Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro

Abstract

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 a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo. 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 a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo. acid ceramidase 1-aminodecylidene bis-phosphonic acid acid sphingomyelinase bisphosphonate collision energy dihydrosphingosine functional inhibitor of acid sphingomyelinase farnesyl pyrophosphate hypoxanthine phosphoribosyltransferase 1 lysosomal acid sphingomyelinase mean fluorescence intensity nitrogen-containing bisphosphonate neutral ceramidase neutral sphingomyelinase Ras-related protein 1A secretory acid sphingomyelinase sphingomyelin phosphodiesterase 1 sphingosine-1-phosphate sphingosine sphingosine kinase Acid sphingomyelinase [human: ASM, sphingomyelin phosphodiesterase 1 (SMPD1); mouse: Asm, Smpd1; hereafter uniformly ASM] is an enzyme located mainly in the lysosomes where it catalyzes the hydrolysis of sphingomyelin, an abundant sphingolipid in eukaryotic membranes, into ceramide and phosphorylcholine with a pH optimum at ∼5. Ceramide and ASM-generated ceramide are important in cell signaling with versatile roles in many physiological and disease-related processes, particularly when ASM is translocated onto the outer leaflet of the plasma membrane upon a variety of stimuli (1Grassmé H. Cremesti A. Kolesnick R. Gulbins E. Ceramide-mediated clustering is required for CD95-DISC formation.Oncogene. 2003; 22: 5457-5470Crossref PubMed Scopus (235) Google Scholar, 2Grassmé, H., V., Jendrossek, A., Riehle, G., von Kurthy, J., Berger, H., Schwarz, M. Weller, R. Kolesnick, and E. Gulbins. 2003. Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts. Nat. Med., 9: 322–330.Google Scholar, 3Herz, J., J., Pardo, H., Kashkar, M., Schramm, E., Kuzmenkina, E., Bos, K. Wiegmann, R. Wallich, P. J. Peters, S. Herzig, . 2009. Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes. Nat. Immunol., 10: 761–768.Google Scholar, 4Teichgräber, V., M., Ulrich, N., Endlich, J., Riethmuller, B., Wilker, C. C., De Oliveira-Munding, A. M. van Heeckeren, M. L. Barr, G. von Kürthy, K. W. Schmid, . 2008. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat. Med., 14: 382–391.Google Scholar, 5Gulbins, E., M., Palmada, M., Reichel, A., Luth, C., Bohmer, D., Amato, C. P. Müller, C. H. Tischbirek, T. W. Groemer, G. Tabatabai, . 2013. Acid sphingomyelinase-ceramide system mediates effects of antidepressant drugs. Nat. Med., 19: 934–938.Google Scholar). Hereditary mutations of ASM lead to the lysosomal storage disease, Niemann-Pick disease (types A and B), which is characterized by progressive accumulation of sphingomyelin and subsequent organ dysfunction (6Schuchman E.H. Wasserstein M.P. Types A and B Niemann-Pick disease.Best Pract. Res. Clin. Endocrinol. 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G. of acid PubMed Scopus Google Scholar). is important for ASM in the of the and also the and the catalytic activity of the enzyme A. K. G. of acid PubMed Scopus Google Scholar). inhibition of ASM been by functional inhibitors of ASM which a of compounds that and other to in the lysosomal are of with the the of ASM and lysosomal to the and of the enzyme and J., M., A., M., Reichel, C. L. T. W. Groemer, G. M. . of functional inhibitors of acid Scholar, J., M., Reichel, C., C., M., T. W. Groemer, and E. Gulbins. inhibitors of acid sphingomyelinase a pharmacological of with Scholar, J., M., Reichel, J., and E. Gulbins. 2008. of functional inhibitors of acid sphingomyelinase a J. Scholar, M. K. of acid sphingomyelinase and in the of the antidepressant PubMed Scopus Google Scholar, R. K. K. 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A. . 2013. in sphingolipid to lysosomal cell death induced by inhibitors of acid Scholar). of effects require doses for the inhibition of the ASM and are not in vivo. In vivo, lead to a of ceramide to the inhibition of the ASM and have been to the observed in ASM in of cystic and V., M., Ulrich, N., Endlich, J., Riethmuller, B., Wilker, C. C., De Oliveira-Munding, A. M. van Heeckeren, M. L. Barr, G. von Kürthy, K. W. Schmid, . 2008. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat. Med., 14: 382–391.Google Scholar, 5Gulbins, E., M., Palmada, M., Reichel, A., Luth, C., Bohmer, D., Amato, C. P. Müller, C. H. Tischbirek, T. W. Groemer, G. Tabatabai, . 2013. Acid sphingomyelinase-ceramide system mediates effects of antidepressant drugs. Nat. Med., 19: 934–938.Google Scholar). In of and a bisphosphonate 1-aminodecylidene bis-phosphonic acid been by C. inhibitors of acid PubMed Scopus Google and S. S. C. and inhibition of acid sphingomyelinase by PubMed Scopus Google Scholar). In we to ARC39 in vitro and in in to provide as a and ASM and from cultured in with 1 and from at and in a and to and to the in the ASM activity and with to have ASM and in the of the of to the of the of by the and of the ARC39 at 1 in and in a for with The in the at and as at 1 in in and to with ASM in ASM for neutral sphingomyelinase and neutral ceramidase in a neutral the protein and at a of to in the ASM for secretory ASM as C. J. to sphingomyelinase and ceramidase efficiently and A. 2017; PubMed Scopus Google C. J. to sphingomyelinase and ceramidase efficiently and A. 2017; PubMed Scopus Google and in a for The of of and of and for at with The by by at for and the and in a in and onto a with in sphingomyelinase and with acid in The a and with The from of of The as C. L. G. T. A for of acid ceramidase activity and of Res. 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ASM is to have a lysosomal and a secretory that from the to Lysosomal ASM activity is in cell and tissue without the need to to the the activity of is in and and requires of to the to the enzyme In a we to the of the inhibitor with ASM. we and directly with to the of the activity of from and not without further of without not We found an inhibition of activity and activity in and In a antidepressant and a as to of does not ASM in in cell in even when applied at to which is a high that is the to cells. the enzyme of we and in in the of ARC39 cell with an abundant ASM activity with not with doses of ARC39 and the activity of ASM, and ARC39 ASM efficiently in a the other The ASM inhibitors and efficiently ASM as and in to activity subsequent to the of protein or other of ASM inhibition in cells, as mRNA or ASM we mRNA and ASM protein levels and with ARC39 in and mRNA not In in cells, we a with ARC39 The ASM protein not the enzyme activity not to induce the of ASM, and by with the inhibitor R. K. K. The antidepressant of lysosomal sphingomyelinase in PubMed Scopus Google Scholar). with ASM inhibition induced by and not by ARC39 that inhibition of ASM catalytic activity is also the mechanism by which ARC39 inhibits ASM in cultured cells. ARC39 the with These from for J., M., A., M., Reichel, C. L. T. W. Groemer, G. M. . of functional inhibitors of acid Scholar). the ASM is and when investigating in activity subsequent to in ASM protein upon with both in cell and tissue C. J. to sphingomyelinase and ceramidase efficiently and A. 2017; PubMed Scopus Google it may not as when investigating inhibitors with a of of the many and which may the the enzyme and the the in an may not the a in ASM and C., H., E., A., and C., . A for acid sphingomyelinase activity in cells. of Scholar). The is a sphingomyelin in which as and as of the results in a in fluorescence and a in to a in intact The only upon the fluorescence can to for in the of the by cells. is an and the the in with the to activity and is activity C., H., E., A., and C., . A for acid sphingomyelinase activity in cells. of Scholar). ARC39 to an dose- and inhibition of ASM activity in intact The and at in ARC39 to a in the of in from from as by further the results with the A also in the we further the lysosomal accumulation of sphingomyelin by with with of ARC39 or of as a In with ARC39 or we accumulation of the sphingomyelin which with that with ARC39 to accumulation of sphingomyelin in the lysosomal lysosomal accumulation of sphingomyelin observed in to in in from that been with of In cells, an in and in sphingomyelin observed A in and in ceramide significant in or of ceramide the levels of in the low and time-dependently in results These in to ARC39 or ARC39 directly with or and enzyme to ARC39 not an or for only and are as as of ARC39 or activity when the of ARC39 been in studies to functional results to the inhibition of ASM activity S. S. C. and inhibition of acid sphingomyelinase by PubMed Scopus Google Scholar, S. Ceramide sphingolipid signaling mediates toxicity signaling in PubMed Scopus Google Scholar, C., C., A. S. H. . as a to tumor cells. 14: Scholar, H. C., G. M., A. C., M. J., C. L. H. P. M. H. and R. A. Acid in and Med., 22: Scholar, J., J., C., J., A., J. C. T. A. . and in and tumor signaling in cells. Scholar, M. J., M., A., V., C., J. W. and W. M. Acid sphingomyelinase mediates of J. Scholar). that adhesion and of is by a of ASM that is from upon with tumor to clustering and of the of tumor in A., K. A., G., M., K. W. Schmid, C. S. . of tumor by acid Med., Scholar). are not the of ASM in and adhesion in vitro and in is upon with A., K. A., G., M., K. W. Schmid, C. S. . of tumor by acid Med., Scholar). a functional in the we with with or without 1 of from and with or without 1 of ARC39 and the adhesion of to and the adhesion of cells. not cell which by 1 of ARC39 with cells. The observed with In the and primary not of cell death with ARC39 even with doses as high as for that a of the The cell not with doses in and with doses which are the required for ASM inhibition mechanism of toxicity by nitrogen-containing is the inhibition of farnesyl pyrophosphate in the E. E. L. C. S. pyrophosphate is the of nitrogen-containing Res. PubMed Scopus Google Scholar, E. E. L. C. S. pyrophosphate pyrophosphate activity with to in vitro and in Res. PubMed Scopus Google Scholar, J. E., S. M., C. D., H. and M. J. for inhibition of farnesyl in vitro and inhibition of in by nitrogen-containing J. Scholar, R. G. the and of Res. PubMed Scopus Google Scholar). we ARC39 by protein in and cells, with doses of we levels of in with cells, particularly with doses that to toxicity at of also in to a that inhibition of at a for the toxicity at with for of a of the the or for of an to accumulation of sphingomyelin and lysosomal and to the of the compartment and ARC39 significant the of or and to a in the of both is with are and lead to an of the lysosomes H., A., M., J. C., A. A. A. . the of as an inhibitor for acid Scholar, H., D., A. M., M., M. S. T. H. A. . 2013. in sphingolipid to lysosomal cell death induced by inhibitors of acid Scholar). ARC39 the of only which to the accumulation of sphingomyelin and a lysosomal and to an even in the of The of many the lysosomal compartment been in S. T. Lysosomal to 2017; Google Scholar). we to ARC39 other lysosomal and induce a which significant accumulation of with the control an in with ARC39 that not show and the of ARC39 in the of ASM inhibition in vivo, ARC39 applied intraperitoneally in at for it that in by the in which to J. P. The tissue of in The effects of and the of J. Metab. PubMed Scopus Google Scholar). the of we that high doses may required to reach sufficient concentrations for ASM inhibition in to the both in a and a high doses to particularly by by and toxicity in of in T. E. The effects of doses of and and in 2003; PubMed Scopus Google and in G. P. J. C. J., J. S. H. S. S. . 2003. with Scholar, L. J. with the of J. 2003; PubMed Scopus Google Scholar). We subtoxic doses to and for to ASM doses are to doses for other relevant We ASM activity the or the short-term with the significant in locally in the peritoneal lavage the may to in the are we to by in the the short-term A relevant and significant in only observed locally in the peritoneal lavage not Inhibition of ASM as an investigational tool and as a therapeutic intervention in the J. A. G. Gulbins E. Acid sphingomyelinase inhibitors ceramide and in cystic J. PubMed Scopus Google Scholar, A., K. A., G., M., K. W. Schmid, C. S. . of tumor by acid Med., Scholar, M., A., C. R. J. M. . inhibition of acid sphingomyelinase by tumor cell Scholar). are as inhibitors of ASM in vitro and in and are and in an and it been at in vitro, that of may other in and in sphingolipid In vivo, lead to a relevant of ceramide to the inhibition of ASM and have been to the observed in ASM in of cystic and V., M., Ulrich, N., Endlich, J., Riethmuller, B., Wilker, C. C., De Oliveira-Munding, A. M. van Heeckeren, M. L. Barr, G. von Kürthy, K. W. Schmid, . 2008. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat. Med., 14: 382–391.Google Scholar, 5Gulbins, E., M., Palmada, M., Reichel, A., Luth, C., Bohmer, D., Amato, C. P. Müller, C. H. Tischbirek, T. W. Groemer, G. Tabatabai, . 2013. Acid sphingomyelinase-ceramide system mediates effects of antidepressant drugs. Nat. Med., 19: 934–938.Google Scholar). is at for to other and ASM In we characterized which is of the ASM inhibitors C. inhibitors of acid PubMed Scopus Google Scholar). We show that ARC39 inhibits both and efficiently and selectively in vitro by directly the catalytic activity without ASM mRNA or protein without of ASM and other acid in the lysosomal sphingolipid catalytic H., A., M., J. C., A. A. A. . the of as an inhibitor for acid that inhibits at in vitro in a We also show that activity is with both and in cells. ARC39 is a it is at physiological it is that The been in studies to to ASM it in and S. S. C. and inhibition of acid sphingomyelinase by PubMed Scopus Google and death signaling S. Ceramide sphingolipid signaling mediates toxicity signaling in PubMed Scopus Google Scholar, C., C., A. S. H. . as a to tumor cells. 14: Scholar, H. C., G. M., A. C., M. J., C. L. H. P. M. H. and R. A. Acid in and Med., 22: Scholar, J., J., C., J., A., J. C. T. A. . and in and tumor signaling in cells. and of in when been with ARC39 M. J., M., A., V., C., J. W. and W. M. Acid sphingomyelinase mediates of J. Scholar). evidence inhibitor reach the compartment and the of effects are to activity of upon onto the outer leaflet of the plasma Here, we provide the evidence of inhibition by ARC39 in vitro, as it a dose- and in ASM activity with a in in intact a of and as by and lysosomal accumulation of The that ARC39 in is with the findings that are to the and other R. H. The of and in cultured PubMed Scopus Google Scholar). by K. of bisphosphonate requires of PubMed Scopus Google the of and by in the of and that compounds in the mechanism of the ASM inhibition in in the compartment with ARC39 is and a of in the is to ASM activity when the inhibitor directly to cell In to a of is required to ASM activity in in to the where are with in the where are the with and a in the to a of ARC39 the and The that inhibition with a inhibitor is even in the in the that the is and is to the mechanism of of by with accumulation in the compartment K. of bisphosphonate requires of PubMed Scopus Google Scholar). we that ARC39 may have other effects the We also provide an functional for ASM inhibition by which platelet- and ASM-promoted adhesion of in vitro. in to a cell ASM activity and also activity A., K. A., G., M., K. W. Schmid, C. S. . of tumor by acid Med., Scholar). when been with of into the M. J., M., A., V., C., J. W. and W. M. Acid sphingomyelinase mediates of J. Scholar). In vitro, the toxicity of ARC39 is low to the of ASM are in the as to and to in and Inhibition of is mechanism of also a of toxicity E. E. L. C. S. pyrophosphate is the of nitrogen-containing Res. PubMed Scopus Google Scholar, E. E. L. C. S. pyrophosphate pyrophosphate activity with to in vitro and in Res. PubMed Scopus Google Scholar, J. E., S. M., C. D., H. and M. J. for inhibition of farnesyl in vitro and inhibition of in by nitrogen-containing J. Scholar, R. G. the and of Res. PubMed Scopus Google Scholar). in which the is located in the it is located in other relevant in the J. E., S. M., C. D., H. and M. J. for inhibition of farnesyl in vitro and inhibition of in by nitrogen-containing J. the the inhibition of in vitro and inhibition of in in In van E. effects of of the of Res. PubMed Scopus Google that the in the by an or the of and the of both and not to the of that ARC39 and lead to the and the of inhibition requires further and we that ARC39 the lysosomal compartment which may to ASM sphingomyelin and a lysosomal of the of with ARC39 or with to when been for with A possible is that sphingomyelin to the lead to an of the lysosomal of the significant accumulation of with upon with the of ARC39 ASM in vitro not in vivo. When high subtoxic doses a relevant in only locally in the peritoneal a ARC39 may have high to and from the plasma and by and also by in the a of PubMed Scopus Google Scholar, A. H. A. H. and plasma protein of Metab. PubMed Scopus Google Scholar, of and the of a in Metab. Google Scholar, of a in Metab. 19: Google Scholar). the other is currently to ASM inhibition in directly without with the tissue sphingomyelin may provide it may not ASM particularly in a short-term In of may an In conclusion, that ARC39 is a and ASM inhibitor in vitro. a need for compounds for in that can reach tissue concentrations relevant to ASM inhibition with to the need for high doses and the of a ARC39 that can into the is currently for tissue in possible B., B., M., M. M. A., D., A. R. G. R. A. . for to the A for lysosomal storage J. Scholar). is particularly when the to the system not the which as a therapeutic in a of disease, a lysosomal storage where a suggested that activity is a therapeutic N., K. A., M., C., W. J. M. J. E. . Acid sphingomyelinase deficiency J. Scholar). The of for the of The and for with and for with the The and for and with

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