Study of Drug Targets Associated With Oncogenesis and Cancer Cell Survival and the Therapeutic Activity of Engineered Ashwagandha Extract Having Differential Withanolide Constitutions.

Ashwagandha (Withania somnifera) has gained worldwide popularity for a multitude of health benefits inclusive of cancer-preventive and curative effects. Despite numerous research data supporting the benefits of this wonder herb, the actual use of ashwagandha for cancer treatment in clinics is limited. The primary reason for this is the inconsistent therapeutic outcome due to highly variable composition and constitution of active ingredients in the plant extract impacting ashwagandha's pharmacology. We investigate here an engineered yield: an ashwagandha extract (Oncowithanib) that has a unique and fixed portion of active ingredients to achieve consistent and effective therapeutic activity. Using the MCF7 cell line, Oncowithanib was studied for its anti-neoplastic efficacy and drug targets associated with cell cycle regulation, translation machinery, and cell survival and apoptosis. Results demonstrate a dose-dependent decline in Oncowithanib-treated MCF7 cell viability and reduced colony-forming ability. Treated cells showed increased cell death as evidenced by enhancement of Caspase 3 enzyme activity and decreased expressions of cell proliferation markers such as Ki67 and Aurora Kinase A. Oncowithanib treatment was also found to be associated with expressional suppression of key cellular kinases such as RSK1, Akt1, and mTOR in MCF7 cells. Our findings indicate that Oncowithanib decreases MCF7 cell survival and propagation, and sheds light on common drug targets that might be good candidates for the development of cancer therapeutics. Further in-depth investigations are required to fully explore the potency and pharmacology of this novel extract. This study also highlights the importance of the standardization of herbal extracts to get consistent therapeutic activity for the disease indication.

Withania somnifera root extract inhibits MGO-induced skin fibroblast cells dysfunction via ECM-integrin interaction.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal, known as Ashwagandha, has long been used in traditional medicine in Ayurveda, India, a representative adaptogen. The main active constituents of W. somnifera are withanolides, and the root is often used as a medicine with a wide range of pharmacological activities, which can be used to treat insomnia, neurasthenia, diabetes mellitus and skin cancer. AIM OF THE STUDY: Whole-component qualitative and quantitative analyses were performed on W. somnifera. We explored the ameliorative effect of the adaptogen representative plant W. somnifera on the senescence events of MGO-injured fibroblasts and its action mechanism and verified the hypotheses that WS can inhibit the accumulation of AGEs and regulate the dynamic balance among the components of the ECM by modulating the expression of integrin ß1 receptor; as a result, WS maintains cellular behavioural and biological functions in a normal range and retards the aging of skin from the cellular level. MATERIALS AND METHODS: In this study, the components of WS were first qualitatively and quantitatively analysed by HPLC fingerprinting and LC-MS detection. Second, a model of MGO-induced injury of CML-overexpressing fibroblasts was established. ELISA was used to detect CML expression and the synthesis of key extracellular matrix ECM protein components COL1, FN1, LM5 and TNC synthesis; CCK-8 was used to detect cell viability; EDU was used to detect cell proliferation capacity; fluorescence was used to detect cell adhesion capacity; and migration assay were used to detect cell migration capacity; qRT-PCR was used to detect the regulatory pathway TGF-ß1 and MMP-2, MMP-9 in ECMs; immunofluorescence was used to detect the expression of ITGB1; and WB was used to detect the expression of COL1, FN1, LM5, Tnc, TGF-ß1, MMP-2, MMP-9 and ITGB1. RESULTS: In total, 27 active ingredients were analysed from WS, which mainly consisted of withanolide components, such as withaferin A and withanolide A. Based on the model of MGO-induced fibroblast senescence injury, WS significantly inhibited CML synthesis. By up-regulating the expression of integrin ß1, it upregulated the expression of the TGF-ß1 gene, which is closely related to the generation of ECMs, downregulated the expression of the MMP-2 and MMP-9 genes, which are closely related to the degradation of ECMs, maintained the dynamic balance of the four types of ECMs, and improved cell viability as well as proliferation, migration and adhesion abilities. CONCLUSIONS: WS can prevent cellular behavioural dysfunction and delay skin ageing by reducing the accumulation of CML, upregulating the expression of the ITGB1 receptor, maintaining the normal function of ECM-integrin receptor interaction and preventing an imbalance between the production and degradation of protein components of ECMs. The findings reported in this study suggest that WS as a CML inhibitor can modulate ECM-integrin homeostasis and has great potential in the field of aging retardation.

A critical assessment of the whole plant-based phytotherapeutics from Withania somnifera (L.) Dunal with respect to safety and efficacy vis-a-vis leaf or root extract-based formulation.

Withania somnifera (L.) Dunal, popularly known as Ashwagandha or Indian ginseng, is well acclaimed for its health-enhancing effects, including its potent immunomodulatory, anti-inflammatory, neuroprotective, and anti-tumorigenic properties. The prime biological effectors of these attributes are a diverse group of ergostane-based steroidal lactones termed withanolides. Withanones and withanosides are distributed differentially across the plant body, whereas withanolides and withanones are known to be more abundant in leaves, while withanosides are found exclusively in the roots of the plants. Standardized W. somnifera extract is Generally Recognized as Safe (GRAS)-affirmed, however, moderate to severe toxic manifestations may occur at high dosages. Withaferin A, which also happens to be the primary bioactive ingredient for the effectiveness of this plant. There have been contrasting reports regarding the distribution of withaferin A in W. somnifera. While most reports state that the roots of the plant have the highest concentrations of this phytochemical, several others have indicated that leaves can accumulate withaferin A in proportionately higher amounts. A comprehensive survey of the available reports suggests that the biological effects of Ashwagandha are grossly synergistic in nature, with many withanolides together mediating the desired physiological effect. In addition, an assorted formulation of withanolides can also neutralize the toxic effects (if any) associated with withaferin A. This mini-review presents a fresh take on the recent developments regarding the safety and toxicity of the plant, along with a critical assessment of the use of roots against leaves as well as whole plants to develop therapeutic formulations. Going by the currently available scientific evidence, it is safe to infer that the use of whole plant formulations instead of exclusively root or leaf recipes may present the best possible option for further exploration of therapeutic benefits from this novel medicinal plant.HighlightsTherapeutic potential of withanolides owes to the presence of α,ß unsaturated ketone which binds to amines, alcohols, and esters and 5ß, 6ß epoxy group which react with side chain thiols of proteins.At concentrations above NOAEL (no observed adverse effect level), the same mechanisms contribute towards toxicity of the molecule.Although withanosides are found exclusively in roots, whole plants have higher contents of withanones and withanolides.Whole plant-based formulations have other metabolites which can nullify the toxicity associated with roots.Extracts made from whole plants, therefore can holistically impart all therapeutic benefits as well as mitigate toxicity.

In Silico Study on the Interactions, Molecular Docking, Dynamics and Simulation of Potential Compounds from Withania somnifera (L.) Dunal Root against Cancer by Targeting KAT6A.

Cancer is characterized by the abnormal development of cells that divide in an uncontrolled manner and further take over the body and destroy the normal cells of the body. Although several therapies are practiced, the demand and need for new therapeutic agents are ever-increasing because of issues with the safety, efficacy and efficiency of old drugs. Several plant-based therapeutics are being used for treatment, either as conjugates with existing drugs or as standalone formulations. Withania somnifera (L.) Dunal is a highly studied medicinal plant which is known to possess immunomodulatory activity as well as anticancer properties. The pivotal role of KAT6A in major cellular pathways and its oncogenic nature make it an important target in cancer treatment. Based on the literature and curated datasets, twenty-six compounds from the root of W. somnifera and a standard inhibitor were docked with the target KAT6A using Autodock vina. The compounds and the inhibitor complexes were subjected to molecular dynamics simulation (50 ns) using Desmond to understand the stability and interactions. The top compounds (based on the docking score of less than -8.5 kcal/mol) were evaluated in comparison to the inhibitor. Based on interactions at ARG655, LEU686, GLN760, ARG660, LEU689 and LYS763 amino acids with the inhibitor WM-8014, the compounds from W. somnifera were evaluated. Withanolide D, Withasomniferol C, Withanolide E, 27-Hydroxywithanone, Withanolide G, Withasomniferol B and Sitoindoside IX showed high stability with the residues of interest. The cell viability of human breast cancer MCF-7 cells was evaluated by treating them with W. Somnifera root extract using an MTT assay, which showed inhibitory activity with an IC50 value of 45 µg/mL. The data from the study support the traditional practice of W. somnifera as an anticancer herb.

Cloning and homologous characterization of geranylgeranyl pyrophosphate synthase (GGPPS) from Withania somnifera revealed alterations in metabolic flux towards gibberellic acid biosynthesis.

MAIN CONCLUSION: Overexpression of a novel geranylgeranyl pyrophosphate synthase gene (WsGGPPS) in planta resulted in increased levels of gibberellic acid and decrease in withanolide content. Withania somnifera (L.) Dunal, the herb from family Solanaceae is one of the most treasured medicinal plant used in traditional medicinal systems owing to its unique stockpile of pharmaceutically active secondary metabolites. Phytochemical and pharmacological studies in this plant were well established, but the genes affecting the regulation of biosynthesis of major metabolites were not well elucidated. In this study cloning and functional characterization of a key enzyme in terpenoid biosynthetic pathway viz. geranylgeranyl pyrophosphate synthase (EC 2.5.1.29) gene from Withania somnifera was performed. The full length WsGGPPS gene contained 1,104 base pairs that encode a polypeptide of 365 amino acids. The quantitative expression analysis suggested that WsGGPPS transcripts were expressed maximally in flower tissues followed by berry tissues. The expression levels of WsGGPPS were found to be regulated by methyl jasmonate (MeJA) and salicylic acid (SA). Amino acid sequence alignment and phylogenetic studies suggested that WsGGPPS had close similarities with GGPPS of Solanum tuberosum and Solanum pennellii. The structural analysis provided basic information about three dimensional features and physicochemical parameters of WsGGPPS protein. Overexpression of WsGGPPS in planta for its functional characterization suggested that the WsGGPPS was involved in gibberellic acid biosynthesis.

Oxidative Stress and AKT-Associated Angiogenesis in a Zebrafish Model and Its Potential Application for Withanolides.

Oxidative stress and the AKT serine/threonine kinase (AKT) signaling pathway are essential regulators in cellular migration, metastasis, and angiogenesis. More than 300 withanolides were discovered from the plant family Solanaceae, exhibiting diverse functions. Notably, the relationship between oxidative stress, AKT signaling, and angiogenesis in withanolide treatments lacks comprehensive understanding. Here, we summarize connecting evidence related to oxidative stress, AKT signaling, and angiogenesis in the zebrafish model. A convenient vertebrate model monitored the in vivo effects of developmental and tumor xenograft angiogenesis using zebrafish embryos. The oxidative stress and AKT-signaling-modulating abilities of withanolides were highlighted in cancer treatments, which indicated that further assessments of their angiogenesis-modulating potential are necessary in the future. Moreover, targeting AKT for inhibiting AKT and its AKT signaling shows the potential for anti-migration and anti-angiogenesis purposes for future application to withanolides. This particularly holds for investigating the anti-angiogenetic effects mediated by the oxidative stress and AKT signaling pathways in withanolide-based cancer therapy in the future.

Production and Structural Diversification of Withanolides by Aeroponic Cultivation of Plants of Solanaceae: Cytotoxic and Other Withanolides from Aeroponically Grown Physalis coztomatl.

Withanolides constitute one of the most interesting classes of natural products due to their diversity of structures and biological activities. Our recent studies on withanolides obtained from plants of Solanaceae including Withania somnifera and a number of Physalis species grown under environmentally controlled aeroponic conditions suggested that this technique is a convenient, reproducible, and superior method for their production and structural diversification. Investigation of aeroponically grown Physalis coztomatl afforded 29 withanolides compared to a total of 13 obtained previously from the wild-crafted plant and included 12 new withanolides, physacoztolides I-M (9-13), 15α-acetoxy-28-hydroxyphysachenolide C (14), 28-oxophysachenolide C (15), and 28-hydroxyphysachenolide C (16), 5α-chloro-6ß-hydroxy-5,6-dihydrophysachenolide D (17), 15α-acetoxy-5α-chloro-6ß-hydroxy-5,6-dihydrophysachenolide D (18), 28-hydroxy-5α-chloro-6ß-hydroxy-5,6-dihydrophysachenolide D (19), physachenolide A-5-methyl ether (20), and 17 known withanolides 3-5, 8, and 21-33. The structures of 9-20 were elucidated by the analysis of their spectroscopic data and the known withanolides 3-5, 8, and 21-33 were identified by comparison of their spectroscopic data with those reported. Evaluation against a panel of prostate cancer (LNCaP, VCaP, DU-145, and PC-3) and renal carcinoma (ACHN) cell lines, and normal human foreskin fibroblast (WI-38) cells revealed that 8, 13, 15, and 17-19 had potent and selective activity for prostate cancer cell lines. Facile conversion of the 5,6-chlorohydrin 17 to its 5,6-epoxide 8 in cell culture medium used for the bioassay suggested that the cytotoxic activities observed for 17-19 may be due to in situ formation of their corresponding 5ß,6ß-epoxides, 8, 27, and 28.

COVID19-inhibitory activity of withanolides involves targeting of the host cell surface receptor ACE2: insights from computational and biochemical assays.

SARS-CoV-2 outbreak in China in December 2019 and its spread as worldwide pandemic has been a major global health crisis. Extremely high infection and mortality rate has severely affected all sectors of life and derailed the global economy. While drug and vaccine development have been prioritized and have made significant progression, use of phytochemicals and herbal constituents is deemed as a low-cost, safer and readily available alternative. We investigated therapeutic efficacy of eight withanolides (derived from Ashwagandha) against the angiotensin-converting enzyme 2 (ACE2) proteins, a target cell surface receptor for SARS-CoV-2 and report results on the (i) computational analyses including binding affinity and stable interactions with ACE2, occupancy of ACE2 residues in making polar and nonpolar interactions with different withanolides/ligands and (2) in vitro mRNA and protein analyses using human cancer (A549, MCF7 and HSC3) cells. We found that among all withanolides, Withaferin-A, Withanone, Withanoside-IV and Withanoside-V significantly inhibited the ACE2 expression. Analysis of withanolides-rich aqueous extracts derived from Ashwagandha leaves and stem showed a higher ACE2 inhibitory potency of stem-derived extracts. Taken together, we demonstrated the inhibitory potency of Ashwagandha withanolides and its aqueous extracts against ACE2.Communicated by Ramaswamy H. Sarma.

Withanolides from Withania somnifera Ameliorate Neutrophil Infiltration in Endotoxin-Induced Peritonitis by Regulating Oxidative Stress and Inflammatory Cytokines.

Identification of novel anti-inflammatory strategies are needed to avoid the side effects associated with the currently available therapies. Use of anti-inflammatory herbal remedies is gaining attention. The purpose of the present investigation was to evaluate the pharmacological potential of the withanolide-rich root extracts of the medical plant Withania somnifera (L.) Dunal using in vivo and in vitro models of endotoxin-induced inflammation and oxidative stress. The pharmacological effects of W. somnifera root extracts were evaluated using a mouse model of endotoxin (lipopolysaccharide)-induced peritonitis and various relevant human cell lines. HPLC analysis of the W. somnifera root extracts identified the presence of various bioactive withanolides. In vivo challenge of mice with endotoxin resulted in the infiltration of various leukocytes, specifically neutrophils, along with monocytes and lymphocytes into the peritoneal cavity. Importantly, prophylactic treatment with W. somnifera inhibited the migration of neutrophils, lymphocytes, and monocytes and decreased the release of interleukin-1ß, TNF-α, and interleukin-6 cytokines into the peritoneal cavity as identified by ELISA. Liver (glutathione peroxidase, glutathione, glutathione disulfide, superoxide dismutase, malondialdehyde, myeloperoxidase) and peritoneal fluid (nitrite) biochemical analysis revealed the antioxidant profile of W. somnifera. Similarly, in human HepG2 cells, W. somnifera significantly modulated the antioxidant levels. In THP-1 cells, W. somnifera decreased the secretion of interleukin-6 and TNF-α. In HEK-Blue reporter cells, W. somnifera inhibited TNF-α-induced nuclear factor-κB/activator protein 1 transcriptional activity. Our findings suggest the pharmacological effects of root extracts of W. somnifera rich in withanolides inhibit neutrophil infiltration, oxidative hepatic damage, and cytokine secretion via modulating the nuclear factor-κB/activator protein 1 pathway.

Improving the inhibition of ß-amyloid aggregation by withanolide and withanoside derivatives.

Here, we have studied the ameliorative effects of Withania somnifera derivatives (Withanolide A, Withanolide B, Withanoside IV, and Withanoside V) on the fibril formation of amyloid-ß 42 for Alzheimer's disease. We analyzed reduction in the aggregation of ß amyloid protein with these Ashwagandha derivatives by Thioflavin T assay in the oligomeric and fibrillar state. We have tested the cytotoxic activity of these compounds against human SK-N-SH cell line for 48 h, and the IC 50 value found to be 28.61 ± 2.91, 14.84 ± 1.45, 18.76 ± 0.76 and 30.14 ± 2.59 µM, respectively. After the treatment of the cells with half the concentration of IC 50 value, there was a remarkable decrease in the number of apoptotic cells stained by TUNEL assay indicating the DNA damage and also observed significant decrease of reactive oxygen species. Also, the binding and molecular stability of these derivatives with amyloid ß was also studied using bioinformatics tools where these molecules were interacted at LVFFA region which is inhibition site of amyloid-ß1 42. These studies revealed that the Withanolides and Withanosides interact with the hydrophobic core of amyloid-ß 1-42 in the oligomeric stage, preventing further interaction with the monomers and diminishing aggregation.

Ashwagandha: Advances in plant biotechnological approaches for propagation and production of bioactive compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera is one of the most extensively delved Ayurvedic medicine. Apart from rejuvenation and increasing longevity, it has several other properties such as immunomodulation, anti-cancer, anti-stress and neuroprotection. Because of its prevailing use and increasing demand, it becomes prudent to scientifically evaluate and document both its propagation and production of desired phytoconstituents. AIM OF THE STUDY: This review aims to highlight the research progress achieved on various biotechnological and tissue culture aspects of Withania somnifera and to cover up-to-date information regarding in-vitro propagation and production of withanolides. MATERIALS AND METHODS: Significant published studies were identified for the years 2000-2018 using Elsevier-Science Direct, Pubmed and Google scholar and several research studies in our laboratory. Following keywords such as "plant extracts", "in vitro cultures", "callus and suspension culture", "micropropagation", "hairy root cultures" were used. Further, "Withania somnifera", "secondary metabolites specially withanolides", "molecular techniques" and "in vitro conservation" were used to cross-reference the keywords. RESULTS: Ashwagandha comprises a broad spectrum of phytochemicals with a wide range of pharmacological properties. W. somnifera seeds have reduced viability and germination rates; thus, its regular cultivation method fails to achieve commercial demands mainly for the production of desired phytoconstituents. Cultivation of plant cells/tissues under in vitro conditions and development of various biotechnological strategies will help to build an attractive alternative to provide adequate quality and quantity raw materials. Recently, a large number of in vitro protocols has developed for W. somnifera not only for its propagation but for the production of secondary metabolites as well. Present work highlights a variety of biotechnological strategies both for prompt propagation and production of different bioactive secondary metabolites. CONCLUSION: The present review focuses on the development and opportunities in various biotechnological approaches to accomplish the global demand of W. somnifera and its secondary metabolites. This review underlines the advances in plant biotechnological approaches for the propagation of W. somnifera and production of its bioactive compounds.

Characterization and overexpression of sterol Δ22-desaturase, a key enzyme modulates the biosyntheses of stigmasterol and withanolides in Withania somnifera (L.) Dunal.

Withanolides constitute an extensive and vital class of metabolites displaying wide array of structural and therapeutic properties with unique side-chain modifications. These show diversified scaffolds and are promising pharmaceutical molecules with well documented anti-inflammatory and anti-cancer properties. Sterols are dynamic class of compounds and essential molecules having structural and functional significance. These contribute to the synthesis of withanolides by providing structural precursors. In this context, we have characterized sterol Δ22-desaturase from Withania somnifera and also functionally validating it by confirming its desaturase nature in conjunction with quantitative real-time expression profiling and metabolite evaluation. Further, transgenic hairy roots of W. somnifera displayed a higher accumulation of stigmasterol and withanolides. The increase in chemical constituents was concomitant with an increased gene copy number predicted via Southern blotting. Additionally, transgenic lines of tobacco over-expressing WsCYP710A11 displayed a substantial increase in its expression, corroborating well with enhanced stigmasterol content. Characterization of CYP710A11 from W. somnifera and its homologous transgenic expression has demonstrated its role in the regulation of withanolides biosynthesis. It also exhibited a differential transcriptional profile in response to exogenous elicitations. These empirical findings suggest the crucial role of CYP710A11 in stigmasterol biosynthesis. This in turn has implications for the overproduction of withanolides via pathway channelling.

Virus-Induced Gene Silencing for Functional Genomics in Withania somnifera, an Important Indian Medicinal Plant.

Virus-induced gene silencing (VIGS) has emerged as a fast and efficient reverse and forward genetics tool to study gene function in model plants as well as in agriculturally important plants. In addition, VIGS approach has been successfully used to provide insights into the role of several genes and regulators involved in plant secondary metabolism. Ashwagandha (Withania somnifera) is an important Indian medicinal plant that accumulates pharmacologically important triterpenoid steroidal lactones, which are collectively termed as withanolides. W. somnifera being a highly recalcitrant plant for genetic transformation, Tobacco rattle virus (TRV)-mediated VIGS was established by our group to facilitate understanding of withanolides' pathway. Here, we describe a detailed procedure to carry out VIGS for gene function studies in W. somnifera.

Withaferin A suppresses breast cancer cell proliferation by inhibition of the two-pore domain potassium (K2P9) channel TASK-3.

Withaferin A (WFA), a C5,C6-epoxy steroidal lactone isolated from the medicinal plant Withania somnifera (L.) Dunal, inhibits growth of tumor cells in different cancer types. However, the mechanisms underlying the effect of WFA on tumor cells are not fully understood. In the present study, we evaluated the blockade of TASK-3 channels by WFA in TASK-3-expressing HEK-293 cells. Explore if the WFA-mediated TASK-3 blockade can be used as a pharmacological tool to decrease the cell viability in cancer cells. A combination of functional experiments (patch-clamp, gene downregulation, overexpression and pharmacological inhibition) and molecular docking analysis were used to get insights into the mechanism by which the inhibition of TASK-3 by WFA affects the growth and viability of cancer cells. Withaferin A was found to inhibit the activity of TASK-3 channels. The inhibitory effect of Withaferin A on TASK-3 potassium currents was dose-dependent and independent of voltage. Molecular modeling studies identified putative WFA-binding sites in TASK-3 channel involved the channel blockade. In agreements with the molecular modeling predictions, mutation of residues F125 to A (F125A), L197 to V (L197 V) and the double mutant F125A-L197 V markedly decreased the WFA-induced inhibition of TASK-3. Finally, the cytotoxic effect of WFA was tested in MDA-MB-231 human breast cancer cells transfected with TASK-3 or shRNA that decreases TASK-3 expression. Together, our results show that the cytotoxic effect of WFA on fully transformed MDA-MB-231 cells depends on the expression of TASK-3. Herein, we also provide insights into the mechanism of TASK-3 inhibition by WFA.

Withanolide-A treatment exerts a neuroprotective effect via inhibiting neuroinflammation in the hippocampus after pilocarpine-induced status epilepticus.

Status epilepticus (SE) is a medical emergency with high mortality and a risk factor for the development of chronic epilepsy. Given that effective treatments for the pathophysiology following SE are still lacking, suppressing pathophysiological mechanisms of SE may be important to inhibit epileptogenesis. Withanolide-A (WA), a major bioactive component of Withania somnifera, is a potential medicinal natural compound showing improvement of some neurological diseases, such as cerebral ischemia. In the present study, we examined whether administration of WA can exert the beneficial effects involved in neuroprotection and anti-inflammatory effects in a mouse model of pilocarpine-induced SE. Our results showed that WA treatment ameliorated SE-induced apoptotic neuronal cell death in the hippocampus. Moreover, WA treatment reduced immunoreactivity of both ionized calcium binding adapter molecule 1-positive microglia/macrophage and glial fibrillary acidic protein-positive reactive astrocytes, and the SE-induced increase in both interleukin-1 ß and tumor necrosis factor in the hippocampus, suggesting that inhibiting pro-inflammatory factors by WA treatment might induce neuroprotection after SE. These results suggest that WA may be useful in improving the treatment efficacy for pathophysiology following SE.

Berry transcriptome: insights into a novel resource to understand development dependent secondary metabolism in Withania somnifera (Ashwagandha).

Withania somnifera (Ashwagandha) is considered as Rasayana in Indian systems of medicine. This study reports a novel transcriptome of W. somnifera berries, with high depth, quality and coverage. Assembled and annotated transcripts for nearly all genes related with the withanolide biosynthetic pathway were obtained. Tissue-wide gene expression analysis reflected almost similar definitions for the terpenoid pathway in leaf, root and berry tissues with relatively higher abundance of transcripts linked to steroid, phenylpropanoid metabolism as well as flavonoid metabolism in berries. The metabolome map generated from the data embodied transcripts from 143 metabolic pathways connected together and mediated collectively by about 1792 unique enzyme functions specific to berry, leaf and root tissues, respectively. Transcripts specific to cytochrome p450 (CYP450), methyltransferases and glycosyltransferases were distinctively located in a tissue specific manner and exhibited a complex network. Significant distribution of transcription factor genes such as MYB, early light inducible protein (ELI), minichromosome maintenance1, agamous, deficiens and serum response factor (MADS) and WRKY etc. was observed, as the major transcriptional regulators of secondary metabolism. Validation of the assembly was ascertained by cloning WsELI, which has a light dependent regulatory role in development. Quantitative expression of WsELI was observed to be considerably modulated upon exposure to abiotic stress and elicitors. Co-relation of over-expression of WsELI, may provide new aspects for the functional role of ELI proteins in plants linked to secondary metabolism. The study offers the first comprehensive and comparative evaluation of W. somnifera transcriptome data between the three tissues and across other members of Solanaceae (Nicotiana, Solanum and Capsicum) with respect to major pathways and their metabolome regulation.

The Natural Compound Withaferin A Covalently Binds to Cys239 of ß-Tubulin to Promote Tubulin Degradation.

Withaferin A (WIT) is a natural product possessing a wide range of pharmacologic activities. Previous studies have reported covalent binding of WIT to tubulin and down-of tubulin protein levels although the underlying mechanisms remain to be established. In the current investigation, we showed that WIT induces down-regulation of tubulin in a post-transcriptional manner, suggestive of direct and potent activity in tubulin degradation. The N,N'-ethylene bis(iodoacetamide) assay and competitive binding experiments with four colchicine site-targeted tubulin inhibitors further revealed that WIT interacts with the colchicine site of tubulin to promote degradation. WIT irreversibly inhibited tubulin polymerization, and mass spectrometry results disclosed binding to cysteine at position 239 (Cys239) and Cys303 sites of ß-tubulin. Interestingly, WIT promoted degradation of the ß-tubulin isoforms containing Cys239 [ß2, ß4, and ß5(ß)] but had no effect on those containing Ser239 (ß3 and ß6). Moreover, a C239S but not C303S mutation in ß-tubulin completely abolished the degradation effect of WIT, suggesting that the Cys239-WIT covalent bond accounts for this activity. Our collective results clearly demonstrate that covalent interactions between WIT and Cys239 of ß-tubulin promote tubulin degradation, supporting its potential utility as a therapeutic compound. SIGNIFICANCE STATEMENT: Withaferin A, a natural product possessing a wide range of pharmacologic activities, covalently binds to Cys239 of ß-tubulin near the colchicine site, and the WIT-Cys239 covalent bond accounts for WIT-induced tubulin degradation, fully clarifying the underlying mechanisms and supporting its potential utility a therapeutic compound.

Profiling withanolide A for therapeutic targets in neurodegenerative diseases.

To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects.

RNAi of Sterol Methyl Transferase1 Reveals its Direct Role in Diverting Intermediates Towards Withanolide/Phytosterol Biosynthesis in Withania somnifera.

The medicinal properties of Ashwagandha (Withania somnifera) are accredited to a group of compounds called withanolides. 24-Methylene cholesterol is the intermediate for sterol biosynthesis and a proposed precursor of withanolide biogenesis. However, conversion of 24-methylene cholesterol to withaferin A and other withanolides has not yet been biochemically dissected. Hence, in an effort to fill this gap, an important gene, encoding S-adenosyl l-methionine-dependent sterol-C24-methyltransferase type 1 (SMT1), involved in the first committed step of sterol biosynthesis, from W. somnifera was targeted in the present study. Though SMT1 has been characterized in model plants such as Nicotiana tabacum and Arabidopsis thaliana, its functional role in phytosterol and withanolide biosynthesis was demonstrated for the first time in W. somnifera. Since SMT1 acts at many steps preceding the withanolide precursor, the impact of this gene in channeling of metabolites for withanolide biosynthesis and its regulatory nature was illustrated by suppressing the gene in W. somnifera via the RNA interference (RNAi) approach. Interestingly, down-regulation of SMT1 in W. somnifera led to reduced levels of campesterol, sitosterol and stigmasterol, with an increase of cholesterol content in the transgenic RNAi lines. In contrast, SMT1 overexpression in transgenic N. tabacum enhanced the level of all phytosterols except cholesterol, which was not affected. The results established that SMT1 plays a crucial role in W. somnifera withanolide biosynthesis predominantly through the campesterol and stigmasterol routes.

Elucidating the active interaction mechanism of phytochemicals withanolide and withanoside derivatives with human serum albumin.

Withania somnifera (Ashwagandha) is an efficient medicinal plant known in Ayurveda and Chinese medicine since ancient times, whose extracts are consumed orally as food supplement or as a health tonic owing to its several restorative properties for various CNS disorders, inflammation, tumour, stress, rheumatism etc. In this study, we have analyzed the binding interaction of four derivatives of Withania somnifera (Withanolide A, Withanolide B, Withanoside IV and Withanoside V) with HSA because of their important pharmacological properties. To unravel the binding between derivatives of Withania somnifera and HSA, fluorescence spectroscopy was used. Binding studies were further studied by molecular docking and dynamics and results confirmed greater stability upon binding of derivatives with HSA. Circular dichroism data illustrated change in the secondary structure of protein upon interaction with these derivatives, particularly the helical structure was increased and ß-sheets and random coils were decreased. Furthermore, morphological and topological changes were observed using AFM and TEM upon binding of ligands with HSA indicating that HSA-withnoside/withanolide complexes were formed. All the results cumulatively demonstrate strong binding of withanosides and withanolides derivatives with serum albumin, which should further be explored to study the pharmacokinetics and pharmacodynamics of these derivatives.