NMS-873 functions as a dual inhibitor of mitochondrial oxidative phosphorylation
Graphical abstract
Introduction
Small-molecule enzyme inhibitors are an important pharmacologic tool for basic research. Prior to the wide-spread use of RNA interference and genome editing techniques, selective pharmacologic inhibitors were the mainstay for perturbing enzymes as a means to inferring their functions in the cell. The relative stability and ease with which most small-molecule inhibitors can be delivered to cells continues to make them an attractive choice for basic science investigators seeking to selectively and specifically block a given enzyme or cellular process.
Despite their advantages, one of the key problems associated with pharmacologic agents is their potential for off-target effects [1]. Because many classes of enzymes share similar structural motifs within their catalytic domain, it is common for small-molecule inhibitors to bind a broad set of related enzymes at various levels of affinity. This challenge has been well documented for compounds that function as competitive inhibitors, especially those that mimic the structure of generic enzyme substrates such as ATP [2,3]. As such it is often difficult to achieve a high degree of selectivity when screening for drugs that inhibit the activity of ATP-dependent enzymes such as kinases and ATPases.
One means by which the specificity and selectivity of small-molecule inhibitors can be improved is to identify compounds that bind to allosteric regulatory sites on a target enzyme rather than to the catalytic domain [[4], [5], [6]]. Because allosteric sites are usually less conserved than the active site across families of enzymes, it is possible to achieve significant selectivity for a specific enzyme within a broader class. The drug NMS-873, which was identified as a low nanomolar concentration inhibitor of the AAA ATPase VCP/p97, provides a good example of this concept [7]. NMS-873 functions by binding to the interface of two adjacent domains within the active hexameric structure of VCP/p97 and leads to an interruption of its catalytic cycle by stabilizing the ADP-bound state. Unlike competitive inhibitors discovered in the same study, NMS-873 showed a high degree of selectivity for VCP/p97 compared to other AAA ATPases and a panel of 53 kinases. The authors attribute this selectivity to fact that NMS-873 is not an ATP mimetic and binds to a site on VCP/p97 that is unique to its functional quaternary structure.
Since its initial discovery, several studies have employed NMS-873 to infer a role for VCP/p97 in a variety of pathologically relevant processes, most notably in oncogenic transformation and viral replication [[8], [9], [10], [11], [12]]. Given the well-described role of VCP/p97 in the ERAD and autophagy pathways, its association with diseases that require alteration of cellular proteostasis is not surprising [13,14]. Since VCP/p97 is overexpressed in various cancers and has been shown to facilitate important pro-survival functions, such as DNA repair after ionizing radiation, NMS-873 would seem to be a logical candidate for further therapeutic development [[15], [16], [17]]. Initial testing of the drug against a panel of 38 different cancer cell lines seemed to support its viability for this purpose [7].
While using NMS-873 as a tool to disrupt ERAD in a study related to GLUT transporter trafficking, we consistently observed acidification of culture media every time the drug was employed. This led us to further explore the broader metabolic impact of NMS-873 in both human and mouse cells. Our investigation uncovered an unexpected off-target effect of NMS-873 on mitochondrial OXPHOS in which both Complex I and ATP synthase were inhibited. This observation points out that NMS-873 may not be as selective an inhibitor as previously thought, and that caution should be taken when interpreting the cytotoxic effects of this drug on cultured cells.
Section snippets
Chemical reagents
All chemical reagents and small-molecule inhibitors used in this study were obtained from Sigma-Aldrich (St. Louis, MO) except antimycin-A1, atractyloside, trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP), nigericin, rotenone, and oligomycin, which were purchased from Cayman Chemical (Ann Arbor, MI). Drugs were dissolved to 250-1000x concentration in the appropriate vehicle solvent (DMSO or water) and stored at −20 °C prior to use in assays.
Cell culture
HK-2 immortalized human kidney cells used in
NMS-873 induces lactic acid fermentation in human renal tubule cells
A core project in our laboratory focuses on the trafficking and function of GLUT/SLC2A family glucose transporters in mammalian cells. While studying the impact of glycosylation on GLUT1 trafficking in HK-2 human renal tubule cells, we utilized the drug NMS-873 as a tool compound to inhibit VCP/p97 because of its well-established role in proteolysis of improperly glycosylated membrane proteins (Fig. 1A). When used alone, NMS-873 has no impact on GLUT1 expression levels and only modestly
Discussion and conclusions
Since the advent of chemotherapy in the early 1940s, the field of clinical oncology has continuously sought to identify new compounds with increased potency toward cancer cells and decreased toxicity toward normal tissues. Much of this work has relied upon well-known functional distinctions between transformed and normal cells, such as differences in growth rate, metabolism, or DNA repair capacity. Drug screening strategies that rely upon these functional categories of distinction, however,
Authors’ contribution
M.B., K.H., P.J., S.K., and B.L. contributed to experimental design/execution and provided data for the manuscript figures. L.L. and B.L. provided the overall study design and coordinated research work performed by student researchers. B.L. produced the manuscript text and figures for publication. M.B. and K.H contributed equally to the work. All authors reviewed and approved the final version of the manuscript.
Declaration of competing interest
No conflicts of interest or disclosures are declared by any author involved in this work.
Acknowledgements
We wish to thank Lori Keen and David Ross (Calvin University) for their help procuring reagents and support work as lab managers of the Biology and Chemistry Departments, respectively. This research was supported by the National Institutes of Health - National Institute of Diabetes and Digestive and Kidney Diseases [Grant 1-R15-DK081931].
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These authors contributed equally to the work.