26 research outputs found

    Crystallographic mining driven computer-guided approach to identify the ASK1 inhibitor likely to perturb the catalytic region

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    The pathological levels of reactive oxygen species (ROS) and oxidative stress has been recognized as a critical driver for inflammatory disorders. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to be activated by intracellular ROS and its inhibition leads to a down regulation of p38-and JNK-dependent signaling. ASK1 inhibitors are reported to have the potential to treat clinically important inflammatory pathologies including liver, pulmonary and renal disorders. In view of its biological and pathological significance, inhibition of ASK1 with small molecules has been pursued as an attractive strategy to combat human diseases such as non-alcoholic steatohepatitis (NASH). Despite several ASK1 inhibitors being developed, the failure in Phase 3 clinical trials of most advanced candidate selonsertib’s, underscores to discover therapeutic agents with diverse chemical moiety. Here, by using structural pharmacophore and enumeration strategy on mining co-crystals of ASK1, different scaffolds were generated to enhance the chemical diversity keeping the critical molecular interaction in the catalytic site intact. A total of 15,772 compounds were generated from diverse chemical scaffolds and were evaluated using a virtual screening pipeline. Based on docking and MM-GBSA scores, a lead candidate, S3C-1-D424 was identified from top hits. A comparative molecular dynamics simulations (MD) of APO, Selonsertib and shortlisted potential candidates combined with pharmacokinetics profiling and thermodynamic analysis, demonstrating their suitability as potential ASK1 inhibitors to explore further for establishment towards hit-to-lead campaign. Communicated by Ramaswamy H. Sarma</p

    Morphology and characterization of fC-MSC (A) Representative photomicrograph (10X, 20 µm) of fC-MSC in culture showing spindle shaped morphology.

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    <p>(B) Representative flow cytometric dot-plots showing that fC-MSC are (a) CD29+/CD45−; (b) CD44+/CD45−; (c) CD73+/CD31−; (d) CD90+/HLA-DR−; (e) CD105+/HLA-DR−. (C) Representative photomicrographs (10X, 20 µm) showing differentiation of fC-MSC into Osteoblasts (a-i: differentiated cells positive for Alizarin red stain, and a-ii: control cells negative for Alizarin red stain) and Adipocytes (b-i: differentiated cells positive for oil red O stain, and b-ii: control cells negative for oil red O stain).</p

    Effect of fC-MSC on LV functions: Bar diagrams showing ejection fraction, end systolic volume, end diastolic volume, and left ventricular myo-mass, measured at 1 week after MI (before fC-MSC therapy) and 4 weeks after fC-MSC therapy using gated SPECT analysis.

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    <p>Values shown are mean ± SEM (n = 6); *P<0.05, *P<0.01, *P<0.001 saline group after cell therapy vs before cell therapy (within the group);<sup> #</sup>P<0.05,<sup> #</sup>P<0.01, <sup>#</sup>P<0.001 fC-MSC group after cell therapy vs before cell therapy (within the group);<sup> †</sup>P<0.05, <sup>†</sup>P<0.01, <sup>†</sup>P<0.001 fC-MSC group (after cell therapy) vs saline group (after cell therapy).</p

    Gated SPECT analysis of fC-MSC therapy in rats with MI.

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    <p>***P<0.001,</p><p>**P<0.01 = Healthy Control Vs MI Baseline;</p>††<p>P<0.01 = MI Baseline Vs MI+Saline;</p>‡‡‡<p>P<0.001 <sup>‡‡</sup>P<0.01,</p>‡<p>P<0.05 = MI baseline Vs MI+fC-MSC;</p>§§§<p>P<0.001,</p>§§<p>P<0.01 = MI+Saline Vs MI+fC-MSC.</p

    fC-MSC inhibit the apoptosis in rats with acute myocardial injury.

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    <p>(a) Representative immunofluorescence photomicrographs of saline and fC-MSC treated hearts 4 weeks after fC-MSC therapy showing (a-i & b-i) Overlay; (a-ii & b-ii) Tunel positive cells (green dye) counter stain with (a-iii & b-iii) Hoechst dye respectively (B) TUNEL apoptotic index showing significant decrease in apoptotic cells in fC-MSC treated compared to saline treated hearts. Values shown are mean ± SEM (n = 6). **P<0.01 fC-MSC treated vs saline treated hearts. (C) Representative immune-blots showing expression of BAX and BCL2 in saline and fC-MSC treated rats and (D) their relative density. Densitometric analysis was applied for comparison of relative protein expression. Values expressed Mean ± SE (n = 6), *P<0.05, **P<0.01, ***P<0.001: fC-MSC treated vs. saline treated group.</p

    Effect of fC-MSC on LV functions: Bar diagrams showing ejection fraction, end systolic volume, end diastolic volume, and left ventricular myo-mass, measured at 1 week after MI (before fC-MSC therapy) and 4 weeks after fC-MSC therapy using gated SPECT analysis.

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    <p>Values shown are mean ± SEM (n = 6); *P<0.05, *P<0.01, *P<0.001 saline group after cell therapy vs before cell therapy (within the group);<sup> #</sup>P<0.05,<sup> #</sup>P<0.01, <sup>#</sup>P<0.001 fC-MSC group after cell therapy vs before cell therapy (within the group);<sup> †</sup>P<0.05, <sup>†</sup>P<0.01, <sup>†</sup>P<0.001 fC-MSC group (after cell therapy) vs saline group (after cell therapy).</p

    Effect of fC-MSC on LV perfusion: Representative SPECT perfusion images and polar-maps obtained at 1 week after MI (before fC-MSC therapy) and 4 weeks after fC-MSC therapy.

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    <p>(A) Serial 99mTc-sestamibi perfusion images obtained in SPECT short axis (SA), horizontal long axis (HLA), vertical long axis (VLA) of MI hearts treated with saline and fC-MSC (B) Corresponding polar-maps. The perfusion images (A) and the polar-maps (B) show a myocardial-flow defect in the anterolateral wall of left ventricle in both the groups. However, the hearts treated with fC-MSC demonstrate a smaller ischemic lesion (region of deficit) and a better perfusion in the MI segments.</p

    <i>In</i><i>vivo</i> differentiation of fC-MSC in to cardiomyocytes, smooth muscle and endothelial cells. Representative immunofluorescence photomicrographs (40X; 20 µm) of fC-MSC differentiation into cardiovascular lineage cells observed in MI rat models 4 weeks after fC-MSC administration.

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    <p>Row (i): cardiomyocyte showing immunofluorescence staining for (a) Troponin-T; (b) PKH26 dye; (c) Hoechst dye; (d) Overlay of a, b & c images. Row (ii): endothelial cells showing immunofluorescence staining for (a) CD31; (b) PKH26 dye; (c) Hoechst dye; (d) Overlay of a, b & c images. Row (iii): smooth muscle cells showing immunofluorescence staining for (a) SM-MHC; (b) PKH26 dye; (c) Hoechst dye; (d) Overlay of a, b & c images.</p
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