Discovery of Novel Inhibitors for Mycobacterium tuberculosis D-alanine: D-alanine Ligase through Virtual Screening

Tuberculosis (TB), although not as common in high-income countries due to vaccines and antibiotic therapy, remains one of the highest causes of death in low-income countries. However, overuse of antibiotics has led to the emergence of antibiotic-resistant strains of the disease. To offset these issues, potential small molecule drug compounds must be identified to combat this bacteria in order to further the drug development process. Tuberculosis can be caused by a variety of other strains of the bacterium, but Mycobacterium tuberculosis is the most common and the most virulent. The D-alanine:D-alanine ligase (Ddl) enzyme is an attractive target for drug discovery as it catalyzes a crucial reaction that is necessary for the survival of the bacteria: the formation of the D-alanyl-D-alanine dipeptide from adenosine triphosphate (ATP) and two D-alanine amino acids. This dipeptide complex then plays a pivotal role in crosslinking peptidoglycan chains during the assembly of the bacterial cell wall. Drugs that target Ddl currently exist on the market, but antibiotic resistance has reduced their efficacy. A crystallography structure of Ddl from M. tuberculosis(PDB ID: 3LWB) was used in conjunction with the virtual screening program GOLD (Genetic Optimization for Ligand Docking by the Cambridge Crystallographic Data Center) and ICM (Internal Coordinate Mechanics by Molsoft) to search for ligands that had a predicted strong binding affinity to the enzyme's active site. Both the D-alanine and the ATP sites were targeted in this study. Various ligand libraries were screened and ranked based on binding interactions with the active site. The docking poses of the top-ranking ligands were further verified through the molecular visualization program PyMol (Schrodinger). The highest-ranking ligand targeting the ATP site from GOLD, SAM001246657, came from the National Institutes of Health's Clinical Collection library (NIH) and received an overall fitness score of 104.4. The highest-ranking compound from the ICM screen targeting the ATP site was SPB06710SC from the HitFinder 9 library and received a score of -36.32. For the D-alanine site, the highest docking ligand from GOLD was ZINC14539627 with a score of 100.77. From ICM, the highest scoring ligand was ZINC11535505 with a score of -32.27. Both ligands are from the Zinc ChemBridge library. The inhibitory effects of these top-scoring ligands may be further validated through wet-lab research using binding assays and enzyme inhibition assays. © FASEB.