Structural requirements of ceramide and sphingosine based inhibitors of mitochondrial ceramidase

The effects of structural analogues of ceramide on rat brain mitochondrial ceramidase (mt-CDase) were investigated. Design of target compounds was mai nly based on modifications of the key elements in ceramide and sphingosine, including stereochemistry, the primary and secondary hydroxyl groups, the trans double bond in the sphingosine backbone, and the amide bond. Mt-CDase was inhibited by (1) all stereoisomers of D-erythro-ceramide (D-e-Cer) wit h an IC50 of 0.11, 0.21, and 0.26 mol % for the L-threo, D-threo, and L-ery thro isomers, respectively; (2) all stereoisomers of sphingosine with IC50 ranging from 0.04 to 0.14 mol %. N-methyl-D-erythro-sphingosine (N-Me-Sph, IC50 0.13 mol %); and (3) D-erythro-urea-C-16-ceramide (C-16-urea-Cer IC50 0.33 mol %). The enzyme was not inhibited by N-methyl ceramiae (N-Me-C-16-C er), 1-O-methyl ceramide (1-O-Me-C-16-Cer), 3-O-methyl ceramide (3-O-Me-C-1 6-Cer), cis-D-erythro ceramide (CiS-D-e-C-16-Cer) and 3-O-methyl-D-erythro- sphingosine (3-O-Me-Sph). It was less potently inhibited by D-erythro-sphin ganine (D-e-dh-Sph, IC50 0.20 mol %), D-erythro-dehydro sphingosine (D-e-de h-Sph, IC50 0.25 mol %), (2S)-3-keto-sphinganine (3-keto-dh-Sph, IC50 0.34 mol % (2S) 3-keto-ceramide (3-keto-C-16-Cer, IC50 0.60 mol %), and ceramine (C-18-ceramine, IC50 0.62 mol % 1-O-methyl-D-erythro-sphingosine (1-O-Me-S ph), cis-D-erythro-sphingosine (cis-D-e-Sph), (2S)-3-keto-sphingosine (3-ke to-Sph), (2S)-3-keto-dehyrosphingosine (3-keto-deh-Sph), and N,N-dimethyl-D -erythro-sphingosine (NN-diMe-Sph) were weak inhibitors whereas ceramide-1- phosphate (Cer-1-P) and sphingosine-1-phosphate (Sph-1-P) stimulated the en zyme. Thus, for inhibition, the enzyme requires the primary and secondary h ydroxyl groups, the C4-C5 double bond, the trans configuration of this doub le bond, and the NH-protons from either the amide of ceramide or the amine of sphingosine. Therefore, these results provide important information on t he requirements for ceramide-enzyme interaction, and they suggest that liga nd interaction with the enzyme occurs in a high affinity low specificity ma nner, in contrast to catalysis which is highly specific for D-erythro-ceram ide (D-e-Cer) but occurs with a lower affinity. In addition, this study ide ntifies two competitive inhibitors of mt-CDase; urea-ceramide (C-16-urea-Ce r) and cerarnine (C-18-ceramine) that may be further developed and used to understand the mechanism of mt-CDase in vitro and in biologic responses.