Small-Molecule Modulators of the ATPase VCP/p97 Affect Specific p97 Cellular Functions

Area of Science:

• Molecular and Cellular Biology
• Biochemistry
• Chemical Biology

Background:

• Valosin-containing protein (VCP), also known as p97, is an AAA+ ATPase crucial for cellular processes like protein homeostasis and cell division.
• Existing VCP/p97 inhibitors target the main ATPase domain, leading to cell death, limiting their therapeutic potential.
• The regulatory domain of VCP/p97, comprising N-terminal and D1 ATPase domains, presents an alternative target for modulating its function.

Purpose of the Study:

• To identify novel small molecules targeting the regulatory domain of VCP/p97.
• To characterize the mechanism of action and cellular effects of these new compounds.
• To explore the role of VCP/p97 in specific cellular processes, such as mitotic spindle orientation.

Main Methods:

• Utilized PNA-encoded chemical libraries for high-throughput screening.
• Performed ATPase assays to assess the catalytic activity of VCP/p97 in the presence of compounds.
• Employed photoaffinity labeling to map the binding site of the identified small molecules.
• Investigated the impact of compounds on VCP/p97-dependent reporter degradation and mitotic spindle orientation in HeLa cells.

Main Results:

• Identified two novel small molecules, NW1028 and NW1030, that bind to the regulatory domain of VCP/p97.
• NW1028 inhibits a VCP/p97-dependent reporter degradation, while NW1030 increases it, indicating distinct functional modulation.
• ATPase assays confirmed that NW1028 and NW1030 do not inhibit the main catalytic ATPase domain of VCP/p97.
• Photoaffinity labeling mapped the binding site to a cleft between the N-terminal and D1 ATPase domains.
• Demonstrated a role for VCP/p97 in regulating mitotic spindle orientation in HeLa cells using these compounds.

Conclusions:

• Discovered two unique small molecules, NW1028 and NW1030, that selectively target the regulatory domain of VCP/p97.
• These compounds offer a new avenue for modulating VCP/p97 functions without inducing general cytotoxicity.
• The study reveals a novel function for VCP/p97 in the regulation of mitotic spindle orientation, elucidated through the use of these specific inhibitors.