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Thirty three derivatives of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine analogues were synthesized by molecular modification of a reported antimycobacterial molecule (GSK163574A). Compounds were evaluated in vitro against actively replicative and nutrient starved non-replicative Mycobacterium tuberculosis (MTB), enzymatic screening and cytotoxicity against RAW 264.7 cell line. Among the compounds, 2-ethyl-N-phenethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (5c) was found to be the most active compound against non-replicative MTB with 2.7 log reduction of bacteria at 10μg/mL and was more potent than isoniazid (1.2 log reduction) and rifampicin (2.0 log reduction) at same dose level. Compound 5c also showed activity against MTB alanine dehydrogenase enzyme with IC 50 of 1.82±0.42μM and showed 25% cytotoxicity against RAW 264.7 cell line at 50μg/mL., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosisl-alanine dehydrogenase (MTB l-AlaDH) is one of the important drug targets for treating latent/persistent tuberculosis. In this study we used crystal structure of the MTB l-AlaDH bound with cofactor NAD(+) as a structural framework for virtual screening of our in-house database to identified new classes of l-AlaDH inhibitor. We identified azetidine-2,4-dicarboxamide derivative as one of the potent inhibitor with IC50 of 9.22±0.72μM. Further lead optimization by synthesis leads to compound 1-(isonicotinamido)-N(2),N(4)-bis(benzo[d]thiazol-2-yl)azetidine-2,4-dicarboxamide (18) with l-AlaDH IC50 of 3.83±0.12μM, 2.0log reduction in nutrient starved dormant MTB model and MIC of 11.81μM in actively replicative MTB., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the present study, we used crystal structure of MTB L-AlaDH protein complex with N6-methyl adenosine for structure based virtual screening of in house database to identify new small molecule inhibitors for MTB-L-AlaDH. Two molecules identified as better leads and were modified synthetically to obtain thirty novel analogues belonging to 2-iminothiazolidine-4-ones and 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamides. Among the screened compounds four (4n, 4o, 12 and 14) emerged as potent inhibitors displaying IC50 values ranging from 0.58 ± 0.02 to 1.74 ± 0.03 μM against MTB-L-AlaDH and were non-cytotoxic at 50 μM. Some of these synthesized compounds also exhibited good activity against nutrient starved dormant MTB cells. The most potent inhibitors were found to stabilize the protein which was confirmed biophysically through differential scanning fluorimetry., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Mycobacterium tuberculosis (MTB) the etiological agent of tuberculosis (TB) survives in the human host for decades evading the immune system in a latent or persistent state. The Rv2780 (ald) gene that codes for L-alanine dehydrogenase (L-AlaDH) enzyme catalyzes reversible oxidative deamination of L-alanine to pyruvate and is overexpressed under hypoxic and nutrient starvation conditions in MTB. At present, as there is no suitable drug available to treat dormant tuberculosis; it is essential to identify drug candidates that could potentially treat dormant TB. Availability of crystal structure of MTB L-AlaDH bound with co-factor NAD+ facilitated us to employ structure-based virtual screening approach to obtain new hits from a commercial library of Asinex database using energy-optimized pharmacophore modeling. The resulting pharmacophore consisted of three hydrogen bond donor sites (D) and two hydrogen bond acceptor sites (A). The database compounds with a fitness score more than 1.0 were further subjected to Glide high-throughput virtual screening and docking. Thus, we report the identification of best five hits based on structure-based design and their in vitro enzymatic inhibition studies revealed IC₅₀ values in the range of 35-80 μM., (Copyright © 2013 Elsevier Inc. All rights reserved.)