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

Abstract

Background: 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 Bos taurus 20S proteasome and multiple template homology modelled structure of 20S proteasome of Homo sapiens) using molecular docking and molecular dynamics simulation studies.

Results: 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.

Conclusions: 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.

Figure 1

Structures of withanolides. (A) WA falls under the family of compounds known as withanolides which are a group of naturally occurring C28- steroidal lactones built on an intact or rearranged ergostane framework, in which C-22 and C- 26 are appropriately oxidized to form a six-membered lactone ring. The basic skeleton shown here is designated as the withanolide skeleton defined as a 22-hydroxyergostan-26-oic acid-26,22-lactone. (B) Structure of WA. It contains two sites which are prone to nucleophilic attack: A six membered δ-valero lactone ring containing a carbocyclic ester group and a three membered epoxy ring.

Figure 2

Docking representations of WA into b20S. (A) Docking of WA into the cavity of b20S. (B) Docked ligand being trapped inside the S1 pocket of the β-5 subunit of receptor mesh.

Figure 3

Interactions of the ligand in different docked conformations of b20S. (A) Docked conformation showing proximity of lactone ring’s carbonyl group of WA to the hydroxyl group of N-terminal Thr1 of β-5 subunit of b20S proteasome. (B) Docked conformation showing proximity of three membered epoxy ring of WA to the hydroxyl group of N-terminal Thr1 of β-5 subunit of b20S proteasome.

Figure 4

Conformation of docked ligand occupying S1 pocket of the modelled h20S.

Figure 5

Positioning of WA in the docked structure of h20S. Lactone ring of WA positions itself quite close to the hydroxyl group of Thr1 of h20S receptor thus making itself prone to the nucleophilic attack by Thr1 of β-5 subunit.

Figure 6

Docking representation of the drug WA inside the cavity of h20S obtained using ParDOCK.

Figure 7

(A) Plot of root mean square deviation (RMSD) of Cα of h20S (protein) and h20S/WA (complex). RMSDs were calculated using the initial structures as templates. For protein (red) the reference is the modelled structure and for complex (blue) the reference is the initial model. The trajectories were captured every 1 ps until the simulation time reached 4000 ps. (B) Plot of total energy of h20S and h20S/WA (complex). The energy trajectories of both the protein (red) and the complex (blue) are stable over the entire length of simulation time.