i SYNTHESIS OF COMPOUNDS ENABLING THE DEVELOPMENT OF CHEMO-ID – A METHOD FOR DETERMINING PROTEIN-DRUG INTERACTIONS IN VIVO

Candidate: Abdullah Al-Ramadhan
Supervisor: Dr. Gagan Gupta and Dr. Russell Viirre

ABSTRACT

Chemo-ID is a novel proposed method for determining in vivo drug-protein interactions, thereby predicting drug side effects, and informing on mechanisms of action. Chemo-ID is based on the well-established method of Bio-ID in which a protein of interest (the bait protein) is fused to a biotin ligase enzyme called BirA. In a Bio-ID experiment, the protein interactome of the bait is identified through the proximity-dependent biotinylation catalyzed by the pendant BirA. In Chemo-ID, the bait protein is replaced with a bait small molecule (bait drug). In a live cell, this construct catalyzes the biotinylation of proteins that closely associate with the bait drug. We envision assembling the bait drug-BirA construct within a live cell via either genetic code expansion or halo-tag conjugation. In the genetic code expansion approach, the BirA protein is expressed in live cells bearing a strained alkyne click reaction partner. The bait drug, modified with an azide group, is then covalently appended via bioorthogonal strain-promoted azide-alkyne cycloaddition. In the halo-tag conjugation, the BirA protein is expressed as a fusion with a protein called HALO, which selectively forms a covalent linkage with the bait drug, modified with a chlorohexyl group. The realization of Chemo-ID requires the chemical synthesis of unnatural amino acids bearing a strained alkyne side chain for incorporation into BirA, as well as the modification of a bait drug with an appropriate tether to an azide or chlorohexyl group. To validate this method, early experiments employ bait drugs with well-studied protein interactions. Future work on this project will entail the exploration of a wider variety of bait drugs and click partners.