Benzothiazole Derivative as a Novel Mycobacterium tuberculosis Shikimate Kinase Inhibitor: Identification and Elucidation of Its Allosteric Mode of Inhibition.

Mycobacterium tuberculosis shikimate kinase (Mtb-SK) is a key enzyme involved in the biosynthesis of aromatic amino acids through the shikimate pathway. Since it is proven to be essential for the survival of the microbe and is absent from mammals, it is a promising target for anti-TB drug discovery. In this study, a combined approach of in silico similarity search and pharmacophore building using already reported inhibitors was used to screen a procured library of 20,000 compounds of the commercially available ChemBridge database. From the in silico screening, 15 hits were identified, and these hits were evaluated in vitro for Mtb-SK enzyme inhibition. Two compounds presented significant enzyme inhibition with IC50 values of 10.69 ± 0.9 and 46.22 ± 1.2 µM. The best hit was then evaluated for the in vitro mode of inhibition where it came out to be an uncompetitive and noncompetitive inhibitor with respect to shikimate (SKM) and ATP, respectively, suggesting its binding at an allosteric site. Potential binding sites of Mtb-SK were identified which confirmed the presence of an allosteric binding pocket apart from the ATP and SKM binding sites. The docking simulations were performed at this pocket in order to find the mode of binding of the best hit in the presence of substrates and the products of the enzymatic reaction. Molecular dynamics (MD) simulations elucidated the probability of inhibitor binding at the allosteric site in the presence of ADP and shikimate-3-phosphate (S-3-P), that is, after the formation of products of the reaction. The inhibitor binding may prevent the release of the product from Mtb-SK, thereby inhibiting its activity. The binding stability and the key residue interactions of the inhibitor to this product complex were also revealed by the MD simulations. Residues ARG43, ILE45, and PHE57 were identified as crucial that were involved in interactions with the best hit. This is the first report of an allosteric binding site of Mtb-SK, which could largely address the selectivity issue associated with kinase inhibitors.

Identification and characterization of novel small molecule inhibitors of the acetyltransferase activity of Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU).

This study aims at identifying novel chemical scaffolds as inhibitors specific to the acetyltransferase domain of a bifunctional enzyme, Escherichia coli GlmU, involved in the cell wall biosynthesis of Gram-negative organisms. A two-pronged approach was used to screen a 50,000 small-molecule library. Using the first approach, the library was in silico screened by docking the library against acetyltransferase domain of E. coli GlmU studies. In the second approach, complete library was screened against Escherichia coli ATCC 25922 to identify the whole cell active compounds. Active compounds from both the screens were screened in a colorimetric absorbance-based assay to identify inhibitors of acetyltransferase domain of E. coli GlmU which resulted in the identification of 1 inhibitor out of 56 hits identified by in silico screening and 4 inhibitors out of 35 whole cell active compounds on Gram-negative bacteria with the most potent inhibitor showing IC50 of 1.40 ± 0.69 µM. Mode of inhibition studies revealed these inhibitors to be competitive with AcCoA and uncompetitive with GlcN-1-P. These selected inhibitors were also tested for their antibacterial and cytotoxic activities. Compounds 5175178 and 5215319 exhibited antibacterial activity that co-related with GlmU inhibition. These compounds, therefore, represent novel chemical scaffolds targeting acetyltransferase activity of E. coli GlmU.

Discovery of new Mycobacterium tuberculosis proteasome inhibitors using a knowledge-based computational screening approach.

Mycobacterium tuberculosis bacteria cause deadly infections in patients [Corrected]. The rise of multidrug resistance associated with tuberculosis further makes the situation worse in treating the disease. M. tuberculosis proteasome is necessary for the pathogenesis of the bacterium validated as an anti-tubercular target, thus making it an attractive enzyme for designing Mtb inhibitors. In this study, a computational screening approach was applied to identify new proteasome inhibitor candidates from a library of 50,000 compounds. This chemical library was procured from the ChemBridge (20,000 compounds) and the ChemDiv (30,000 compounds) databases. After a detailed analysis of the computational screening results, 50 in silico hits were retrieved and tested in vitro finding 15 compounds with IC50 values ranging from 35.32 to 64.15 µM on lysate. A structural analysis of these hits revealed that 14 of these compounds probably have non-covalent mode of binding to the target and have not reported for anti-tubercular or anti-proteasome activity. The binding interactions of all the 14 protein-inhibitor complexes were analyzed using molecular docking studies. Further, molecular dynamics simulations of the protein in complex with the two most promising hits were carried out so as to identify the key interactions and validate the structural stability.

Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus.

Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC50 of 44.24 ± 1.85 µM. Mode of inhibition studies revealed that terreic acid was competitive with AcCoA and uncompetitive with GlcN-1-P. It also exhibited concentration-dependent killing of E. coli ATCC 25922 up to 4× minimum inhibitory concentration and inhibited the growth of biofilms generated by E. coli. Characterization of resistant mutants established mutation in the acetyltransferase domain of GlmU. Terreic acid was also found to be metabolically stable in the in vitro incubations with rat liver microsome in the presence of a NADPH regenerating system. The studies reported here suggest that terreic acid is a potent antimicrobial agent and support that E. coli GlmU acetyltransferase is a molecular target of terreic acid, resulting in its antibacterial activity.

High-throughput screen identifies small molecule inhibitors targeting acetyltransferase activity of Mycobacterium tuberculosis GlmU.

N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a pivotal bifunctional enzyme, its N and C terminal domains catalyzes uridyltransferase and acetyltransferase activities, respectively. Final product of GlmU catalyzed reaction, uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), acts as sugar donor providing GlcNAc residues in the synthesis of peptidoglycan and a disaccharide linker (D-N-GlcNAc-1-rhamnose), the key structural components of Mycobacterium tuberculosis (M. tuberculosis) cell wall. In the present study, we have searched new inhibitors against acetyltransferase activity of M. tuberculosis GlmU. A subset of 1607 synthetic compounds, selected through dual approach i.e., in-silico and whole cell screen against 20,000 compounds from ChemBridge library, was further screened using an in-vitro high throughput bioassay to identify inhibitors of acetyltransferase domain of M. tuberculosis GlmU. Four compounds were found to inhibit GlmU enzyme specific to acetyltransferase activity, with IC50 values ranging from 9 to 70 µM. Two compounds (6624116, 5655606) also exhibited whole cell activity against drug susceptible as well as drug resistant M. tuberculosis. These two compounds also exhibited increased anti-TB activity when tested in combination with rifampicin, isoniazid and ethambutol, however 5655606 was cytotoxic to eukaryotic cell line. These results demonstrate that identified chemical scaffolds can be used as inhibitors of M. tuberculosis cell wall enzyme after optimizations for future anti-TB drug development program.

Synthesis and Biological Evaluation of Polar Functionalities Containing Nitrodihydroimidazooxazoles as Anti-TB Agents.

Novel polar functionalities containing 6-nitro-2,3-dihydroimidazooxazole (NHIO) analogues were synthesized to produce a compound with enhanced solubility. Polar functionalities including sulfonyl, uridyl, and thiouridyl-bearing NHIO analogues were synthesized and evaluated against Mycobacterium tuberculosis (MTB) H37Rv. The aqueous solubility of compounds with MIC values ≤0.5 µg/mL were tested, and six compounds showed enhanced aqueous solubility. The best six compounds were further tested against resistant (Rif(R) and MDR) and dormant strains of MTB and tested for cytotoxicity in HepG2 cell line. Based on its overall in vitro characteristics and solubility profile, compound 6d was further shown to possess high microsomal stability, solubility under all tested biological conditions (PBS, SGF and SIF), and favorable oral in vivo pharmacokinetics and in vivo efficacy.