Probing the anticancer mechanism of prospective herbal drug Withaferin A on mammals: a case study on human and bovine proteasomes

<p>Abstract</p> <p>Background</p> <p>The UPP (ubiquitin proteasome pathway) is the major proteolytic system in the cytosol and nucleus of all eukaryotic cells which regulates cellular events, including mitotis, differentiation, signal transduction, apoptosis, and inflammation. UPP controls activation of the transcriptional factor NF-κB (nuclear factor κB), which is a regulatory protein playing central role in a variety of cellular processes including immune and inflammatory responses, apoptosis, and cellular proliferation. Since the primary interaction of proteasomes occurs with endogenous proteins, the signalling action of transcription factor NF-κB can be blocked by inhibition of proteasomes. A great variety of natural and synthetic chemical compounds classified as peptide aldehydes, peptide boronates, nonpeptide inhibitors, peptide vinyl sulfones and epoxyketones are now widely used as research tools for probing their potential to inhibit proteolytic activities of different proteasomes and to investigate the underlying inhibition mechanisms. The present work reports a bio-computational study carried out with the aim of exploring the proteasome inhibition capability of WA (withaferin A), a steroidal lactone, by understanding the binding mode of WA as a ligand into the mammalian proteasomes (X-ray crystal structure of <it>Bos taurus</it> 20S proteasome and multiple template homology modelled structure of 20S proteasome of <it>Homo sapiens</it>) using molecular docking and molecular dynamics simulation studies.</p> <p>Results</p> <p>One possible mode of action which is proposed here for WA to act as a proteasome inhibitor is by suppression of the proteolytic activity which depends on the N-terminal threonine (Thr1) residue hydroxyl group. Docking studies carried out with herbal ligand WA into the structures of bovine and human proteasomes substantiate that WA has the ability to inhibit activity of mammalian 20S proteasomes by blocking the nucleophilic function of N-terminal Thr1. Results from molecular dynamics simulations in water show that the trajectories of both the native human 20S proteasome and the proteasome complexed with WA are stable over a considerably long time period of 4 ns suggesting the dynamic structural stability of human 20S proteasome/WA complex.</p> <p>Conclusions</p> <p>Inhibition of proteasomal activity are promising ways to retard or block degradation of specific proteins to correct diverse pathologies. Though quite a number of selective and efficient proteasomal inhibitors exist nowadays, their toxic side effects limit their potential in possible disease treatment. Thus there is an indispensable need for exploration of novel natural products as antitumor drug candidates. The present work supports the mammalian proteasomes inhibiting activity of WA along with elucidation of its possible mode of action. Since WA is a small herbal molecule, it is expected to provide one of the modest modes of inhibition along with added favours of ease in oral administration and decreased immunogenicity. The molecular docking results suggest that WA can inhibit the mammalian proteasomes irreversibly and with a high rate through acylation of the N-terminal Thr1 of the β-5 subunit.</p

Hsp90/Cdc37 Chaperone/co-chaperone complex, a novel junction anticancer target elucidated by the mode of action of herbal drug Withaferin A

<p>Abstract</p> <p>Background</p> <p>HSPs (Heat shock proteins) are highly conserved ubiquitous proteins among species which are involved in maintaining appropriate folding and conformation of other proteins and are thus referred to as molecular chaperones. Hsp90 (Heat-shock protein 90 kDa) is one of a group of molecular chaperones responsible for managing protein folding and quality control in cell environment. However it is also involved in the maturation and stabilization of a wide range of oncogenic client proteins which are crucial for oncogenesis and malignant progression. Hsp90 requires a series of co-chaperones to assemble into a super-chaperone complex for its function. These co-chaperones bind and leave the complex at various stages to regulate the chaperoning process. Arresting the chaperone cycle at these stages by targeting different co-chaperone/Hsp90 interactions seems to be quite a viable alternative and is likely to achieve similar consequences as that of Hsp90 direct inhibition with added favors of high specificity and reduced side effect profile. The study conducted here is an attempt to explore the potential of <it>Withania somnifera’s</it> major constituent WA (Withaferin A) in attenuating the Hsp90/Cdc37 chaperone/co-chaperone interactions for enhanced tumor arresting activity and to elucidate the underlying mode of action using computational approaches.</p> <p>Results</p> <p>Formation of active Hsp90/Cdc37 complex is one of the essential steps for facilitation of chaperone client interaction, non-assembly of which can lead to prevention of the chaperone-client association resulting in apoptosis of tumor cells. From our flexible docking analysis of WA into active Hsp90/Cdc37 complex in which key interfacing residues of the complex were kept flexible, disruption of the active association complex can be discerned. While docking of WA into segregated Hsp90 leaves the interface residues untouched. Thus the molecular docking analysis of WA into Hsp90 and active Hsp90/Cdc37 complex conducted in this study provides significant evidence in support of the proposed mechanism of chaperone assembly suppression by inhibition or disruption of active Hsp90/Cdc37 complex formation being accounted by non-assembly of the catalytically active Hsp90/Cdc37 complex. Results from the molecular dynamics simulations in water show that the trajectories of the protein complexed with ligand WA are stable over a considerably long time period of 4 ns, with the energies of the complex being lowered in comparison to the un-docked association complex, suggesting the thermodynamic stability of WA complexed Hsp90/Cdc37.</p> <p>Conclusions</p> <p>The molecular chaperone Hsp90 has been a promising target for cancer therapy. Cancer is a disease marked by genetic instability. Thus specific inhibition of individual proteins or signalling pathways holds a great potential for subversion of this genetic plasticity of cancers. This study is a step forward in this direction. Our computational analysis provided a rationalization to the ability of naturally occurring WA to alter the chaperone signalling pathway. The large value of binding energy involved in binding of WA to the active Hsp90/Cdc37 complex consolidates the thermodynamic stability of the binding. Our docking results obtained substantiate the hypothesis that WA has the potential to inhibit the association of chaperone (Hsp90) to its co-chaperone (Cdc37) by disrupting the stability of attachment of Hsp90 to Cdc37. Conclusively our results strongly suggest that withaferin A is a potent anticancer agent as ascertained by its potent Hsp90-client modulating capability.</p

Ashwagandha Derived Withanone Targets TPX2-Aurora A Complex: Computational and Experimental Evidence to its Anticancer Activity

Cancer is largely marked by genetic instability. Specific inhibition of individual proteins or signalling pathways that regulate genetic stability during cell division thus hold a great potential for cancer therapy. The Aurora A kinase is a Ser/Thr kinase that plays a critical role during mitosis and cytokinesis and is found upregulated in several cancer types. It is functionally regulated by its interactions with TPX2, a candidate oncogene. Aurora A inhibitors have been proposed as anticancer drugs that work by blocking its ATP binding site. This site is common to other kinases and hence these inhibitors lack specificity for Aurora A inhibition in particular, thus advocating the need of some alternative inhibition route. Previously, we identified TPX2 as a cellular target for withanone that selectively kill cancer cells. By computational approach, we found here that withanone binds to TPX2-Aurora A complex. In experiment, withanone treatment to cancer cells indeed resulted in dissociation of TPX2-Aurora A complex and disruption of mitotic spindle apparatus proposing this as a mechanism of the anticancer activity of withanone. From docking analysis, non-formation/disruption of the active TPX2-Aurora A association complex could be discerned. Our MD simulation results suggesting the thermodynamic and structural stability of TPX2-Aurora A in complex with withanone further substantiates the binding. We report a computational rationale of the ability of naturally occurring withanone to alter the kinase signalling pathway in an ATP-independent manner and experimental evidence in which withanone cause inactivation of the TPX2-Aurora A complex. The study demonstrated that TPX2-Aurora A complex is a target of withanone, a potential natural anticancer drug

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A

Abstract Background Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug. Results Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured. Conclusions We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus.</p

Identification, characterization and quantification of new impurities by LC–ESI/MS/MS and LC–UV methods in rivastigmine tartrate active pharmaceutical ingredient

Six impurities were detected at trace level in rivastigmine tartrate drug substance by a newly developed high performance liquid chromatography method. Three impurities were characterized rapidly and three impurities were found to be unknown. The unknown impurities were enriched and identified with a combination of semi-preparative HPLC and LC/MS/MS techniques. Proposed structures were further confirmed by characterization using NMR, FT-IR, and EA techniques of impurity standards. Based on the spectroscopic, spectrometric and elemental analysis data unknown impurities were characterized as 3-[1-(dimethylamino)ethyl]phenyl N-ethyl- N-methyl carbamate N-oxide, ethyl-methyl-carbamic acid 4-(1-dimethylamino-ethyl)-phenyl ester and ethyl-methyl-carbamic acid 2-(1-dimethylamino-ethyl)-phenyl ester. A plausible mechanism for the formation of these impurities is also proposed. The method was validated according to ICH guidelines for fourteen impurities to demonstrate specificity, precision, linearity, accuracy and stability indicating nature of the method. Regression analysis showed correlation coefficient value greater than 0.999 for rivastigmine tartrate and its impurities. Accuracy of the method was established based on the recovery obtained between 93.41 and 113.33% for all impurities

Identification and structural elucidation of two process impurities and stress degradants in darifenacin hydrobromide active pharmaceutical ingredient by LC-ESI/MSn

The present study describes the identification and characterization of two process impurities and major stress degradants in darifenacin hydrobromide using high performance liquid chromatography (HPLC) analysis. Forced degradation studies confirmed that the drug substance was stable under acidic, alkaline, aqueous hydrolysis, thermal and photolytic conditions and susceptible only to oxidative degradation. Impurities were identified using liquid chromatography coupled with ion trap mass spectrometry (LC-MS/MS n ). Proposed structures were unambiguously confirmed by synthesis followed by characterization using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR) and elemental analysis (EA). Based on the spectroscopic, spectrometric and elemental analysis data, the unknown impurities were characterized as 2-{1-[2-(2,3-dihydrobenzofuran-5-yl)-2-oxo-ethyl]-pyrrolidin-3-yl}-2,2-diphenylacetamide (Imp-A), 2-[1-(2-benzofuran-5-yl-ethyl)-pyrrolidin-3-yl]-2,2-diphenylacetamide (Imp-B), 2-{1-[2-(2,3-dihydrobenzofuran-5-yl)-ethyl]-1-oxy-pyrrolidin-3-yl}-2,2-diphenylacetamide (Imp-C) and 2-{1-[2-(7-bromo-2,3-dihydrobenzofuran-5-yl)-ethyl]-pyrrolidin-3-yl}-2,2-diphenylacetamide (Imp-D). Plausible mechanisms for the formation and control of these impurities have also been proposed. The method was validated as per regulatory guidelines to demonstrate specificity, sensitivity, linearity, precision, accuracy and the stability-indicating nature. Regression analysis showed a correlation coefficient value greater than 0.99 for darifenacin hydrobromide and its impurities. The accuracy of the method was established based on the recovery obtained between 86.6 and 106.7% for all impurities. The study discloses the identification and characterization of two process impurities and two oxidative stress degradants by LC-ESI/MS n

Highly efficient, selective, sensitive and stability indicating RP-HPLC–UV method for the quantitative determination of potential impurities and characterization of four novel impurities in eslicarbazepine acetate active pharmaceutical ingredient by LC/ESI-IT/MS/MS

► First report on a stability indicating validated LC–UV method for the quantification of potential impurities. ► Four novel impurities were identified and characterized. ► Minimum detectable limits of impurities were found to be as low as 0.005%. ► New LC–UV method was found to be giving excellent resolution for 15 impurities. A novel, sensitive, selective and stability indicating LC–UV method was developed for the determination of potential impurities of eslicarbazepine acetate. High performance liquid chromatographic investigation of eslicarbazepine acetate laboratory sample revealed the presence of several impurities. Three impurities were characterized rapidly and four impurities were found to be unknown. The unknown impurities were identified by liquid chromatography coupled with electrospray ionization, ion trap mass spectrometry (LC/ESI-IT/MS/MS). Structural confirmation of these impurities was unambiguously carried out by synthesis followed by characterization using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (FT–IR) and mass spectrometry (MS). Based on the spectroscopic, spectrometric and elemental analysis data unknown impurities were characterized as 5-acetyl-5,11-dihydro-10 H-dibenzo [ b, f]azepin-10-one, N-acetyl-5 H-dibenzo[ b, f]azepine-5-carboxamide, 5-acetyl-10,11-dihydro-5 H-dibenzo[ b, f]azepin-10-yl acetate and 5-acetyl-5 H-dibenzo[ b, f]azepin-10-yl acetate. The newly developed LC–UV method was validated according to ICH guidelines considering eleven potential impurities and four new impurities to demonstrate specificity, precision, linearity, accuracy and stability indicating nature of the method. The newly developed method was found to be highly efficient, selective, sensitive and stability indicating. A plausible pathway for the formation of four new impurities is proposed