Abstract
Prior to COVID-19, tuberculosis (TB) was the leading infectious killer in the world. In 2023, 10.8 million people were infected with TB and 1.25 million people died. TB is projected to return as the leading infectious killer this year. This remains a persistent problem due to rising drug-resistance rates. Currently, treatment success rates for multi-drug resistant (MDR) strains of TB are at a concerning 68%. Additionally, the US is not immune to the impact of TB as the largest outbreak in modern history was recently reported in Kansas City, Kansas earlier this year. Given these significant concerns, it is critical to discover new effective treatments for TB. Blocking mycobacterial cell wall biosynthesis is a clinically validated method as three of the four first-line anti-tubercular therapies target the cell wall. Peptidoglycan is an essential component of many bacteria for the cell wall. This project was designed to develop novel inhibitors of a peptidoglycan biosynthesis enzyme, the D-alanine: D-alanine ligase (Ddl), by using structure-based drug design. Ddl is the drug target for D-cycloserine, a second-line therapy option for TB. However, its clinical use is limited due to the severe central nervous system toxicity. This is due to its interactions with the N-methyl-D-aspartate (NMDA) receptor. Through in-depth analysis of Ddl and the NMDA receptor, new potential scaffolds for a novel inhibition mechanism have been designed by using previous kinetic and crystallography studies of the dead-end complex: Ddl-ATP-D-Ala-D-Ala.
After careful comparison of Ddl to close homolog kinase enzymes, we designed novel fragment-like compounds that mimic the previously reported dead-end complex with Ddl. A molecular model used for docking simulations was developed achieving 80% accuracy at confirming the top five binding compounds according to nanoDSF thermal unfolding experiments. While these compounds lacked anti-microbial activity, 870D, 800D, 860D, A7, and 820D were the top performing due to increase thermal shifts compared to the original dipeptide, D-alanyl-d-alanine. These likely mimicked the dead-end complex but with stronger binding interactions. Thus, further evaluation and expansion upon these fragments to develop these into potent small molecule drugs is warranted to develop a potential new class of anti-tubercular agents.