Adenosine triphosphate (
ATP)-competitive p97 inhibitor CB-5339, the successor of
CB-5083, is being evaluated in Phase 1 clinical trials for anti-
cancer therapy. Different modes-of-action p97 inhibitors such as allosteric inhibitors are useful to overcome drug-induced resistance, one of the major problems of targeted
therapy. We previously demonstrated that allosteric p97 inhibitor
NMS-873 can overcome CB-5083-induced resistance in HCT116. Here we employed chemical proteomics and drug-induced thermal
proteome changes to identify drug targets, in combination with drug-resistant cell lines to dissect on- and off-target effects. We found that
NMS-873 but not
CB-5083 affected glycometabolism. By establishing NMS-873-resistant HCT116 cell lines and performing both cell-based and proteomic analysis, we confirmed that
NMS-873 dysregulates glycometabolism in a p97-independent manner. We then used
proteome integral solubility alteration with a temperature-based method (PISA T) to identify NDUFAF5 as one of the potential targets of
NMS-873 in the mitochondrial complex I. We also demonstrated that glycolysis inhibitor 2-DG enhanced the anti-proliferative effect of
NMS-873. The polypharmacology of
NMS-873 can be advantageous for anti-
cancer therapy for
colon cancer.