title: Pharmacological characterization of synthetic serine palmitoyltransferase inhibitors by biochemical and cellular analyses.
abstract: Human serine palmitoyltransferase (SPT) is a PLP-dependent enzyme residing in the endoplasmic reticulum. It catalyzes the synthesis of 3-ketodihydrosphingosine (3-KDS) from the substrates palmitoyl-CoA and l-serine. It is a rate-limiting enzyme for sphingolipid synthesis in cells. In the present study, we characterized and pharmacologically profiled a series of tetrahydropyrazolopyridine derivatives that potently inhibit human SPT enzymatic activity, including two cell-active derivatives and one fluorescent-labelled derivative. These SPT inhibitors exhibited dual inhibitory activities against SPT2 and SPT3. We used a fluorescent-labelled probe to molecularly assess the inhibitory mechanism and revealed its binding to the SPT2 or SPT3 subunit in the small subunit (ss) SPTa/SPT1/SPT2/or ssSPTa/SPT1/SPT3 functional complexes. One of the SPT inhibitors exhibited a significantly slow dissociation from the SPT complex. We confirmed that our SPT inhibitors suppressed ceramide content in non-small-cell lung cancer cell line, HCC4006, by performing a target engagement analysis. The potency of ceramide reduction correlated to that observed in a recombinant SPT2 enzyme assay. We thus elucidated and provided a fundamental understanding of the molecular mode of action of SPT inhibitors and developed potent, cell-active SPT inhibitors that can be used to clarify the biological function of SPT.
authors.affiliation: Biomolecular Research Laboratories, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan. Electronic address: ryutaro.adachi@takeda.com.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Adachi
authors.affiliation: Oncology Drug Discovery Unit, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Yasutomi
authors.initials: Y
authors.lastname: Asano
authors.affiliation: Biomolecular Research Laboratories, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Ogawa
authors.affiliation: Biomolecular Research Laboratories, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Motomi
authors.initials: M
authors.lastname: Oonishi
authors.affiliation: Biomolecular Research Laboratories, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Yukiya
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Biomolecular Research Laboratories, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kawamoto
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy; D045744:Cell Line, Tumor; D002518:Ceramides / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000138:chemical synthesis*; D006801:Humans; D008175:Lung Neoplasms; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051102:Serine C-Palmitoyltransferase / Q000037:antagonists & inhibitors*
chemical_list: D002518:Ceramides; D004791:Enzyme Inhibitors; D011720:Pyrazoles; D011725:Pyridines; C118531:pyrazolopyridine; D051102:Serine C-Palmitoyltransferase
keywords: Cancer; Ceramide; Myriocin; Serine palmitoyltransferase inhibitor; Slow dissociation; Time-resolved fluorescence energy transfer assay