CB-5083: A Selective, Orally Bioavailable p97 AAA-ATPase Inhibitor

Executive Summary: CB-5083 is a potent, selective inhibitor of the AAA ATPase p97, also known as valosin-containing protein (VCP), exhibiting an IC₅₀ of 15.4 nM for wild-type p97 in biochemical assays (APExBIO). The compound competitively binds to the second ATPase domain of p97, disrupting protein degradation and inducing the unfolded protein response (UPR) in cancer cells (Carrasquillo Rodríguez et al. 2024). In vitro, CB-5083 causes dose-dependent accumulation of poly-ubiquitinated proteins and apoptosis in human cell lines, including HEK293T, A549, and HCT116 (APExBIO). In vivo, oral dosing in mouse xenograft models leads to significant inhibition of tumor growth and UPR activation. CB-5083 is highly soluble in DMSO (≥20.65 mg/mL) and ethanol (≥4.4 mg/mL), but insoluble in water, and should be stored at -20°C for stability (APExBIO).

Biological Rationale

The AAA ATPase p97/VCP is a central regulator of the ubiquitin-proteasome system and endoplasmic reticulum-associated degradation (ERAD) (Carrasquillo Rodríguez et al. 2024). p97 coordinates extraction of misfolded proteins from the ER membrane for proteasomal degradation, maintaining protein homeostasis and organelle function. Disruption of p97 activity impairs protein quality control and triggers ER stress and UPR, processes implicated in cancer cell survival and apoptosis. Recent studies highlight crosstalk between protein homeostasis and ER lipid metabolism, with p97 activity influencing both proteostasis and lipid droplet formation (Carrasquillo Rodríguez et al. 2024). Small molecule p97 inhibitors like CB-5083 enable targeted interrogation of these pathways in experimental and translational oncology.

Mechanism of Action of CB-5083

CB-5083 selectively inhibits p97 by engaging its second ATPase domain (D2), competing with ATP for binding (APExBIO). The inhibitor is highly selective for p97 over other AAA ATPases, with no significant off-target activity at concentrations up to 10 μM. Biochemical assays demonstrate an IC₅₀ of 15.4 nM against purified human p97 under standard buffer conditions (50 mM Tris-HCl, pH 8.0, 5 mM MgCl₂, 25°C), using ATPase activity as a readout. In cell-based models, CB-5083 treatment results in rapid accumulation of poly-ubiquitinated substrates and disruption of protein degradation, leading to activation of the UPR and caspase-dependent apoptosis at micromolar concentrations. This mechanism underpins its antitumor efficacy in preclinical models.

Evidence & Benchmarks

• CB-5083 exhibits an IC₅₀ of 15.4 nM for wild-type p97 in vitro ATPase assays (50 mM Tris-HCl, pH 8.0, 5 mM MgCl₂, 25°C) (APExBIO).
• CB-5083 induces dose-dependent accumulation of poly-ubiquitinated proteins in HEK293T, A549, and HCT116 cells (1–10 μM, 24 hr) (APExBIO).
• Oral administration of CB-5083 (10–30 mg/kg, qd, p.o.) in mouse xenograft models of lung carcinoma, colorectal adenocarcinoma, and multiple myeloma leads to significant tumor growth inhibition and UPR activation (APExBIO).
• CB-5083 disrupts ER-associated degradation and protein quality control, as reflected by increased ER stress and apoptotic markers in treated cancer cells (Carrasquillo Rodríguez et al. 2024).
• CB-5083 advances to phase 1 clinical trials for multiple myeloma and solid tumors, confirming translational potential (APExBIO).

This article extends and clarifies mechanistic insights from "CB-5083: Disrupting Protein Homeostasis and ER Lipid Regulation" by providing quantitative benchmarks and detailed experimental conditions for reproducibility. For strategic workflow guidance, see also "CB-5083: Selective p97 Inhibitor for Protein Homeostasis Research", which this article updates with recent in vivo efficacy data.

Applications, Limits & Misconceptions

CB-5083 is widely used in translational cancer research to study protein homeostasis, UPR induction, and apoptosis in solid and hematologic malignancies. It is suitable for in vitro biochemical and cellular assays, as well as in vivo mouse xenograft studies. CB-5083 is a key tool for dissecting the role of p97 in ER stress, proteostasis, and the interplay with lipid metabolism. However, its utility is limited by solubility constraints (insoluble in water; use DMSO or ethanol), and it is not recommended for chronic in vivo studies beyond acute tumor models due to potential off-target ER stress effects at high doses. CB-5083 is not a pan-AAA ATPase inhibitor and does not effectively inhibit unrelated ATPases at relevant concentrations.

Common Pitfalls or Misconceptions

• CB-5083 is not a general proteasome inhibitor; it acts upstream by inhibiting p97/VCP-mediated substrate extraction.
• The compound should not be stored in solution for long periods; use fresh aliquots due to stability concerns (APExBIO).
• CB-5083 is ineffective in cell-free proteasome assays lacking p97.
• Water-based buffers are unsuitable for CB-5083 due to insolubility; always dissolve in DMSO or ethanol.
• Chronic administration in animal models may trigger off-target ER stress responses not observed in short-term dosing.

Workflow Integration & Parameters

For in vitro assays, dissolve CB-5083 in DMSO (≥20.65 mg/mL) or ethanol (≥4.4 mg/mL) and dilute to working concentrations (10–1000 nM for cell-based; 1–100 nM for biochemical). Avoid freeze-thaw cycles and use fresh solutions. In vivo, oral gavage is recommended (10–30 mg/kg/day, typically for 7–21 days) in mouse xenograft models. Store solid material at -20°C. CB-5083 can be used alongside proteasome inhibitors or ER stress markers for combinatorial studies. For troubleshooting and maximizing reproducibility, consult the B6032 kit instructions and recent methodology reviews (APExBIO).

This article updates practical integration strategies described in "CB-5083: Unraveling p97 Inhibition for Advanced Cancer and Metabolic Disease" by emphasizing validated dosing, solubility, and storage parameters.

Conclusion & Outlook

CB-5083, available from APExBIO as product B6032, is a benchmark tool for dissecting p97/AAA ATPase signaling, protein homeostasis disruption, and tumor growth inhibition in cancer models. Its nanomolar potency, selectivity, and oral bioavailability support both mechanistic studies and translational research. Ongoing clinical and preclinical results affirm its value for investigating ER quality control, caspase signaling, and the intersection of proteostasis and lipid metabolism. For up-to-date protocols, reference the CB-5083 product page and curated literature.