Protein function modifications for therapeutic purposes are typically accomplished with small molecule drugs that bind in deep hydrophobic pockets of proteins. For protein-protein interactions occurring over an extended shallow area, it is challenging to have small molecules binding to the surface area and thereby affecting protein functions. Ras -Sos is a classic example of extensive protein-protein surface interaction, and therefore it remains challenging to inhibit KRas- Sos interactions. KRas is activated by Sos, and it is critical to develop technologies to inhibit interactions of oncogenic mutant KRas with Sos selectively. One such promising technology is the Chemical Epitope Targeting technology, developed for designing peptide ligands with high affinity and specificity against specific regions of an intracellular protein that may be inaccessible to small molecules or antibodies. This technology involves using proximity-catalyzed reaction for screening a specific region of the target protein to isolate peptide ligands. The PI proposes to streamline this technology to make it more accessible and user-friendly, and then to tailor this technology to successfully target mutant KRas(G12V) protein, specifically at the Ras-Sos interface. The technology will be streamlined by developing and characterizing synthetic macrocyclic one-bead-one compound (OBOC) peptide libraries that can be cleaved by one-step light exposure for seamless sequencing by tandem Mass Spectrometry (MS/MS) (Aim 1). Inverse Electron Demand Diels-Alder (IEDDA) reaction between tetrazine and alkene will be used for the screening process rather than the currently used azide-alkyne cycloaddition. The PI shall demonstrate, as a proof-of-concept, that IEDDA reaction between substituted tetrazine and an alkene can be proximity-catalyzed by a protein. KRas(G12C) or KRas mutants with one Cys will be modified at Cys with tetrazine. The labeling of the complex with an alkene containing Sos-helix peptide, known to bind at the Ras- Sos interface, will be monitored using tandem Mass Spectrometry (Aim 2). The optimal temperature for minimal background IEDDA reaction between tetrazine and alkene will be identified, to minimize background for the screen in the next step. A Chemical Epitope Targeting screen against the Chemical Epitope, KRas(G12V) complexed to a GDP- alkene small molecule, will be performed using S,S-tetrazine cyclized OBOC peptide libraries (Aim 3). Screening for inhibition of KRas(G12V)-Sos interaction using a split luciferase platform will follow.
Aims 1 and 2 should be achieved, and Aim 3 be initiated within the grant period. This project, being multidisciplinary, will allow the PI to train undergraduate and graduate students at Clark University in a variety of chemical and biochemical techniques.

Public Health Relevance

We are developing a technology that allows one to isolate cyclic peptide binders to specific regions of proteins. Our long- term objective is to isolate cyclic peptides that inhibit the interaction of oncogenic protein KRas(G12V) with Sos selectively. Such peptides can be used as tools in chemical biology and can serve as pre-drug leads.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
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Synthetic and Biological Chemistry A Study Section (SBCA)
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Fabian, Miles
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Clark University (Worcester, MA)
Schools of Arts and Sciences
United States
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