Substrate capture mechanism provides a mode for inhibition

Abstract

Mycobacteria tuberculosis (Mtb), the causative agent of tuberculosis, is responsible for more death in the world today than any other bacteria. As part of the Tuberculosis Structural Genomics Consortium (TBSGC), our research group previously determined the structure of anthranilate phosphoribosyl transferase (AnPRT) from Mtb. AnPRT is the second enzyme in the tryptophan biosynthetic pathway and was identified as a potential drug target through gene knockout experiments, which resulted in a strain of Mtb that was essentially avirulent even in immunodeficient mice. AnPRT catalyses a reaction between anthranilate and phosphoribosylpyrophosphate (PRPP), and the crystal structure of Mtb-AnPRT was originally determined with and without PRPP (PDB ID: 1ZVW and 2BPQ, respectively). In silico docking was used to predict the binding motif of anthranilate, the second substrate, surprisingly predicted two sites despite a 1:1 reaction ratio with PRPP. Previously, 165 compounds were screened for inhibitory action against Mtb-AnPRT. The most potent of these compounds was co-crystallized with Mtb-AnPRT and PRPP. One compound had a bianthranilate character and the 2.0 Å resolution structure of this inhibitor bound to Mtb-AnPRT (PDB ID: 3QQS) was determined by molecular replacement using the Mtb-AnPRT structure without PRPP bound (PDB ID: 1ZVW) as a search model. Interestingly, the structure revealed multiple binding motifs for the inhibitor, two of which were consistent with the previously predicted binding motifs for anthranilate. Forty analogues of this potent Mtb-AnPRT inhibitor were subsequently assayed for activity against the enzyme, several of which showed were found to be more potent inhibitors. This new series of inhibitors were docked into the 3QQS structure, providing insights for the development of more potent inhibitors. Such techniques will continue to drive design of increasingly potent inhibitors against Mtb-AnPRT for future development of a new anti-tuberculosis agent.