A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

Abstract

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes-primarily those involved in macromolecular synthesis-are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α-β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

Allosteric inhibitors of Mycobacterium tuberculosis tryptophan synthase

“…M. tuberculosis TrpA (α subunit) and TrpB (β subunit) were coexpressed from individual vectors in E. coli and recombinant proteins were purified and crystallized as described previously . Crystals of complexes were produced by soaking TrpAB with two previously published in vivo and in vitro inhibitors of Mt TrpAB (GSK1 and GSK2) .…”

“…Tryptophan synthase was studied extensively biochemically, structurally and computationally for the past 60 years, but most research focused on a model system from Salmonella typhimurium and orthologs from Escherichia coli and Pyrococcus furiosus . Only recently the field has expanded to include TrpAB from other organisms . In M. tuberculosis TrpAB research the major breakthrough involved developing recombinant coexpression system in E. coli , purification of fully active αββα complex and obtaining crystals that diffracted to high resolution and permitted detailed visualization of enzyme inhibitor interactions and insight into the catalytic mechanism .…”

“…This was specifically proposed for the allosteric sites. We have previously reported an inhibitor BRD4592 that selectively targets Mt TrpAB and binds to a new allosteric site with high affinity . Another group discovered and partly characterized compounds belonging to two distinct chemical classes.…”

“…Here we present crystal structures of Mt TrpAB with two inhibitors from that research, termed GSK1 and GSK2, at 2.4 Å resolution. As BRD4592, these compounds are strong inhibitors . The MICs for GSK1 and GSK2 were determined in M. tuberculosis H37Rv to be 0.76 and 1.1 μM, respectively, and for BRD4592 in various M. tuberculosis strains they are in the range of 1.6–3 μM .…”

The Molecular Industrial Revolution: Automated Synthesis of Small Molecules

“…[6] This is because this class of chemical matter can also perform many frontier functions that have been minimally utilized to date. These include modulating protein-protein interactions, [7] allosterically modifying protein function, [8] acting as prostheses on the molecular scale, [9] serving as next-generation biological probes, [10] enabling miniaturized diagnostics, [11] harvesting sunlight. [12] transducing energy, [13] emitting light, [14] initiating self-healing, [15] acting as molecular magnets, [16] acting as redox flow batteries, [17] enabling next generation computing, [18] and superconducting.…”