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.

Review on target domains and natural compound-based inhibitors of fatty acid synthase for anticancer drug discovery.

Cancer cells require a higher amount of energy in the form of fatty acids for their uncontrolled proliferation and growth. Fatty acid synthase (FASN) plays a crucial role in the synthesis of palmitate, which is involved in most of the critical malignant pathways. Hence, by targeting FASN, tumour growth can be controlled. By designing and developing FASN inhibitors with catalytic domain specificity, safe and potential anticancer drugs can be achieved. The article draws light towards the catalytic domains of FASN, their active site residues and interaction of some of the reported natural FASN inhibitors (resveratrol, lavandulyl flavonoids, catechins, stilbene derivatives, etc). The rationality (structure-activity relationship) behind the variation in the activity of the reported natural FASN inhibitors (butyrolactones, polyphenolics, galloyl esters and thiolactomycins) has also been covered. Selective, safe and potentially active FASN inhibitors could be developed by: (i) having proper understanding of the function of all catalytic domains of FASN (ii) studying the upstream and downstream FASN regulators (iii) identifying cancer-specific FASN biomarkers (that are non-essential/absent in the normal healthy cells) (iv) exploring the complete protein structure of FASN, e-screening of the compounds prior to synthesis and study their ADME properties (v) predicting the selectivity based on their strong affinity at the catalytic site of FASN.

New synthetic coumarinolignans as attenuators of pro-inflammatory cytokines in LPS-induced sepsis and carrageenan-induced paw oedema models.

OBJECTIVES: The aim of the study was to explore the inhibition efficacy of new synthetic coumarinolignans (SCLs) against the secretion of pro-inflammatory cytokines in two in vivo models of inflammation. METHODS: Four SCLs 1-4 were screened for their pro-inflammatory cytokine inhibitory potential through oral administration at a dose of 50 mg/kg body weight in lipopolysaccharide-induced mouse endotoxaemia and carrageenan-induced mouse paw oedema models. Levels of pro-inflammatory cytokines (IL-1ß, TNFα and IL-6) in blood and paw tissue samples were estimated using ELISA. Paw oedema was measured using a plethysmometer. Results were compared with a natural coumarinolignan, cleomiscosin A (5), and the structure-activity relationship (SAR) was interpreted. RESULTS AND DISCUSSION: Compound 2 had the greatest potential in the endotoxaemia model, exhibiting 66.41%, 62.56% and 43.15% inhibition of plasma IL-1ß, TNFα and IL-6 secretions, respectively. Further dose-dependent study revealed its anti-inflammatory potential even at dose of 10 mg/kg body weight with 24.42% decline in the level of IL-1ß. Nevertheless, SCLs 1, 3 and 4 showed marked inhibitory activity with 57.54%, 51.48% and 62.46% reduction in the levels of IL-1ß, respectively. Moreover, compound 2 decreased the plasma TNFα and IL-1ß levels to 50.03% and 36.58% along with the reduction of paw oedema volume in the local inflammation induced by carrageenan. All compounds including cleomiscosin A (5) were more effective against IL-1ß. By studying SAR, the presence of dihydroxyl groups in the phenyl ring of lignans was identified to be essential for the activity. Also, esterification of lignans and presence of a 4-methyl substituent in the coumarin nucleus were found to play some role in enhancing the activity. CONCLUSION: All four SCLs, especially compound 2, have shown vast potential to emerge out as promising anti-inflammatory drugs.

Attenuation of TNF-α secretion by L-proline-based cyclic dipeptides produced by culture broth of Pseudomonas aeruginosa.

To identify small molecule inhibitors of TNF-α, bioassay- and LC-MS-guided chemical investigation on EtOAc extract of Pseudomonas aeruginosa ABS-36 culture broth (EEPA) was performed, which yielded four proline-based cyclic dipeptides, cyclo(Gly-l-Pro) (1), cyclo(l-Pro-l-Phe) (2), cyclo(trans-4-hydroxy-l-Pro-l-Phe) (3) and cyclo(trans-4-hydroxy-l-Pro-l-Leu) (4). Compounds 1 and 3 exhibited potent inhibition of TNF-α release with IC50 values of 4.5 and 14.2µg/mL, respectively, while EEPA showed IC50 of 38.8µg/mL under lipopolysaccharide treated RAW 264.7 cell ELISA assay. Also, marked attenuation of mRNA-expression of TNF-α was shown by all compounds. In vivo testing in rats of EEPA and chemically synthesized 4 validated significant TNF-α reduction with 51% (500mg/kg) and 79% (50mg/kg), respectively. In addition, all compounds exhibited significant diminution of IL-1ß and IL-6 mRNA-expression levels and NO production. All samples displayed only weak toxicity to lipopolysaccharide-induced RAW 264.7 cells.

Cytotoxic constituents of Abutilon indicum leaves against U87MG human glioblastoma cells.

The study was aimed to identify cytotoxic leads from Abutilon indicum leaves for treating glioblastoma. The petroleum ether extract, methanol extract (AIM), chloroform and ethyl acetate sub-fractions (AIM-C and AIM-E, respectively) prepared from AIM were tested for cytotoxicity on U87MG human glioblastoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These extracts exhibited considerable activity (IC50 values of 42.6-64.5 µg/mL). The most active AIM-C fraction was repeatedly chromatographed to yield four known compounds, methyl trans-p-coumarate (1), methyl caffeate (2), syringic acid (3) and pinellic acid (4). Cell viability assay of 1-4 against U87MG cells indicated 2 as most active (IC50 value of 8.2 µg/mL), whereas the other three compounds were much less active. Interestingly, compounds 1-4 were non-toxic towards normal human cells (HEK-293). The content of 2 in AIM-C was estimated as 3% by HPLC. Hence, presence of some more active substances besides methyl caffeate (2) in AIM-C is anticipated.