Nicotinamide phosphoribosyltransferase is a molecular target of potent anticancer agents identified from phenotype-based drug screening.

Phenotypic screening in drug discovery has been revived with the expectation of providing promising lead compounds and drug targets and improving the success rate of drug approval. However, target identification remains a major bottleneck in phenotype-based drug discovery. We identified the lead compounds K542 and K405 with a selective inhibition of cell viability against sphingosine-1-phosphate lyase 1 (SGPL1)-transduced ES-2 cells by phenotypic screening. We therefore performed an in vivo pharmacological examination and observed the antitumor activity of K542 in an HT-1080 tumor-bearing mouse xenograft model. SGPL1 was expected to be a therapeutic target in some cancers, suggesting that these lead molecules might be promising candidates; however, their mechanisms of action still remain unexplained. We therefore synthesized the affinity probe Ind-tag derived from K542 and identified the proteins binding to Ind-tag via a pull-down experiment. Proteomics and biochemical analyses revealed that the target molecule of these lead compounds was Nicotinamide phosphoribosyltransferase (NAMPT). We established K542-resistant DLD-1 and HT-1080 cells, and genetic analyses of these cells identified a missense mutation in the NAMPT-encoding gene. This enzymatic experiment clearly showed that K393 exerts enzymatic inhibition against NAMPT. These proteomics, genetics and biochemical analyses clarified that compounds K542 and K405 were NAMPT inhibitors.

Preclinical and first-in-human phase I studies of KW-2450, an oral tyrosine kinase inhibitor with insulin-like growth factor receptor-1/insulin receptor selectivity.

Numerous solid tumors overexpress or have excessively activated insulin-like growth factor receptor-1 (IGF-1R). We summarize preclinical studies and the first-in-human study of KW-2450, an oral tyrosine kinase inhibitor with IGF-1R and insulin receptor (IR) inhibitory activity. Preclinical activity of KW-2450 was evaluated in various in vitro and in vivo models. It was then evaluated in a phase I clinical trial in 13 patients with advanced solid tumors (NCT00921336). In vitro, KW-2450 inhibited human IGF-1R and IR kinases (IC50 7.39 and 5.64 nmol/L, respectively) and the growth of various human malignant cell lines. KW-2450 40 mg/kg showed modest growth inhibitory activity and inhibited IGF-1-induced signal transduction in the murine HT-29/GFP colon carcinoma xenograft model. The maximum tolerated dose of KW-2450 was 37.5 mg once daily continuously; dose-limiting toxicity occurred in two of six patients at 50 mg/day (both grade 3 hyperglycemia) and in one of seven patients at 37.5 mg/day (grade 3 rash). Four of 10 evaluable patients showed stable disease. Single-agent KW-2450 was associated with modest antitumor activity in heavily pretreated patients with solid tumors and is being further investigated in combination therapy with lapatinib/letrozole in patients with human epidermal growth factor receptor 2-postive metastatic breast cancer.

Effect of 5-fluorouracil and epidermal growth factor on cell growth and dihydropyrimidine dehydrogenase regulation in human uterine cervical carcinoma SKG-IIIb cells.

We previously demonstrated that epidermal growth factor (EGF) induces a decrease in dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), in EGF receptor (EGFR)-positive human SKG-IIIb uterine cervical carcinoma cells, and thereby increased the sensitivity of the cells to 5-FU. In the present study, we examined the individual and combined effects of 5-FU and EGF on growth and DPD activity in SKG-IIIb cells, and also investigated the mode of regulation of DPD activity. The cells showed sensitivity to 5-FU, and growth was stimulated by EGF. When the agents were used in combination, the sensitivity of SKG-IIIb cells to 5-FU was increased roughly sixfold at maximum, as judged in terms of the 50% growth-inhibitory concentration. We then examined the effects of 5-FU and EGF on DPD. Either agent inhibited DPD activity and protein expression in a concentration-dependent manner. Expression of DPD mRNA was concentration-dependently inhibited by treatment with 5-FU and by EGF at a concentration that strongly stimulated cell growth. Further, combination treatment inhibited DPD activity, as well as DPD protein and mRNA expression, more strongly than did treatment with 5-FU or EGF alone. These results suggest that inhibition of DPD activity by EGF or 5-FU is regulated at least at the level of protein expression and that regulation via mRNA is also involved. The above findings indicate that 5-FU and EGF act synergistically in suppressing DPD activity and that the use of 5-FU against tumors in which EGF plays an important role would maximize the potential of 5-FU as an anticancer drug.