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

Abstract

Background: 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 Withania somnifera's 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.

Results: 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.

Conclusions: 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.

Figure 1

Structures of withanolides. (A) Withaferin A 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 structure is designated as the withanolide skeleton defined as a 22-hydroxyergostan-26-oic acid-26,22-lactone. (B) Structure of withaferin A

Figure 2

Docking representations of Withaferin A. (A) Ligand docked into the Hsp90 receptor cavity (B) Docked Ligand inside the pocket of Hsp90 receptor mesh

Figure 3

Interactions of docked withaferin A with Hsp90 receptor. (A) H-Bond interactions of the docked ligand with Hsp90 residues. (B) Docked withaferin A forming vdw interactions with the hydrophobic residues of Hsp90.

Figure 4

Interactions of docked withaferin A with Hsp90 receptor. The docked withaferin A does not interact with any of the Cdc37 interacting residues of Hsp90.

Figure 5

H-bonding interactions present in the docked complex of withaferin A with active Hsp90/Cdc37 complex using flexible docking. Polar hydroxyl terminal group of withaferin A captivates amino group of Gln 208 of Cdc37 to form H-bond with itself, thus disrupting the earlier present Gln 208 - Ser 113H-bond. WA also disrupts the H-bond between Gln 133-Arg 166 by forming H-bond with Arg 166 of Cdc37. WA also forms H-bonds with Asp 54 and Asp 106 of Hsp90.

Figure 6

(A) Plot of root mean square deviation (RMSD) of Cα of Hsp90/Cdc37 (protein) and Hsp90/Cdc37/WA (complex). RMSDs were calculated using the initial structures as templates. For protein (red) the reference is the PDB structure and for complex (blue) the reference is the initial docked structure. The trajectories were captured every 2.5 ps until the simulation time reached 4000 ps. (B) Plot of total energy of Hsp90/Cdc37 and Hsp90/Cdc37/WA (complex) The energy trajectories of both the protein (red) and the complex (blue) are stable over the entire length of simulation time.

Figure 7

(A) Plot of root mean square deviation (RMSD) of Cα of Hsp90 (protein) and Hsp90/ WA (complex). RMSDs were calculated using the initial structures as templates. For protein (red) the reference is the PDB structure and for complex (blue) the reference is the initial docked structure. The trajectories were captured every 2.5 ps until the simulation time reached 3200 ps. (B) Plot of total energy of Hsp90 and Hsp90/WA (complex) The energy trajectories of both the protein (red) and the complex (blue) are stable over the entire length of simulation time.