Abstract 1131 Structure-Based Virtual Screening for novel Allosteric Inhibitors of 3-Oxoacyl ACP Reductase (FabG) in Plasmodium falciparum

This is the authors' abstract. We don't add key points for this paper.

Malaria is a devastating disease, affecting hundreds of millions of individuals and causing hundreds of thousands of deaths annually, particularly in populations that are marginalized with little access to quality healthcare. Recent growing resistance to antimalarial therapies necessitates the discovery of novel pharmaceutical compounds. Plasmodium falciparum, the malaria parasite, employs the type II Fatty Acid Biosynthesis pathway, which is essential for its survivability and not present in humans. Enzymes participating in this pathway are therefore appealing drug targets due to the selectivity. One such enzyme, 3-Oxoacyl-ACP Reductase (FabG), catalyzes the reaction between NADPH and 3-Oxoacyl-Acyl-Carrier-Protein, the first step in fatty acid elongation. There are no current clinically approved pharmaceuticals that target FabG in this organism, leaving it a prime target for novel inhibitors. Furthermore, we targeted a lesser known conserved cryptic allosteric binding site between two FabG monomers, as identified by Cukier et. al. Small molecules binding to this site would induce conformational changes across the protein, displacing key residues and making it impossible for NADPH to bind. In order to identify potential small molecule inhibitors, virtual screening was conducted using the ligand docking software GOLD against a homology model of PfFabG (template PDB: 4BNT) at the allosteric binding site. A control docking against fragments that docked well into the allosteric site of Pseudomonas aeruginosa FabG, as well as randomly chosen controls, revealed that hydrophobic interactions were the main determinant of ligand binding, as opposed to hydrogen bonding, as also observed by Cukier et. al. When screened against a subset from the ChemBridge Diversity library, several compounds displayed promising scores, including ZINC__12 (Score: 104.11) and ZINC__14 (Score: 104.03). Notably, these scores were higher than for the known positive control ligand (PDB identifier 36I) from the crystal structure of the homologous protein (PDB identifier 4BNY): (Score: 76.22). To validate these potential hit compounds, the coding DNA sequence of PfFabG was expressed via autoinduction with lactose as the inducer in recombinant Bl21 E. coli bacteria and purified by Ni-NTA column chromatography with a 6xHIS tag. The identity of the protein was confirmed with an SDS-PAGE gel, which resulted in a clear 33 kDa band. The melting temperature of the protein was determined through Differential Scanning Fluorimetry (DSF). Through the computational results, we have identified ZINC__12, ZINC__14, and other compounds as possible novel inhibitor candidates against PfFabG, which we will further validate in vitro with enzymatic assays against the purified and characterized PfFabG protein. Ultimately, they may serve as candidates for further antimalarial research and drug development.