Mariah (Riah) Culpepper

Mariah, a freshman studying Biology at Duke University, characterized SpyTag/SpyCatcher technology and optimized conditions of a high-throughput screening assay that will be utilized in cancer drug discovery.

A new approach uses non-oncogenic mutations to identify targets that cause synthetic lethality when disrupted with tumor-specific dependency. The Broad Institute's Cancer Dependency Map identified protein A as a synthetic lethal target in drug-resistant cancer cells. We aim to identify small molecules that disrupt the A-B-C complex.

People at the Broad Institute truly radiate passion in their work. This passion is tangible in their efforts to be better scientists and to support future leaders. This constant encouragement of growth allowed me and my cohort to blossom in these nine weeks. The passion found in every lab reignited my love for learning and a need to be better, not only for my science but for myself. Words don’t do justice to how much the Broad Summer Research Program shaped me into becoming the scientist and person I once only dared to be. I am forever appreciative of every experience along the way. Small molecules are tested in various biochemical and biophysical assays to be later optimized for in vivo studies. A Time-resolved Fluorescence Energy Transfer (TR-FRET) assay was developed for high-throughput screening (HTS) for hit identification within the Broad Institute’s compound library. Protein A will only bind to protein C when bound to protein B. When the donor fluorophore on protein A and the acceptor fluorophore (ATTO647) on protein C interact within 5-10 nm, a fluorescent signal will indicate a full A-B-C complex assembly. When protein C is displaced by a small molecule, the signal will decrease. An ideal labeling strategy would need to bring the donor/acceptor pair close enough to signal, be small enough to minimize background fluorescence and be cost-effective for HTS.

Past commercially available labeling strategies did not meet these parameters, but SpyTag/SpyCatcher technology was made in-house to link protein C and the acceptor fluorophore. SpyTag/SpyCatcher covalently binds to protein C, increasing the signal-to-background ratio in the assay. The first goal of this assay optimization was to express, purify, and characterize the SpyTag/SpyCatcher technology. The second goal of this study was to optimize the labeling reaction of protein C-SpyTag-SpyCatcher and the ATTO647 acceptor dye for HTS.

Project: Optimizing HTS Assay with Integration of SpyTag/SpyCatcher Technology

Mentors: Vanessa Chirino & Cerise Raffier

PI: The Center for the Development of Therapeutics (CDoT)