Computational analysis followed by in vitro studies to explore cytokines (TNF-α, IL-6 and IL-1ß) inhibition potential of the new natural molecule polonilignan.

Targeting pro-inflammatory cytokines and their production is found to be of therapeutic benefit for the regulation of inflammation in various chronic autoimmune diseases. Our continued efforts to discover small molecular-weight pro-inflammatory cytokine inhibitors resulted in identifying a novel natural lignan molecule named polonilignan, isolated from the culture broth extract of an endophytic fungus Penicillium polonicum. An in silico study (molecular docking, ADME predictions, binding free energy calculation and molecular dynamics simulation) of the polonilignan over the pro-inflammatory cytokines proteins TNF-α, IL-6 and IL-1ß was performed using Schrodinger LLC software to understand the binding interactions, drug-like properties, and stability of the ligand-protein complex. Further, in-vitro testing of inhibition of TNF-α, IL-6 and IL-1ß by polonilignan was carried out using ELISA and RT-PCR on LPS-induced RAW 264.7 cell lines along with the testing of nitrite production effect (Griess assay) and cytotoxicity (MTT) analysis. Under the computational study, polonilignan revealed good docking scores, binding interactions, and stability under MDS and desirable in silico ADME results over the proteins TNF-α, IL-1ß and IL-6. Poloniligan showed significant inhibition of IL-1ß, IL-6 and TNF-α with IC50 values of 2.01 µM, 6.59 µM and 42.10 µM, respectively. Also, it reduced the translocation of the NF-κB subunit p65 to the nucleus (confocal microscopy). The mRNA expression levels of pro-inflammatory markers IL-1ß, TNF-α and IL-6 levels were lowered significantly (p < .001) by the compound, and the diminution was higher with IL-1ß. Further, the lignan was non-cytotoxic and effective in attenuating nitrite release (IC50 48.56 µM). Thus, polonilignan has been identified as a new pan-cytokine and NO inhibitor, it is recommended to optimise a method for the synthesis of this small molecular weight lignan and explore its pharmacokinetic characteristics, toxicity and therapeutic effect under various chronic inflammatory disease models.

Computational study on natural molecules targeting ß-ketoacyl reductase domain of fatty acid synthase for the identification of selective inhibitors.

Fatty acid synthase (FASN) enzyme is a lipid metabolism protein that provides the essential nutrients to cancer cells through de novo lipogenesis. Also, it plays a key role in other disease conditions, including obesity and inflammation. Hence, targeting the ß-ketoacyl reductase (KR) domain of FASN protein, an in-silico study was performed on some selective bioactive natural molecules following a repurposing strategy to identify FASN inhibitors. A molecular docking study followed by Absorption, Distribution, Metabolism and Excretion (ADME) predictions, binding free energy calculations, and molecular dynamics (MD) simulations were performed against FASN protein (PDB ID:6NNA) using Schrodinger Drug Discovery Software. Compounds rutin, trans-chlorogenic acid, norbergenin, myricetin, quercetin, physalolactone, quercetin-3-O-galactoside, kaempferol, asperulosidic acid, luteolin, curcumin, 12-deoxywithastramonolide, pedunculoside, hernandifoline, and withafastuosin E were identified as hits, presenting better docking scores (-16.2, -14.1, -12.3, -12.1, -12.0, -11.3, -10.3, -9.8, -9.3, -9.2, -9.1, -8.5, -8.4, -8.3, -7.9, respectively) and hydrogen bond interactions with Ser 2021 and Tyr 2034 amino acids of the KR domain of FASN. The MD simulations study of top five hits in complex with protein 6NNA uncovered the significant interactions leading to the stabilization of ligand with Root Mean Square Deviation (RMSD) below 5.00 Å and the stability was further validated by evaluating the root mean square fluctuation, solvent accessible surface area, and radius of gyration graphs. Also, the FASN inhibition effect of top four hits (50 µM) was >50% when corroborated using High Performance Liquid Chromatography HPLC-based estimation of palmitic acid in MCF-7 cells. Thus rutin, trans-chlorogenic acid, norbergenin, and myricetin are proposed as prospective FASN inhibitory leads. Overall, the results provided useful modifications in pharmacophoric features that could improve the inhibitory effect.Communicated by Ramaswamy H. Sarma.

Novel benzimidazole-triazole hybrids as apoptosis inducing agents in lung cancer: Design, synthesis, 18F-radiolabeling & galectin-1 inhibition studies.

In this study, we have synthesized a new series of benzimidazole-triazole hybrids as galectin-1 (gal-1) mediated apoptosis-inducing agents, and evaluated for their potential anticancer activity against a panel of human cancer cell lines viz. breast cancer (MCF-7 and MDA-MB-231) lung cancer (A-549 and NCI-H460), and human keratinocyte cancer (HaCaT), using MTT assay. The target compound 7c exhibited an excellent growth inhibition against lung cancer (A-549 and NCI-H460) cells with an IC50 value of 0.63 ± 0.21 µM, and 0.99 ± 0.01 µM respectively. The target compound 7c also showed a significant growth inhibition against breast cancer (MCF-7 and MDA-MB-23) with an IC50 value of 1.3 ± 0.18 µM, and 0.94 ± 0.02 µM respectively. In addition, the radiochemical synthesis has been performed using fluorine-18 radionuclide in the GE Tracer-lab FX2N module to prove the target compound 7c as a PET imaging agent. In the final stage, the 18F-7c target compound was successfully purified with 60% ethanol in water. The radiochemical purity was achieved >95% using HPLC, and the residual solvent DMF limit was around 78 ± 3 ppm confirmed by GC analysis. Further, the apoptosis induction by 7c in lung cancer (A-549) cells was confirmed as a result of the decrease in MMP levels, increased percentage of apoptotic cells, and sub G1 phase arrest by JC-1 staining, DAPI staining, annexin V-FITC/PI, and flow cytometric analysis. In addition, the target compound 7c significantly reduced the gal-1 protein levels in a dose-dependent manner as confirmed by ELISA studies. The protein binding studies like Surface Plasmon Resonance (SPR) and Fluorescence Spectroscopy (FS) studies indicated that the target compound 7c is capable of binding to gal-1 with an equilibrium constant (KD) value of 1.19E-06 M, and binding constant (Ka) of 9.5 × 103 M-1 respectively. The in-silico computational studies also revealed possible interactions and pharmacokinetic properties (ADMET) of compound 7c with the binding domain of gal-1. Therefore, the novel benzimidazole-triazole hybrids as apoptosis-inducing agents in lung cancer would be potential cytotoxic and PET imaging agents via gal-1.