Usta J, El Bawab S, Roddy P, Szulc Z M, Hannun A, Bielawska A
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA.
Biochemistry. 2001 Aug 14;40(32):9657-68. doi: 10.1021/bi010535k.
The effects of structural analogues of ceramide on rat brain mitochondrial ceramidase (mt-CDase) were investigated. Design of target compounds was mainly 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) with an IC50 of 0.11, 0.21, and 0.26 mol % for the L-threo, D-threo, and L-erythro 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-C16-ceramide (C16-urea-Cer IC50 0.33 mol %). The enzyme was not inhibited by N-methyl ceramide (N-Me-C16-Cer), 1-O-methyl ceramide (1-O-Me-C16-Cer), 3-O-methyl ceramide (3-O-Me-C16-Cer), cis-D-erythro ceramide (cis-D-e-C16-Cer) and 3-O-methyl-D-erythro-sphingosine (3-O-Me-Sph). It was less potently inhibited by D-erythro-sphinganine (D-e-dh-Sph, IC50 0.20 mol %), D-erythro-dehydro sphingosine (D-e-deh-Sph, IC50 0.25 mol %), (2S)-3-keto-sphinganine (3-keto-dh-Sph, IC50 0.34 mol %), (2S) 3-keto-ceramide (3-keto-C16-Cer, IC50 0.60 mol %), and ceramine (C18-ceramine, IC50 0.62 mol %), 1-O-methyl-D-erythro-sphingosine (1-O-Me-Sph), cis-D-erythro-sphingosine (cis-D-e-Sph), (2S)-3-ketosphingosine (3-keto-Sph), (2S)-3-keto-dehyrosphingosine (3-keto-deh-Sph), and N,N-dimethyl-D-erythrosphingosine (N,N-diMe-Sph) were weak inhibitors whereas ceramide-1-phosphate (Cer-1-P) and sphingosine-1-phosphate (Sph-1-P) stimulated the enzyme. Thus, for inhibition, the enzyme requires the primary and secondary hydroxyl groups, the C4-C5 double bond, the trans configuration of this double bond, and the NH-protons from either the amide of ceramide or the amine of sphingosine. Therefore, these results provide important information on the requirements for ceramide-enzyme interaction, and they suggest that ligand interaction with the enzyme occurs in a high affinity low specificity manner, in contrast to catalysis which is highly specific for D-erythro-ceramide (D-e-Cer) but occurs with a lower affinity. In addition, this study identifies two competitive inhibitors of mt-CDase; urea-ceramide (C16-urea-Cer) and ceramine (C18-ceramine) that may be further developed and used to understand the mechanism of mt-CDase in vitro and in biologic responses.
研究了神经酰胺结构类似物对大鼠脑线粒体神经酰胺酶（mt-CDase）的影响。目标化合物的设计主要基于对神经酰胺和鞘氨醇关键元素的修饰，包括立体化学、伯羟基和仲羟基、鞘氨醇主链中的反式双键以及酰胺键。mt-CDase受到以下物质的抑制：（1）D-赤藓糖神经酰胺（D-e-Cer）的所有立体异构体，L-苏阿糖型、D-苏阿糖型和L-赤藓糖型异构体的IC50分别为0.11、0.21和0.26 mol%；（2）鞘氨醇的所有立体异构体，IC50范围为0.04至0.14 mol%，N-甲基-D-赤藓糖鞘氨醇（N-Me-Sph，IC50 0.13 mol%）；以及（3）D-赤藓糖脲-C16-神经酰胺（C16-脲-Cer，IC50 0.33 mol%）。该酶不受N-甲基神经酰胺（N-Me-C16-Cer）、1-O-甲基神经酰胺（1-O-Me-C16-Cer）、3-O-甲基神经酰胺（3-O-Me-C16-Cer）、顺式-D-赤藓糖神经酰胺（顺式-D-e-C16-Cer）和3-O-甲基-D-赤藓糖鞘氨醇（3-O-Me-Sph）的抑制。D-赤藓糖鞘氨醇（D-e-dh-Sph，IC50 0.20 mol%）、D-赤藓糖脱氢鞘氨醇（D-e-deh-Sph，IC50 0.25 mol%）、（2S）-3-酮鞘氨醇（3-酮-dh-Sph，IC50 0.34 mol%）、（2S）3-酮神经酰胺（3-酮-C16-Cer，IC50 0.60 mol%）和神经胺（C18-神经胺，IC50 0.62 mol%）对该酶的抑制作用较弱，1-O-甲基-D-赤藓糖鞘氨醇（1-O-Me-Sph）、顺式-D-赤藓糖鞘氨醇（顺式-D-e-Sph）（2S）-3-酮鞘氨醇（3-酮-Sph）、（2S）-3-酮脱氢鞘氨醇（3-酮-deh-Sph）和N，N-二甲基-D-赤藓糖鞘氨醇（N，N-二甲基-Sph）是弱抑制剂；而神经酰胺-1-磷酸（Cer-1-P）和鞘氨醇-1-磷酸（Sph-1-P）则刺激该酶。因此，对于抑制作用，该酶需要伯羟基和仲羟基、C4-C5双键、该双键的反式构型以及来自神经酰胺酰胺或鞘氨醇胺的NH质子。因此，这些结果提供了关于神经酰胺与酶相互作用要求的重要信息，并且表明配体与酶的相互作用以高亲和力低特异性的方式发生，这与对D-赤藓糖神经酰胺（D-e-Cer）具有高度特异性但亲和力较低的催化作用形成对比。此外，本研究鉴定出两种mt-CDase的竞争性抑制剂；脲神经酰胺（C16-脲-Cer）和神经胺（C18-神经胺），它们可能会进一步开发并用于了解mt-CDase在体外和生物反应中的机制。
