaCa-2 cells. We then screened a second small-molecule library based on M1 and generated additional hits which decreased mutant KRAS signaling. Likewise, we generated two derivatives of compound 11 which were tested and gave insights to critical functional groups. Combining our results with detailed structural analysis, we are able to describe key ligand-receptor interactions that correlate with activity. Thus, showing that our screening techniques were very successful at generating KRAS binders that have effects on signaling in cells. To our knowledge compound 11 is the first known nanomolar binder of KRAS that disrupts interaction with CRaf resulting in decreased p-ERK levels and cell proliferation. Therefore, compound 11 is a promising hit for the development of novel non-covalent KRAS inhibitors.


KRAS, K-RAS, K-ras, drug design, molecular dynamics, biophysics, RAS inhibitor, KRAS inhibitor, small molecule



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