| N-1-(5-phosphoribosyl)-ATP transferase (ATP-PRTase/HisG) of Mycobacterium tuberculosis (M. tuberculosis) was cloned from genomic DNA with a 6-His tag at the N-terminus and overexpressed and crystallized in the presence or absence of the natural inhibitors, AMP and histidine in different conditions respectively. The crystal structures of ATP-PRTase were solved from electron density maps calculated by multiple-wavelength anomalous diffraction (MAD) methods. The structure has three distinct domains (I, II, and III). The active site is formed between the domain I and II, which is widely exposed to solvent. A large conformational change is induced by histidine binding on domain III. This allosteric inhibition mechanism seems to limit substrate accessibility to the active site by inducing a "closed" conformation of the inactive hexameric enzyme. In addition, this mechanism allows additional inter-subunit interactions to AMP, which is bound far from the histidine-binding site. This explains synergistic inhibition between the natural inhibitors and is consistent with previous biochemical evidences (Morton and Parsons 1977) that demonstrated enhanced inhibition in the presence of the both inhibitors.; Based on the identified active site of the structures solved, a high-throughput virtual screening was performed to discover potential inhibitors that might be novel antitubercular drugs through a lead optimization process. Screening of more than 600,000 small molecules was accomplished using the docking program GOLD in a dramatically short period of time via a grid-computing system (more than 300 times faster than a single MIPS R12000 CPU from Silicon Graphics) without pre-process filtering step. The results from the screening were rescored using consensus scoring method and were docked again by a different docking program FLEXX to improve hit rate. The drug-likeness rules developed by Christopher Lipinski were used to distinguish non-drug-like molecules from the hits. Ultimately, all the hits selected based on the docking and consensus scores as well as drug-likeness rules were scrutinized visually in the binding sites. All the hits were eliminated but thirteen compounds from Chembridge screening set, four of which exhibited inhibition of enzyme activity at 10 muM. IC 50 values of two of them were near 5 mum. (Abstract shortened by UMI.)... |
