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Highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors.

机译:用于快速筛选内皮素转化酶抑制剂的高灵敏度和选择性荧光测定法。

摘要

The highly potent vasoconstrictor peptide endothelin (ET) is generated from an inactive precursor, big endothelin (bET), by endothelin-converting enzyme (ECE). ECE is a phosphoramidon-sensitive zinc metallopeptidase, which is closely related to neprilysin (neutral endopeptidase). It is possible that compounds which inhibit the formation of ET may be used as new drugs for the treatment of cardiovascular diseases. Such an approach requires a fast, simple and selective assay to measure ECE activity, allowing rapid screening of inhibitors. We describe here two new ECE substrates based on the concept of 'intramolecularly quenched fluorescence' which may fulfill this aim. One, S(1) [Pya(21)-Nop(22)-bET-1(19--35)], is the (19--35) fragment of the natural peptide big-ET-1(1--38), which is modified by introducing the fluorescent amino acid, pyrenylalanine (Pya), in position 21 and a quencher, p-nitrophenylalanine (Nop), in position 22. The second substrate (S(2)) is a small peptide, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-NH(2), from a biased substrate peptide library. The recombinant, hECE-1c, cleaved both Pya(21)-Nop(22)-bET-1(19--35) and the natural substrate selectively between residues 21 and 22, whereas cleavage occurred between alanine and phenylalanine in the small peptide. In both cases, this generated intense fluorescence emission. The synthesis and kinetic parameters of these substrates are described. These assays, which can be used directly on tissue homogenates, are the most sensitive and selective described to date for ECE, and are easily automated for a high-throughput screening of inhibitors.
机译:高效的血管收缩肽内皮素(ET)是由无活性的前体大内皮素(bET)通过内皮素转化酶(ECE)生成的。 ECE是磷酰胺敏感的锌金属肽酶,与中性溶酶(中性内肽酶)密切相关。抑制ET形成的化合物有可能被用作治疗心血管疾病的新药。这种方法需要一种快速,简单和选择性的测定方法来测量ECE活性,从而可以快速筛选抑制剂。我们在这里基于“分子内猝灭的荧光”概念描述了两种新的ECE底物,可以实现这一目标。其中一个S(1)[Pya(21)-Nop(22)-bET-1(19--35)]是天然肽big-ET-1(1--的(19--35)片段38),通过在位置21引入荧光氨基酸pyr基丙氨酸(Pya)和在位置22引入淬灭剂对硝基苯丙氨酸(Nop)进行修饰。第二种底物(S(2))是小肽, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-NH(2),来自有偏见的底物肽库。重组hECE-1c可以选择性地在残基21和22之间切割Pya(21)-Nop(22)-bET-1(19--35)和天然底物,而在小肽中的丙氨酸和苯丙氨酸之间发生了切割。 。在两种情况下,这都会产生强烈的荧光发射。描述了这些底物的合成和动力学参数。这些测定法可直接用于组织匀浆,是迄今为止针对ECE所描述的最灵敏和选择性最高的测定法,并且易于自动化进行抑制剂的高通量筛选。

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