Insights into the potential of withanolides as Phosphodiesterase-4 (PDE4D) inhibitors.

Medicinal herbs have been used as traditional medicines for centuries. The molecular mechanism of action of their bioactive molecules against various diseases or therapeutic targets is still being explored. Here, the active compounds (withanolides) of a well-known Indian medicinal herb, Ashwagandha (Withania somnifera), have been studied for their most potential therapeutic targets and their mechanism of action using ligand-based screening and receptor-based approaches. Ligand-based screening predicted the six top therapeutic targets, namely, Protein kinase C alpha (PRKCA), Protein kinase C delta (PRKCD), Protein kinase C epsilon (PRKCE), Androgenic Receptor (AR), Cycloxygenase-2 (PTGS-2) and Phosphodiesterase-4D (PDE4D). Further, when these predictions were validated using receptor-based studies, i.e. molecular docking, molecular dynamics simulation and free energy calculations, it was found that PDE4D was the most potent target for four withanolides, namely, Withaferin-A, 17-Hydroxywithaferin-A, 27-Hydroxywithanone and Withanolide-R. These compounds had a better binding affinity and similar interactions as that of an already known inhibitor (Zardaverine) of PDE4D. These results warrant further in-vitro and in-vivo investigations to examine their therapeutic potential as an inhibitor of PDE4D.Communicated by Ramaswamy H. Sarma.

Withanone and Withaferin-A are predicted to interact with transmembrane protease serine 2 (TMPRSS2) and block entry of SARS-CoV-2 into cells.

Coronavirus disease 2019 (COVID-19) initiated in December 2019 in Wuhan, China and became pandemic causing high fatality and disrupted normal life calling world almost to a halt. Causative agent is a novel coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV). While new line of drug/vaccine development has been initiated world-wide, in the current scenario of high infected numbers, severity of the disease and high morbidity, repurposing of the existing drugs is heavily explored. Here, we used a homology-based structural model of transmembrane protease serine 2 (TMPRSS2), a cell surface receptor, required for entry of virus to the target host cell. Using the strengths of molecular docking and molecular dynamics simulations, we examined the binding potential of Withaferin-A (Wi-A), Withanone (Wi-N) and caffeic acid phenethyl ester to TPMRSS2 in comparison to its known inhibitor, Camostat mesylate. We found that both Wi-A and Wi-N could bind and stably interact at the catalytic site of TMPRSS2. Wi-N showed stronger interactions with TMPRSS2 catalytic residues than Wi-A and was also able to induce changes in its allosteric site. Furthermore, we investigated the effect of Wi-N on TMPRSS2 expression in MCF7 cells and found remarkable downregulation of TMPRSS2 mRNA in treated cells predicting dual action of Wi-N to block SARS-CoV-2 entry into the host cells. Since the natural compounds are easily available/affordable, they may even offer a timely therapeutic/preventive value for the management of SARS-CoV-2 pandemic. We also report that Wi-A/Wi-N content varies in different parts of Ashwagandha and warrants careful attention for their use.Communicated by Ramaswamy H. Sarma.

Computational and in vitro experimental analyses of the anti-COVID-19 potential of Mortaparib and MortaparibPlus.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has become a global health emergency. Although new vaccines have been generated and being implicated, discovery and application of novel preventive and control measures are warranted. We aimed to identify compounds that may possess the potential to either block the entry of virus to host cells or attenuate its replication upon infection. Using host cell surface receptor expression (angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS2)) analysis as an assay, we earlier screened several synthetic and natural compounds and identified candidates that showed ability to down-regulate their expression. Here, we report experimental and computational analyses of two small molecules, Mortaparib and MortaparibPlus that were initially identified as dual novel inhibitors of mortalin and PARP-1, for their activity against SARS-CoV-2. In silico analyses showed that MortaparibPlus, but not Mortaparib, stably binds into the catalytic pocket of TMPRSS2. In vitro analysis of control and treated cells revealed that MortaparibPlus caused down-regulation of ACE2 and TMPRSS2; Mortaparib did not show any effect. Furthermore, computational analysis on SARS-CoV-2 main protease (Mpro) that also predicted the inhibitory activity of MortaparibPlus. However, cell-based antiviral drug screening assay showed 30-60% viral inhibition in cells treated with non-toxic doses of either MortaparibPlus or Mortaparib. The data suggest that these two closely related compounds possess multimodal anti-COVID-19 activities. Whereas MortaparibPlus works through direct interactions/effects on the host cell surface receptors (ACE2 and TMPRSS2) and the virus protein (Mpro), Mortaparib involves independent mechanisms, elucidation of which warrants further studies.

Molecular mechanism of anti-SARS-CoV2 activity of Ashwagandha-derived withanolides.

COVID-19 caused by SARS-CoV-2 corona virus has become a global pandemic. In the absence of drugs and vaccine, and premises of time, efforts and cost required for their development, natural resources such as herbs are anticipated to provide some help and may also offer a promising resource for drug development. Here, we have investigated the therapeutic prospective of Ashwagandha for the COVID-19 pandemic. Nine withanolides were tested in silico for their potential to target and inhibit (i) cell surface receptor protein (TMPRSS2) that is required for entry of virus to host cells and (ii) viral protein (the main protease Mpro) that is essential for virus replication. We report that the withanolides possess capacity to inhibit the activity of TMPRSS2 and Mpro. Furthermore, withanolide-treated cells showed downregulation of TMPRSS2 expression and inhibition of SARS-CoV-2 replication in vitro, suggesting that Ashwagandha may provide a useful resource for COVID-19 treatment.

Pharmacokinetic characterization of a natural product-inspired novel MEK1 inhibitor E6201 in preclinical species.

PURPOSE: E6201 is a natural product-inspired novel inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 (MEK1) and other kinases and is currently under development as an anticancer (parenteral administration) and antipsoriasis agent (topical application). In vitro and in vivo preclinical studies were performed to characterize the pharmacokinetics of E6201. Allometric scaling was applied to predict human pharmacokinetics of E6201. METHODS: In vitro metabolism studies for CYP induction and CYP inhibition were conducted using human hepatocytes and microsomes, respectively. Metabolic stability using microsomes and protein-binding studies using pooled plasma were performed for mice, rats, dogs, and human. Pharmacokinetics of E6201 and its isomeric metabolite, ER-813010, in mice, rats, and dogs was determined following single IV administration of E6201 at three dose levels. Bioanalysis was performed using LC/MS/MS. Pharmacokinetic parameters were determined using non-compartmental analysis, and allometric scaling with a two-compartment model was used to predict E6201 pharmacokinetics in humans. RESULTS: E6201 showed high plasma protein binding (>95%), and metabolic stability half-life ranged from 36 to 89 min across species. In vitro CYP inhibition (CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A) and CYP induction (CYP1A, 3A, 2C9, and 2C19) suggested no inhibitory or induction effect on the tested human CYPs up to 10 µM of E6201. Pharmacokinetics of E6201 in mice, rats, and dogs was characterized by mean clearance ranging from 3.45 to 10.92 L/h/kg, distribution volume ranging from 0.63 to 13.09 L/kg, and elimination half-life ranging from 0.4 to 1.6 h. ER-813010 was detected in all species with metabolite to parent exposure ratio (AUC(R)) ranging from 3.1 to 33.4% and exhibited fast elimination (<3 h). The allometry predicted high clearance and large volume of distribution of E6201 in humans and was in general in good agreement with the observed first human subject pharmacokinetics. CONCLUSIONS: E6201 exhibited high clearance, high to moderate distribution, and fast elimination in preclinical species. In vitro results suggested that E6201 has low risk of drug-drug interactions due to CYP inhibition and induction in humans. In the first-in-man study, E6201 exhibited high clearance, which was well predicted by allometric scaling.

Ascorbate sustains neutrophil NOS expression, catalysis, and oxidative burst.

Previous studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guinea pigs to further assess the implication of ascorbate deficiency in vivo on neutrophil ascorbate and tetrahydrobiopterin content, NOS expression/activity, phagocytosis, and respiratory burst. Ascorbate deficiency significantly reduced ascorbate and tetrahydrobiopterin amounts, NOS expression/activity, and NO as well as free radical generation in neutrophils from scorbutics. Ascorbate and tetrahydrobiopterin supplementation in vitro, though, significantly enhanced NOS catalysis in neutrophil lysates and NO generation in live cells, but could not restore them to control levels. Although phagocytic activity remained unaffected, scorbutic neutrophils were compromised in free radical generation. Ascorbate-induced free radical generation was NO dependent and prevented by NOS and NADPH oxidase inhibitors. Augmentation of oxidative burst with dehydroascorbate (DHA) was counteracted in the presence of glucose (DHA uptake inhibitor) and iodoacetamide (glutaredoxin inhibitor), suggesting the importance of ascorbate recycling in neutrophils. Ascorbate uptake was, however, unaffected among scorbutic neutrophils. These observations thus convincingly demonstrate a novel role for ascorbate in augmenting both NOS expression and activity in vivo, thereby reinforcing oxidative microbicidal actions of neutrophils.

Pattern profiling of the herbal preparation picroliv using liquid chromatography-tandem mass spectrometry.

At present, the construction of chromatographic fingerprints of complex herbal preparations in combination with mass spectrometry plays an important role in their development and standardization as potential therapeutic agents. Picroliv, an extract from roots and rhizomes of Picrorhiza kurroa, is a herbal hepatoprotective developed by CDRI. We report for the first time pattern profiling of various constituents of picroliv along with a precise and accurate method to estimate relative concentration of major components in the preparation by liquid chromatography-tandem mass spectrometry. In total, 27 components could be detected in multiple reaction monitoring (MRM) mode out of which fourteen could be quantified in terms of their relative concentration. Seven components were structurally correlated and confirmed based on the fragmentation pattern and information available in literature. The detection was carried out using MRM in negative ionization mode with analytes quantified from the summed total ion value of their most intense molecular ion transitions. The separation of various components was achieved using a gradient elution on RP-18 column with acetonitrile and Milli-Q water as mobile phase at a flow rate of 1.0 ml/min. The method was validated in terms of linearity, accuracy and precision (within- and between-assay variation) for 5 days. Linearity range was different for various components depending upon their sensitivity and abundance in the herbal preparation. Within- and between-assay accuracy (%bias) and precision (%R.S.D.) were within acceptable limits. The method was successfully applied to detect and determine relative concentrations of various components in two different batches of picroliv.