The substrate capture mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase provides a mode for inhibition.
Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5'-phosphoribosyl 1'-pyrophosphate (PRPP) and anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5-119 μM, with the strongest inhibition displayed by a bianthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme's activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action.
Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland , 3 Symonds Street, Auckland 1142, New Zealand.
Alina
A
Castell
Francesca L
FL
Short
Genevieve L
GL
Evans
Tammie V M
TV
Cookson
Esther M M
EM
Bulloch
Dmitri D A
DD
Joseph
Clare E
CE
Lee
Emily J
EJ
Parker
Edward N
EN
Baker
J Shaun
JS
Lott
D000877:Anthranilate Phosphoribosyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D000995:Antitubercular Agents / Q000494:pharmacology; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism
D000995:Antitubercular Agents; D004791:Enzyme Inhibitors; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D010754:Phosphoribosyl Pyrophosphate; D000877:Anthranilate Phosphoribosyltransferase