Toward the Discovery of Small Molecules that Target Adenosine 5'- Phosphosulfate Reductase.

Abstract

Adenosine-5’-phosphosulfate reductase (APR) is an iron-sulfur protein that catalyzes the first committed step in the de novo biosynthesis of cysteine in mycobacteria. APR is a validated target for the development of new antitubercular agents, particularly for the treatment of latent infection. The goal of this research is to develop small-molecule inhibitors of APR that serve as tools for biological discovery or as leads for drug discovery. Toward this end, we have combined virtual ligand screening (VLS), rational structure-based design, and enzyme mechanism analysis. Through VLS and experimental testing, we have identified the first nonphosphate-based inhibitors of APR and discovered an additional ligand-binding site, which could be exploited for the design of bifunctional small-molecules. To facilitate the development of potent and specific inhibitors of APR, we have also probed the molecular determinants that underlie binding and specificity via a series of substrate and product analogs. Our findings reveal a critical role for the α-phosphate group and provide evidence for ligand-specific conformational states within the C-terminal domain. In addition, we demonstrate that a conserved histidine within the flexible segment plays an essential role in substrate binding and in closure of the active site lid. Subsequently, a structure-based approach was utilized in the design and synthesis of an irreversible cysteine-targeted inhibitor of APR, which locks the enzyme in its closed, inactive state. Finally, we have investigated the function of the iron-sulfur cluster in APR and provide kinetic evidence that the cofactor plays an essential role in substrate specificity of sulfonucleotide reductases. Collectively, these data further our understanding of the APR reaction mechanism and pave the way for development of new inhibitors to target this therapeutically important class of enzymes.