Phosphatidylinositol-3,5-bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Acid sphingomyelinase (ASMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of NiemannPick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membraneassociated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of ASMase, L-alpha-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P(2)), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant K-i for the new ASMase inhibitor PtdIns3,5P(2) is 0.53 muM as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human ASMase purified from insect cells. Even at concentrations of up to 50 muM, PtdIns3,5P(2) neither decreased plasma membrane associated, magnesiumdependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases beta-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositolbisphosphates were studied to elucidate structureactivity relationships for ASMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent ASMase inhibitors optimised for diverse applications.