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

Determination of Antioxidant Capacity and Free Radical Scavenging Activity of Milk from Native Cows (Bos Indicus), Exotic Cows (Bos Taurus), and Riverine Buffaloes (Bubalus Bubalis) Across Different Lactation Stages

The aim of this study was to evaluate comparative changes in total antioxidant capacity and free radical scavenging activity of milk during lactation in different cattle types and buffaloes. Milk samples from a total of 96 healthy animals of Sahiwal cows (Indian native cattle), Karan Fries cows (Cross-bred), Holstein Frisian cows (exotic cattle) and Murrah buffaloes (Riverine buffaloes) were collected at different lactation stages; early lactation (5-15 days), peak (30-60 days), mid (100-140 days) and late lactation (>215 days). The total antioxidant capacity (TAC) of milk was measured by ferric reducing/antioxidant power assay (FRAP) and free radical scavenging activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. TAC in milk was higher during early lactation. Similar results were observed for DPPH radical scavenging activity of the samples. The data suggested that milk during the early lactation period of dairy cows and buffaloes had higher content of antioxidants in comparison to other stages of lactation

Seasonal effect and long-term nutritional status following exit from a Community-Based Management of Severe Acute Malnutrition program in Bihar, India.

BACKGROUND/OBJECTIVES: Children aged 6 months to 5 years completing treatment for severe acute malnutrition (SAM) in a Médecins Sans Frontières Community Management of Acute Malnutrition (CMAM) program in Bihar, India, showed high cure rates; however, the program suffered default rates of 38%. This report describes the nutritional status of 1956 children followed up between 3 and 18 months after exiting the program. SUBJECTS/METHODS: All children aged 6-59 months discharged as cured with mid-upper arm circumference (MUAC) ⩾120 mm or who defaulted from the program with MUAC <115 mm were traced at 3, 6, 9, 12 and 18 months (±10 days) before three exit reference dates: first at the end of the food insecure period, second after the 2-month food security and third after the 4-month food security. RESULTS: Overall, 68.7% (n=692) of defaulters and 76.2% (n=1264) of children discharged as cured were traced. Combined rates of non-recovery in children who defaulted with MUAC <115 mm were 41%, 30.1%, 9.9%, 6.1% and 3.6% at 3, 6, 9, 12 and 18 months following exit, respectively. Combined rates of relapse among cured cases (MUAC ⩾120 mm) were 9.1%, 2.9%, 2.1%, 2.8% and 0% at 3, 6, 9, 12 and 18 months following discharge, respectively. Prevalence of undernutrition increased substantially for both groups traced during low food security periods. Odds of death were much higher for children defaulting with MUAC <110 mm when compared with children discharged as cured, who shared the same mortality risk as those defaulting with MUAC 110-<115 mm. CONCLUSIONS: Seasonal food security predicted short-term nutritional status after exit, with relapse rates and non-recovery from SAM much higher during food insecurity. Mortality outcomes suggest that a MUAC of 110 mm may be considered an appropriate admission point for SAM treatment programs in this context

Structural and Molecular Analysis of a Protective Epitope of Lyme Disease Antigen OspA and Antibody Interactions

The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 based on computational predictions on the crystal structure of the complex, and experimentally tested for in-vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA-2 light chain, N52 on the LA-2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA-2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for design of vaccines against Lyme disease

Politics of #LoSha: using naming and shaming as a feminist tool on Facebook

This chapter examines the new feminist intervention in India against sexual harassment (SH) through the online weapon of anonymously listing sexual offenders. The publication of the list on Facebook—known as the List of Shame (or #LoSha)—was inspired by the #metoo campaign following the Hollywood Weinstein affair and was composed through a collection of first-hand survivor narratives. A list of 70 names of alleged academic sexual offenders was first shared by a lawyer based in the US, and became viral on Facebook. This chapter will look at how this campaign used naming as a risk-taking tool to point at the lack of institutional frameworks within academic spaces. In doing so, it successfully used the online space of Facebook to create a feminist debate around the issue of sexual harassment transcending geographical and hierarchical barriers and to raise questions regarding the viability of the established feminist recourses against SH. Using the methodological tool of situated critique (Bannerji, Thinking Through: Essays on Feminism, Marxism, and Anti-Racism. Toronto: Women’s Press, 1995), in this chapter I will utilize my own experience of participating in the list as well as in the larger feminist debate to discuss the politics of risk-taking and solidarity and the implications of list-activism. In doing so, it has re-established the role of cyberfeminism (Daniels, Women’s Studies Quarterly, 37 (1 & 2): 101–124, 2009) in India and surfaced a new intersectional autocritique of the academia based on caste, class and gender. Though questions regarding the method remain, the use of Facebook for providing survivors a voice with anonymity promises new boundaries of empowerment and fear