Computational Study Reveals PARP1 and P2Y1 Receptors as Prospective Targets of Withaferin-A for Cardiovascular Diseases

Background: Cardiovascular Diseases (CVDs) remain the leading cause of death worldwide, which urges for effective strategies of prevention and treatment. Withaferin-A (WFA), the key metabolite identified in Withania somnifera, has been known for its cardioprotective properties. Although it has been traditionally employed to treat cardiovascular ailments for several decades, its exact mechanism of action still remains unexplained Objective: The current study modelled and scored the interactions of WFA with nine prospective protein-targets associated with cardiovascular diseases through molecular docking and DSX-scoring. Methods: Molecular docking was carried out using Autodock and DSX-scoring was carried out using DSX standalone software. WFA was observed to favorably interact with six targets before DSX-based rescoring, but only with Poly (ADP-Ribose) Polymerase-1 and P2Y Purinoceptor-1 after DSX-based rescoring. The spatial orientation, physicochemical properties and structural features of Withaferin-A were compared with that of these approved drugs by pharmacophore modeling and hierarchical clustering Results: The results of molecular docking, DSX-based rescoring and complete pharmacophore modeling together revealed that PARP1 and P2Y1 receptor could be prospective targets of WFA for the treatment of CVD. Conclusion: Simulation using GROMACS has revealed that WFA forms a more stable complex with PARP1 and will be useful in developing the broad-spectrum drugs against cardiovascular diseases. Further computational studies through machine learning and network pharmacology methods can be carried out to improve Withaferin-A compound features by incorporating additional functional groups necessary for molecular recognition of the target genes in network responsible for cardiovascular diseases.

Withaferin A: From Ancient Remedy to Potential Drug Candidate

Withaferin A (WA) is a pivotal withanolide that has conquered a conspicuous place in research, owning to its multidimensional biological properties. It is an abundant constituent in Withania somnifera Dunal. (Ashwagandha, WS) that is one of the prehistoric pivotal remedies in Ayurveda. This article reviews the literature about the pharmacological profile of WA with special emphasis on its anticancer aspect. We reviewed research publications concerning WA through four databases and provided a descriptive analysis of literature without statistical or qualitative analysis. WA has been found as an effective remedy with multifaceted mechanisms and a broad spectrum of pharmacological profiles. It has anticancer, anti-inflammatory, antiherpetic, antifibrotic, antiplatelet, profibrinolytic, immunosuppressive, antipigmentation, antileishmanial, and healing potentials. Evidence for wide pharmacological actions of WA has been established by both in vivo and in vitro studies. Further, the scientific literature accentuates the role of WA harboring a variable therapeutic spectrum for integrative cancer chemoprevention and cure. WA is a modern drug from traditional medicine that is necessary to be advanced to clinical trials for advocating its utility as a commercial drug.

Analysis Of Anticancer Activity And Its Molecular Interaction Mechanism Of Withanone, An Active Ingredient Of Withania Somnifera Using Molecular Docking

Withania somnifera is an annual evergreen shrub from the Solanaceae family, commonly known as Indian ginseng or Ashwagandha. The plant is mainly found in Asia and Africa regions. In the traditional Indian medicinal system ayurveda, Withania somnifera is used as a rejuvenator and sold in many countries as a dietary supplement. Withanolides are the major phytochemical constituent group found in the Withania somnifera, among which withaferin A and withanone, are considered to be major withanolides, which believed to be involved in majority of biological activity of Withania somnifera. Various studies including both in vitro and in vivo have reported regarding the anticancer potential of Withania somnifera. Along with the anticancer activity of W.somnifera, the anticancer efficacy of one of its major ingredients Withaferin A is also studied previously. This study aimed to analyse the anticancer potential of another major Withanolide present in W. Somnifera, Withanone. The study used Molecular Docking method to find the molecular binding affinity of Withanone towards various cancer proteins. The four major cancer proteins were B-cell lymphoma- extra large (Bcl-xL), Cellular FLICE (c-FLIP), Glutathione Reductase (GR) and Glutathione S- Transferases (GST). The protein structure obtained from the protein data bank and the structure of the molecule obtained from pubchem were modified and prepared for Docking studies with the help of MGL Tools. The protein ligand interaction study was conducted using the software, Autodock vina. The already known anticancer standard, 5-FluoroUracil is used as standard for comparison. Output obtained from the study is visualised using molecular visualiser tool, Pymol. Like the Withaferin A, Withanone also exhibited promising anticancer activity while studied using molecular docking methods.

Phosphocatalytic Kinome Activity Profiling of Apoptotic and Ferroptotic Agents in Multiple Myeloma Cells

Through phosphorylation of their substrate proteins, protein kinases are crucial for transducing cellular signals and orchestrating biological processes, including cell death and survival. Recent studies have revealed that kinases are involved in ferroptosis, an iron-dependent mode of cell death associated with toxic lipid peroxidation. Given that ferroptosis is being explored as an alternative strategy to eliminate apoptosis-resistant tumor cells, further characterization of ferroptosis-dependent kinase changes might aid in identifying novel druggable targets for protein kinase inhibitors in the context of cancer treatment. To this end, we performed a phosphopeptidome based kinase activity profiling of glucocorticoid-resistant multiple myeloma cells treated with either the apoptosis inducer staurosporine (STS) or ferroptosis inducer RSL3 and compared their kinome activity signatures. Our data demonstrate that both cell death mechanisms inhibit the activity of kinases classified into the CMGC and AGC families, with STS showing a broader spectrum of serine/threonine kinase inhibition. In contrast, RSL3 targets a significant number of tyrosine kinases, including key players of the B-cell receptor signaling pathway. Remarkably, additional kinase profiling of the anti-cancer agent withaferin A revealed considerable overlap with ferroptosis and apoptosis kinome activity, explaining why withaferin A can induce mixed ferroptotic and apoptotic cell death features. Altogether, we show that apoptotic and ferroptotic cell death induce different kinase signaling changes and that kinome profiling might become a valid approach to identify cell death chemosensitization modalities of novel anti-cancer agents.

Preparation, Characterization, and Pharmacological Investigation of Withaferin-A Loaded Nanosponges for Cancer Therapy; In Vitro, In Vivo and Molecular Docking Studies

The rapidly growing global burden of cancer poses a major challenge to public health and demands a robust approach to access promising anticancer therapeutics. In parallel, nanotechnology approaches with various pharmacological properties offer efficacious clinical outcomes. The use of new artificial variants of nanosponges (NS) as a transporter of chemotherapeutic drugs to target cells has emerged as a very promising tool. Therefore, in this research, ethylcellulose (EC) NS were prepared using the ultrasonication assisted-emulsion solvent evaporation technique. Withaferin-A (WFA), an active ingredient in Withania somnifera, has been implanted into the nanospongic framework with enhanced anticancer properties. Inside the polymeric structure, WFA was efficiently entrapped (85 ± 11%). The drug (WFA) was found to be stable within polymeric nanosponges, as demonstrated by Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) studies. The WFA-NS had a diameter of 117 ± 4 nm and zeta potential of −39.02 ± 5.71 mV with a polydispersity index (PDI) of 0.419 ± 0.073. In addition, scanning electron microscopy (SEM) revealed the porous surface texture of WFA-NS. In vitro anticancer activity (SRB assay) results showed that WFA–NS exhibited almost twice the anticancer efficacy against MCF-7 cells (IC50 = 1.57 ± 0.091 µM), as quantified by flow cytometry and comet tests. Moreover, fluorescence microscopy with DAPI staining and analysis of DNA fragmentation revealed apoptosis as a mechanism of cancer cell death. The anticancer activity of WFA-NS was further determined in vivo and results were compared to cisplatin. The anticancer activity of WFA-NS was further investigated in vivo, and the data were consistent to those obtained with cisplatin. At Day 10, WFA-NS (10 mg/kg) significantly reduced tumour volume to 72 ± 6%, which was comparable to cisplatin (10 mg/kg), which reduced tumour volume to 78 ± 8%. Finally, the outcomes of molecular modeling (in silico) also suggested that WFA established a stable connection with nanosponges, generating persistent hydrophobic contacts (polar and nonpolar) and helping with the attractive delayed-release features of the formulation. Collectively, all the findings support the use of WFA in nanosponges as a prototype for cancer treatment, and opened up new avenues for increasing the efficacy of natural product-derived medications.

Withaferin A Promotes White Adipose Browning and Prevents Obesity Through Sympathetic Nerve-Activated Prdm16-FATP1 Axis

The increasing prevalence of obesity has resulted in demands for the development of new effective strategies for obesity treatment. The Withaferin A (WA) shows a great potential for prevention of obesity by sensitizing leptin signaling in the hypothalamus. However, the mechanism underlying the weight- and adiposity-reducing effects of WA remains to be elucidated. Here, we report that WA treatment induced white adipose tissue (WAT) browning, elevated energy expenditure (EE), decreased respiratory exchange ratio (RER), and prevented high-fat diet (HFD)-induced obesity. The sympathetic chemical denervation dampened the WAT browning and also impeded the reduction of adiposity in WA-treated mice. WA markedly up-regulated the levels of Prdm16 and FATP1 (Slc27a1) in the inguinal WAT (iWAT), and this was blocked by sympathetic denervation. Prdm16 or FATP1 knockdown in iWAT abrogated the WAT browning-inducing effects of WA, and restored the weight gain and adiposity in WA-treated mice. Together, these findings suggest that WA induces WAT browning through the sympathetic nerve-adipose axis; and the adipocytic Prdm16-FATP1 pathway mediates the promotive effects of WA on white adipose browning.

Withaferin A Promotes White Adipose Browning and Prevents Obesity Through Sympathetic Nerve-Activated Prdm16-FATP1 Axis

The increasing prevalence of obesity has resulted in demands for the development of new effective strategies for obesity treatment. The Withaferin A (WA) shows a great potential for prevention of obesity by sensitizing leptin signaling in the hypothalamus. However, the mechanism underlying the weight- and adiposity-reducing effects of WA remains to be elucidated. Here, we report that WA treatment induced white adipose tissue (WAT) browning, elevated energy expenditure (EE), decreased respiratory exchange ratio (RER), and prevented high-fat diet (HFD)-induced obesity. The sympathetic chemical denervation dampened the WAT browning and also impeded the reduction of adiposity in WA-treated mice. WA markedly up-regulated the levels of Prdm16 and FATP1 (Slc27a1) in the inguinal WAT (iWAT), and this was blocked by sympathetic denervation. Prdm16 or FATP1 knockdown in iWAT abrogated the WAT browning-inducing effects of WA, and restored the weight gain and adiposity in WA-treated mice. Together, these findings suggest that WA induces WAT browning through the sympathetic nerve-adipose axis; and the adipocytic Prdm16-FATP1 pathway mediates the promotive effects of WA on white adipose browning.