Lead identification and optimization of bacterial glutamate racemase inhibitors.

Mycobacterium tuberculosis glutamate racemase is an essential enzyme involved in peptidoglycan synthesis and conserved in most bacteria. Small molecule inhibitors were reported on other bacterial species whereas in M. tuberculosis it wasn't explored much. In this study we have screened in house compound library using fluorescence thermal shift assay and enzyme inhibition assay, form this (1-(3-(benzo[d]thiazol-2-yl)phenyl)-3-(p-tolyl)thiourea) was identified as lead compound with IC50 19.47 ±â€¯0.81 µM. Further lead optimization by synthesis resulted in twenty-three compounds, of which Compound 25 has shown more efficacy compared to lead 1 showing non-competitive mode of inhibition with IC50 1.32 ±â€¯0.43 µM. It also showed significant activity (represented in log reduction) in nutrient starved dormant M. tuberculosis model (2.1), M. tuberculosis biofilm assay (2.0) and in vivo M. marinum infected zebrafish model (3.5).

Crosstalk between CD4 T cells and synovial fibroblasts from human arthritic joints promotes hyaluronan-dependent leukocyte adhesion and inflammatory cytokine expression in vitro.

The content and organization of hyaluronan (HA) in the extracellular matrix (ECM) have been identified as strong indicators of inflammation in joint disease, although the source and role of HA as an effector of inflammation is not clear. In this study, we established co-cultures of activated human CD4 T cells with fibroblast-like synoviocytes (FLS) from osteoarthritis (OA) and rheumatoid arthritis (RA) subjects and examined the role of HA in promoting inflammatory events. Co-cultures of RA FLS with activated CD4 T cells generated an HA-enriched ECM that promoted enhanced monocyte adhesion compared to co-cultures of OA FLS with activated CD4 T cells. In addition, both OA FLS and RA FLS co-cultures with activated CD4 T cells elicited significant increases in the expression of IL1ß, TNF, and IL6, with the increase in IL6 expression most prominent in RA co-cultures. Blocking HA synthesis and accumulation with 4-methylumbelliferone reduced expression of IL6, IL1ß, and TNF in both OA FLS and RA FLS co-cultures. The increase in HA synthesis in the co-cultures was mimicked by IL6 trans-signaling of FLS in the absence of CD4 T cells. Inhibition of HA synthesis blocked the increase in IL6 by RA FLS mediated by IL6 trans-signaling, suggesting that the HA synthetic pathway may be a key mediator in IL6 expression by FLS. Overall, our study indicates that HA-enriched ECM generated by co-cultures of activated CD4 T cells with FLS from human joints creates a pathogenic microenvironment by promoting adhesion of leukocytes and expression of inflammatory cytokines including IL6.

Identification and development of novel indazole derivatives as potent bacterial peptidoglycan synthesis inhibitors.

Background: Tuberculosis is well-known airborne disease caused by Mycobacterium tuberculosis. Available treatment regimen was unsuccessful in eradicating the deaths caused by the disease worldwide. Owing to the drawbacks such as prolonged treatment period, side effects, and drug tolerance, there resulted in patient noncompliance. In the current study, we attempted to develop inhibitors against unexplored key target glutamate racemase. Methods: Lead identification was done using thermal shift assay from in-house library; inhibitors were developed by lead derivatization technique and evaluated using various biological assays. Results: In indazole series, compounds 11 (6.32 ± 0.35 µM) and 22 (6.11 ± 0.51 µM) were found to be most promising potent inhibitors among all. These compounds also showed their inhibition on replicating and nonreplicating bacteria. Conclusion: We have developed the novel inhibitors against M. tuberculosis capable of inhibiting active and dormant bacteria, further optimization of inhibitor derivatives can results in better compounds for eradicating tuberculosis.

Identification and development of benzoxazole derivatives as novel bacterial glutamate racemase inhibitors.

In the present study, we attempted to develop novel class of Mycobacterium tuberculosis (Mtb) inhibitors by exploring the pharmaceutically underexploited enzyme targets which are majorly involved in cell wall biosynthesis of mycobacteria. For this purpose glutamate racemase was selected which racemizes d-glutamate from l-glutamate, a key step in peptidoglycan synthesis. Furthermore, enzyme is neither expressed nor its product, d-glutamate is produced in mammals, and hence inhibiting this enzyme will have no vulnerable effect in host organism. A library of our in-house compounds were screened against glutamate racemase using a biophysical technique; thermal shift assay and further by enzyme inhibition assay to identify Lead 1 molecule. Lead 1 optimization and expansion resulted in twenty four compounds. Among the synthesized compounds twelve compounds shown good enzyme inhibition than Lead 1 (IC50 20.07 ±â€¯0.29 µM). Among all the compounds; compound 22 (IC50 1.1 ±â€¯0.52 µM) showed potent non-competitive mode of inhibition in enzyme assay. Further showed good susceptibility (in replicating bacteria) of MIC 8.72 µM and bactericidal time dependant kill on dormant culture. It also exhibited significant activity in Mtb nutrient starvation model (2.5) and Mtb biofilm model (2.4) and in vivo M. marinum infected Zebra fish model studies (3.6) reduction at logarithmic scale.

Pharmacological evaluation of novel alagebrium analogs as methylglyoxal scavengers in vitro in cardiac myocytes and in vivo in SD rats.

BACKGROUND: Methylglyoxal (MG) is a byproduct of glucose metabolism and an inducer of advanced glycation end products (AGEs). AGEs are implicated in the pathogenesis of diabetes as well as hypertension. Most of the currently available MG scavengers are non-specific and have other effects as well. Alagebrium (ALA), developed by Alteon Corporation is a MG scavenger. Thus the aim of the present study was to investigate the potential of novel ALA analogs as possible MG scavengers and whether they could prevent any deleterious effects of MG. METHODS AND RESULTS: MG levels were measured by HPLC. The different biochemical and molecular parameters were measured by assay kits, RT-PCR and immunocytochemistry. Out of the 15 ALA analogs tested in vitro, compound no. 13 was found to be an effective inhibitor of MG in a concentration and time dependent manner. Compound no. 13 significantly attenuated the MG levels in vitro in MG treated cultured H9C2 cardiomyocytes as well as in vivo in MG treated SD rats. MG induced oxidative stress and apoptosis were attenuated by pretreatment of H9C2 cardiac myocytes with compound no. 13. MG induced cardiac hypertrophy and apoptosis were also attenuated by treating MG treated SD rats with compound no. 13. CONCLUSION: Our results indicate compound 13 as an effective inhibitor of MG in vitro in cultured cardiomyocytes and in vivo in SD rats and thus it may prove very useful in blocking the multiple deleterious effects of MG, including AGEs and vascular complications of diabetes.