A multifaceted approach to identify non-specific enzyme inhibition: Application to Mycobacterium tuberculosis shikimate kinase

Abstract

Single dose high-throughput screening (HTS) followed by dose-response evaluations is a common strategy for the identification of initial hits for further development. Early identification and exclusion of false positives is a cost-saving and essential step in early drug discovery. One of the mechanisms of false positive compounds is the formation of aggregates in assays. This study evaluates the mechanism(s) of inhibition of a set of 14 compounds identified previously as actives in Mycobacterium tuberculosis (Mt) cell culture screening and in vitro actives in Mt shikimate kinase (MtSK) assay. Aggregation of hit compounds was characterized using multiple experimental methods, LC-MS, HNMR, dynamic light scattering (DLS), transmission electron microscopy (TEM), and visual inspection after centrifugation for orthogonal confirmation. Our results suggest that the investigated compounds containing oxadiazole-amide and aminobenzothiazole moieties are false positive hits and non-specific inhibitors of MtSK through aggregate formation.

Mycobacterium tuberculosis Shikimate Pathway Enzymes as Targets for the Rational Design of Anti-Tuberculosis Drugs

“…Recently, six new compounds were characterized as MtSK inhibitors-manzamine A, 8-hydroxymanzamine A, manzamine E, manzamine F, 6-deoxymanzamine X and 6-cyclohexamidomanzamine A. Inhibition studies demonstrated that these molecules show a mixed noncompetitive type of inhibition [127]. Another study showed a collection of 14 hits (out of 404 compounds) containing oxadiazole-amide and aminobenzothiazole scaffolds, which displayed >90% inhibition at concentrations below 50 μM against MtSK [123,128] Experimental and computational studies have shown that catalysis may occur under specific conditions. The LID and SB domains are tightly closed presenting two possible arrangement: (a) the guanidinium group of the essential arginine, located in the LID domain, is close of the γ-phosphate of ATP for its activation and (b) the two substrates are very close together but isolated from the solvent environment for the reaction.…”

“…Recently, six new compounds were characterized as MtSK inhibitors-manzamine A, 8-hydroxymanzamine A, manzamine E, manzamine F, 6-deoxymanzamine X and 6-cyclohexamidomanzamine A. Inhibition studies demonstrated that these molecules show a mixed noncompetitive type of inhibition [127]. Another study showed a collection of 14 hits (out of 404 compounds) containing oxadiazole-amide and aminobenzothiazole scaffolds, which displayed >90% inhibition at concentrations below 50 µM against MtSK [123,128]. Compounds with aromatic modifications at the C5 hydroxyl group of shikimic acid present different inhibition constants against the MtSK and H. pylori SK (HpSK).…”

Production and Synthetic Modifications of Shikimic Acid

“…The several reported crystal structures of this enzyme, either as binary complexes with ATP, ADP, or shikimic acid in the active site, or as ternary complexes, have allowed the structure-base design of inhibitors. Although several classes of inhibitors have been reported, − only substrate mimetics will be herein considered due to their structural similarities with shikimic acid.…”

Theoretical characterization of the shikimate 5-dehydrogenase reaction from Mycobacterium tuberculosis by hybrid QC/MM simulations and quantum chemical descriptors

“…This bacterium is the causative agent of tuberculosis which, according to the World Health Organization, is the most fatal infectious disease with over one million deaths per year [28]. Gene disruption experiments have demonstrated that the SKM pathway is essential for the viability of M. tuberculosis [29] and, as a result, substantial efforts have been made to develop inhibitors against its constituent enzymes, including 3-dehydroquinate dehydratase [34], shikimate kinase [2,24,43], and EPSP synthase [40].…”