ABSTRACT
The AAA+ ATPase p97 (valosin-containing protein, VCP) is a master regulator of protein homeostasis and therefore represents a novel target for cancer therapy. Starting from a known allosteric inhibitor, NMS-873, we systematically optimized this scaffold, in particular, by applying a benzene-to-acetylene isosteric replacement strategy, specific incorporation of F, and eutomer/distomer identification, which led to compounds that exhibited nanomolar biochemical and cell-based potency. In cellular pharmacodynamic assays, robust effects on biomarkers of p97 inhibition and apoptosis, including increased levels of ubiquitinated proteins, CHOP and cleaved caspase 3, were observed. Compound (R)-29 (UPCDC-30766) represents the most potent allosteric inhibitor of p97 reported to date.
ABSTRACT
Pet dogs with naturally occurring cancers play an important role in studies of cancer biology and drug development. We assessed tolerability, efficacy, and pharmacokinetic/pharmacodynamic relationships with a first-in-class small molecule inhibitor of valosin-containing protein (VCP/p97), CB-5339, administered to 24 tumor-bearing pet dogs. Tumor types assessed included solid malignancies, lymphomas, and multiple myeloma. Through a stepwise dose and schedule escalation schema, we determined the maximum tolerated dose to be 7.5 mg/kg when administered orally on a 4 days on, 3 days off schedule per week for 3 consecutive weeks. Adverse events were minimal and mainly related to the gastrointestinal system. Pharmacokinetic/pharmacodynamic data suggest a relationship between exposure and modulation of targets related to induction of the unfolded protein response, but not to tolerability of the agent. An efficacy signal was detected in 33% (2/6) of dogs with multiple myeloma, consistent with a mechanism of action relating to induction of proteotoxic stress in a tumor type with abundant protein production. Clinical trials of CB-5339 in humans with acute myelogenous leukemia and multiple myeloma are ongoing.
Subject(s)
Antineoplastic Agents , Lymphoma , Multiple Myeloma , Valosin Containing Protein , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Dogs , Enzyme Inhibitors/therapeutic use , Lymphoma/drug therapy , Lymphoma/pathology , Lymphoma/veterinary , Maximum Tolerated Dose , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Multiple Myeloma/veterinary , Unfolded Protein Response , Valosin Containing Protein/antagonists & inhibitorsABSTRACT
BACKGROUND: Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack. CONCLUSIONS/SIGNIFICANCE: Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.