Abstract
Tuberculosis is a common, deadly, and contagious disease that is caused by the Gram positive bacterium Mycobacterium tuberculosis. In order to better treat TB, novel antibiotics with shorter treatment periods and improved activity against drug-resistant and latent TB infections are urgently needed. The redox cofactor menaquinone is an essential component of the electron transport chain in M. tuberculosis and enzymes in the biosynthetic pathway for this cofactor are promising targets for anti-TB drug discovery. 1,4-Dihydroxy-2-naphthoyl-CoA synthase (MenB), is the sixth enzyme in the menaquinone biosynthesis pathway, and catalyzes the formation of DHNA-CoA from o-succinylbenzoate-CoA via Claisen condensation. In order to develop novel inhibitors of MenB, a high-throughput screen was performed at Harvard Medical School and a series of 2-amino-4-aryl-4-oxo-butanoic acids were identified as a promising scaffold for subsequent structure-activity relationship studies. However, further research revealed that these compounds were not stable in aqueous buffer and degraded to (E)-4-aryl-4-oxo-but-2-enoic acids, which subsequently reacted with coenzyme A in situ to form the corresponding 4-aryl-2-CoA-4-oxo-butanoic acids adducts. These coenzyme A adducts are potent inhibitors of MenB, with Ki values as low as 49 nM. Further experiments have subsequently been performed to decrease the polarity and increase the in vitro antibacterial activity of the CoA adducts. Target validation experiments performed with S. aureus demonstrate that these compounds inhibit bacterial growth through a direct effect on menaquinone biosynthesis. In separate studies, a series of OSB-CoA analogues were rationally designed and successfully synthesized in order to probe the mechanism of the MenB catalyzed reaction. In particular, an OSB-N-CoA has been successfully used as a ligand for structural studies, leading to the first X-ray structure of MenB in which the entire active site can be visualized. A new MenB reaction mechanism has been proposed based on the structural data together with site-directed mutagenesis.
