User talk:Anshika Malik

From Proteopedia

Summary

The crystal structure of the human Hsp90 N-terminal domain in complex with the small-molecule inhibitor SNX-2112 (PDB ID: 6LTK) reveals the molecular basis of Hsp90 inhibition. SNX-2112 binds deeply within the ATP-binding pocket of Hsp90, blocking its chaperone function. Since Hsp90 is essential for the stability of multiple oncogenic proteins, its inhibition leads to cancer cell death.

This structure supports the development of Hsp90 inhibitors as potential therapeutic agents for non-small cell lung cancer (NSCLC).

The structure and biological activity of SNX-2112 are described in the paper:

Complex Crystal Structure Determination and In Vitro Anti-Non-Small Cell Lung Cancer Activity of Hsp90N Inhibitor SNX-2112 Frontiers in Molecular Biosciences (2021) DOI: 10.3389/fmolb.2021.670964

Structure

The three-dimensional structure of the human Hsp90 N-terminal domain in complex with SNX-2112 was determined by X-ray crystallography at a resolution of 2.14 Å (PDB ID: 6LTK).

SNX-2112 binds in the N-terminal ATP-binding pocket of Hsp90, occupying the site normally used by ATP. This binding blocks the ATP-dependent chaperone activity of Hsp90.

The binding site is stabilized through:

• Hydrogen bonding interactions
• Water-mediated interactions
• Hydrophobic contacts
• A halogen bond involving the fluorine atom of SNX-2112

This structure provides a molecular-level explanation for the strong inhibitory activity of SNX-2112.

Function

Hsp90 is an essential molecular chaperone involved in the folding, stabilization, and activation of many cellular proteins, including proteins involved in cancer progression.

Binding of SNX-2112 to the N-terminal domain prevents ATP binding and inhibits Hsp90’s chaperone function, leading to degradation of client oncoproteins.

Disease Relevance

The study investigated the anti-cancer activity of SNX-2112 in non-small cell lung cancer (NSCLC) cell lines including A549, H1299, and H1975.

SNX-2112 demonstrated:

• Decreased cell viability
• Significant induction of apoptosis
• Alteration of the cell cycle

These findings support the therapeutic potential of Hsp90 inhibitors in the treatment of NSCLC.

Key Structural Interactions

Important residues involved in SNX-2112 binding include:

Hydrogen bonds:

• D93
• K58
• S52
• Y139

Hydrophobic interactions:

• M98
• L103
• L107
• F138
• W162

Additional interactions:

• π–π stacking with F138
• Water-mediated hydrogen bond network
• Halogen bond between fluorine of SNX-2112 and backbone of G135

The strong binding affinity of SNX-2112 for Hsp90N was confirmed by biophysical techniques.

Experimental Methods

• The Hsp90N protein was expressed in E. coli BL21 (DE3)
• Purification was carried out using Ni-NTA affinity chromatography and gel filtration
• Crystallization was performed using the hanging-drop vapor diffusion method at 4°C
• The structure was solved by molecular replacement
• Cell viability was measured using CCK-8 assay
• Apoptosis was measured using Annexin V-FITC/PI staining
• Cell cycle analysis was performed by flow cytometry
• Binding affinity was measured using a thermal shift assay and isothermal titration calorimetry (ITC)

Reference

Zhao, D. et al., 2021. Complex Crystal Structure Determination and In Vitro Anti-Non-Small Cell Lung Cancer Activity of Hsp90N Inhibitor SNX-2112. Frontiers in Molecular Biosciences. DOI: 10.3389/fmolb.2021.670964