Pharmacological characterization of synthetic serine palmitoyltransferase inhibitors by biochemical and cellular analyses

• Published in: Biochemical and biophysical research communications Vol. 497; no. 4; pp. 1171 - 1176
• Main Authors: Adachi, Ryutaro, Asano, Yasutomi, Ogawa, Kazumasa, Oonishi, Motomi, Tanaka, Yukiya, Kawamoto, Tomohiro
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.03.2018
• Subjects: 60 APPLIED LIFE SCIENCES; BIOLOGICAL FUNCTIONS; Cancer; Ceramide; ENDOPLASMIC RETICULUM; ENZYMES; LUNGS; Myriocin; SERINE; Serine palmitoyltransferase inhibitor; Slow dissociation; TIME RESOLUTION; Time-resolved fluorescence energy transfer assay; Slow dissociation; Ceramide; Time-resolved fluorescence energy transfer assay; Myriocin; Serine palmitoyltransferase inhibitor; Cancer
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2016.12.182

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. •Tetrahydropyrazolopyridine derivatives were characterized as SPT inhibitors.•The SPT inhibitors exhibited dual inhibitory activities against SPT2 and SPT3.•A SPT inhibitor bound to the SPT2 or SPT3 subunit in SPT enzyme complexes.•One of the SPT inhibitors exhibited a significantly slow dissociation from SPT.•The SPT inhibitors suppressed ceramide content in a non-small-cell lung cancer cell line.