A Second, Druggable Binding Site in UDP‐Galactopyranose Mutase from Mycobacterium tuberculosis?

Abstract

UDP-galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non-substrate-like inhibitor, MS-208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS-208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS-208 binds at an allosteric binding site (A-site) instead of the enzyme active site (S-site). A candidate A-site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A-site. Further molecular dynamics studies led us to propose that MS-208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A-site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.

Natural and Synthetic Flavonoids as Potent Mycobacterium tuberculosis UGM Inhibitors

“…These two different resultss uggest two possible behaviors of 4 as aU GM inhibitor.E ither it acts as ac ompetitive inhibitor ( 29) > 1000 > 100 7ombuin (31) > 1000 > 100…”

“…This potential allosterics ite has recently been study by Pinto et al [31] They described, through docking calculations, saturation transfer difference (STD) NMR spectroscopy,a nd mutagenesis, the potentialb inding of am ixed inhibitori nt his second pocket of the opened form of Mt UGM. It has therefore been proposed that binding of this allosterics ite would induce am ajor structuralm odification of the substrate actives ite, preventinge nzyme turnover ( Figure 5).…”

Conformational Control of UDP-Galactopyranose Mutase Inhibition

“…Specifically, a small-molecule heterocycle (MS-208) that differs in structure from the inhibitors described herein was reported to inhibit MtUGM ( K i ∼400 μM). 32 The kinetics of inhibition together with computational and mutagenesis studies suggested that MS-208 does not compete directly with the substrate UDP-Gal p but rather binds in an allosteric site. The presence of multiple subsites highlights the flexibility of UGM.…”

“…Inhibitor trapping of UGM in an inactive conformation could be an effective inhibition strategy. 32 Indeed, many kinase inhibitors bind to and stabilize an inactive conformation of their target enzymes 53-56 . Our data thus far suggest inhibitors that can occupy the closed, oxidized state of the enzyme might be especially effective.…”

“…30 Studies of the M. tuberculosis UGM with a non-substrate inhibitor suggest there is an allosteric binding pocket near the active site and that occupation of this pocket prevents loop closure. 32 Eukaryotic UGMs also adopt multiple conformations, and they have a second mobile flap containing an asparagine residue involved in substrate recognition. 33, 34 Additionally, molecular dynamics studies indicate a third mobile loop near the active site in the Trypanosoma cruzi UGM (TcUGM).…”