Mette Knak Christensen

Anticancer agent CHS-828 inhibits cellular synthesis of NAD

Malignant cells display increased demands for energy production and DNA repair. Nicotinamide adenine dinucleotide (NAD) is required for both processes and is also continuously degraded by cellular enzymes. Nicotinamide phosphoribosyltransferase (Nampt) is a crucial factor in the resynthesis of NAD, and thus in cancer cell survival. Here, we establish the cytotoxic mechanism of action of the small molecule inhibitor CHS-828 to result from impaired synthesis of NAD. Initially, we detected cross-resistance in cells between CHS-828 and a known inhibitor of Nampt, FK866, a compound of a structurally different class. We then showed that nicotinamide protects against CHS-828-mediated cytotoxicity. Finally, we observed that treatment with CHS-828 depletes cellular NAD levels in sensitive cancer cells. In conclusion, these results strongly suggest that, like FK866, CHS-828 kills cancer cells by depleting NAD.

Target enzyme mutations are the molecular basis for resistance towards pharmacological inhibition of nicotinamide phosphoribosyltransferase

BackgroundInhibitors of nicotinamide phosphoribosyltransferase (NAMPT) are promising cancer drugs currently in clinical trials in oncology, including APO866, CHS-828 and the CHS-828 prodrug EB1627/GMX1777, but cancer cell resistance to these drugs has not been studied in detail.MethodsHere, we introduce an analogue of CHS-828 called TP201565 with increased potency in cellular assays. Further, we describe and characterize a panel of cell lines with acquired stable resistance towards several NAMPT inhibitors of 18 to 20,000 fold compared to their parental cell lines.ResultsWe find that 4 out of 5 of the resistant sublines display mutations of NAMPT located in the vicinity of the active site or in the dimer interface of NAMPT. Furthermore, we show that these mutations are responsible for the resistance observed. All the resistant cell lines formed xenograft tumours in vivo. Also, we confirm CHS-828 and TP201565 as competitive inhibitors of NAMPT through docking studies and by NAMPT precipitation from cellular lysate by an analogue of TP201565 linked to sepharose. The NAMPT precipitation could be inhibited by addition of APO866.ConclusionWe found that CHS-828 and TP201565 are competitive inhibitors of NAMPT and that acquired resistance towards NAMPT inhibitors can be expected primarily to be caused by mutations in NAMPT.

Synthesis and Antitumor Effect in Vitro and in Vivo of Substituted 1,3-Dihydroindole-2-ones

Optimization of the anticancer activity for a class of compounds built on a 1,3-dihydroindole-2-one scaffold was performed. In comparison with recently published derivatives of oxyphenisatin the new analogues exhibited an equally potent antiproliferative activity in vitro and improved tolerability and activity in vivo. The best compounds from this series showed low nanomolar antiproliferative activity toward a series of cancer cell lines (compound (S)-38: IC(50) of 0.48 and 2 nM in MCF-7 (breast) and PC3 (prostate), respectively) and potent antitumor effects in well tolerated doses in xenograft models. The racemic compound (RS)-38 showed complete tumor regression at a dose of 20 mg/kg administered iv on days 1 and 7 in a PC3 rat xenograft.

Nicotinamide Phosphoribosyltransferase Inhibitors, Design, Preparation, and Structure–Activity Relationship

Existing pharmacological inhibitors for nicotinamide phosphoribosyltransferase (NAMPT) are promising therapeutics for treating cancer. By using medicinal and computational chemistry methods, the structure-activity relationship for novel classes of NAMPT inhibitors is described, and the compounds are optimized. Compounds are designed inspired by the NAMPT inhibitor APO866 and cyanoguanidine inhibitor scaffolds. In comparison with recently published derivatives, the new analogues exhibit an equally potent antiproliferative activity in vitro and comparable activity in vivo. The best performing compounds from these series showed subnanomolar antiproliferative activity toward a series of cancer cell lines (compound 15: IC50 0.025 and 0.33 nM, in A2780 (ovarian carcinoma) and MCF-7 (breast), respectively) and potent antitumor in vivo activity in well-tolerated doses in a xenograft model. In an A2780 xenograft mouse model with large tumors (500 mm(3)), compound 15 reduced the tumor volume to one-fifth of the starting volume at a dose of 3 mg/kg administered ip, bid, days 1-9. Thus, compounds found in this study compared favorably with compounds already in the clinic and warrant further investigation as promising lead molecules for the inhibition of NAMPT.

LC–MS/MS assay for the quantitation of the HDAC inhibitor belinostat and five major metabolites in human plasma

The histone deacetylase inhibitor belinostat is being evaluated clinically as a single agent in the treatment of peripheral T-cell lymphomas and in combination with other anticancer agents to treat a wide range of human cancers including acute leukemias and solid tumors. To determine the pharmacokinetics of belinostat in the NCI ODWG liver dysfunction study, we developed and validated an LC-MS/MS assay for the quantitation of belinostat and five major metabolites in 0.05 mL human plasma. After protein precipitation, chromatographic separation was achieved with a Waters Acquity BEH C18 column and a linear gradient of 0.1% formic acid in acetonitrile and water. Detection with an ABI 4000Q mass spectrometer utilized both electrospray positive and negative mode ionization. The assay was linear from 30 to 5000 ng/mL for all six analytes and proved to be accurate (92.0-104.4%) and precise (CV <13.7%), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring parent drug and five major metabolites in plasma from a patient who was administered belinostat IV at a dose of 400 mg/m(2). The LC-MS/MS assay that has been developed will be an essential tool to further define the metabolism and pharmacology of belinostat in the ongoing liver organ dysfunction as well as other studies that investigate belinostat with other anticancer agents.

Novel small molecule drugs inhibit tumor cell metabolism and show potent anti-tumorigenic potential

BackgroundRapidly dividing tumor cells have an increased demand for nutrients to support their characteristic unabated growth; this demand is met by an increased availability of nutrients such as amino acids through vasculogenesis and by the enhanced cellular entry of nutrients through the upregulation of specific transporters. Deprivation of intracellular amino acids or block of amino acid uptake has been shown to be cytotoxic to many established human cancer cell lines in vitro and in human cancer xenograft models.ResultsIn this paper, we provide evidence that the two small molecule oxyphenisatine analogs TOP001 and TOP216 exert their anti-cancer effect by affecting tumor cell metabolism and inducing intracellular amino acid deprivation, leading to a block of cell proliferation. GCN2-mediated phosphorylation of eIF2α as well as mTOR pathway inhibition supports the above notion. In addition, these novel anti-cancer compounds inhibit DNA and protein synthesis and induce apoptosis in a broad spectrum of cancer cell lines. In vivo, the compounds induce tumor stasis and regression in mouse xenograft models of human breast, prostate, ovarian and pancreatic cancer, both when administered intravenously and orally.ConclusionIn conclusion, these small molecules, built on a 1,3-dihydroindole-2-one scaffold, elicit strong anti-proliferative and cytotoxic activity, and importantly, a strong anti-tumorigenicity is observed in in vivo xenograft models of human breast, ovary, prostate and pancreatic cancers encouraging the translation of this class of compounds into the clinic.