Characterization of ARC39, a new inhibitor of acid sphingomyelinase

Abstract

Background: Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or as a therapeutic intervention in many disease processes. Specific and direct inhibitors of ASM are to this date not sufficiently characterized. Aim: This study aims to characterize the bisphosphonate 1-aminodecylidene bis-phosphonic acid (ARC39), a direct inhibitor of ASM, in vitro and in vivo in order to provide useful input about its potential application as a new ASM inhibitor. Results: ARC39 is as a selective inhibitor of ASM in vitro, leading to specific and efficient (>90%) inhibition of both secretory and lysosomal ASM. Investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA, ASM protein and the effect of protease inhibitors on ASM inhibition suggests that direct inhibition of the catalytic activity of ASM is the major mechanism of action of ARC39 in cultured cells, which differs from the functional inhibitors of ASM (FIASMAs). Evidence of a dose- and time-dependent inhibition of lysosomal ASM in intact living cells by ARC39 is provided. Functionally, ARC39 inhibits platelet- and ASM-promoted adhesion of melanoma cells. The toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not lead to pronounced alterations in the lysosomal compartment and does not induce phospholipidosis in vitro. However, when applied i.p. in vivo, even subtoxic high doses administered as a short-term treatment were sufficient to inhibit ASM and induce sphingomyelin accumulation only locally in the peritoneal lavage but not in serum/plasma, liver, spleen or brain. Conclusion: ARC39 is a highly potent and specific ASM inhibitor in vitro. However, it is not well suited for systemic administration in vivo, which requires further investigation with other possible chemical modifications or alternatives.