Modifications to the N-terminus but not the C-terminus of calcitonin gene-related peptide(8-37) produce antagonists with increased affinity.
Modifications to the N-terminus but not the C-terminus of calcitonin gene-related peptide(8-37) produce antagonists with increased affinity.
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DOI：
10.1021/jm020507f
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发表时间：
2003-06
影响因子：
7.3
通讯作者：
D. D. Smith-D.;S. Saha;Guoyong Fang;C. Schaffert;David J. J. Waugh-David-J.-J.-Waugh-2251816870;W. Zeng;G. Tóth;M. Hulce;Peter W. Abel
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文献类型：
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作者：
D. D. Smith-D.;S. Saha;Guoyong Fang;C. Schaffert;David J. J. Waugh-David-J.-J.-Waugh-2251816870;W. Zeng;G. Tóth;M. Hulce;Peter W. Abel
采用固相法合成了17个新的人降钙素基因相关肽（8-37）（hCGRP(8-37)）类似物，并采用半制备阳离子交换和/或反相高效液相色谱法纯化。c端苯丙氨酸被Gly、环己基丙氨酸（Cha）、Tyr、-甲基苯丙氨酸（β - mephe）的所有四种异构体以及1 -和d-四氢异喹啉羧酸（Tic）取代，得到类似物3-11。对于含β - mephe类似物6-9的合成，采用结晶法将β - mephe的四种同分异构体的混合物分离成红血球对映体（2S,3S, 2R,3R）和三对对映体（2S,3R, 2R,3S），然后将其转化为Fmoc衍生物并用于两次单独的合成。每次合成得到两个非对映体肽，用反相高效液相色谱分离得到对映体纯的6-9。Tyr取代Phe对CGRP受体的结合亲和力没有影响。所有其他替代Phe导致结合亲和力的显著降低。事实上，类似物7、9和11没有被观察到结合，它们都在c端位置含有一个d氨基酸残基，其余类似物的结合亲和力比h- α - cgrp低10倍（8-37）。这些数据表明，h- α -CGRP（8-37）的c端位置的构象柔性苯环更适合与CGRP受体高亲和力结合。h- α - cgrp（8-37）和h- β - cgrp（8-37）的n端乙酰化、苯甲酰化和苄酰化分别产生类似物12-14和16-18。通过反相高效液相色谱（RP-HPLC）从各苯基化类似物的树脂裂解粗产物中分离出一个副产物，表征为h- α - cgrp（8-37）和h- β - cgrp（8-37）的二苯基化衍生物（分别为类似物15和19）。氨基酸分析和(1)核磁共振表明，第二个苄基位于His（10）的咪唑环的C4碳上。放射性配体结合实验表明，衍生化n端显著增加了与CGRP受体的结合亲和力。苯甲酰化和二苯甲酰化衍生物的亲和度最高，约为h- α - cgrp的50倍（8-37）。功能实验证实，n端衍生的h- α - cgrp类似物（8-37）是比h- α - cgrp（8-37）更有效的拮抗剂。总之，这些研究强调了h- α -CGRP（8-37）的Phe（37）与CGRP受体结合的重要性，并确定了n端和His（10）是两个可以用于设计对CGRP受体亲和力增加的拮抗剂的位置。
Seventeen novel analogues of human calcitonin gene-related peptide(8-37) (hCGRP(8-37)) were synthesized by solid-phase methods and purified to apparent homogeneity by semipreparative cation exchange and/or reversed-phase high-performance liquid chromatography. The C-terminal Phe was replaced by Gly, cyclohexylalanine (Cha), Tyr, all four isomers of beta-methylphenylalanine (beta-MePhe), and l- and d-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the beta-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of beta-MePhe into the erythro pair of enantiomers (2S,3S, 2R,3R) and the threo pair of enantiomers (2S,3R, 2R,3S), which were then converted to Fmoc derivatives and used in two separate syntheses. Two diastereomeric peptides were obtained from each synthesis and were separated by RP-HPLC to yield enantiomerically pure 6-9. Substitution of Tyr for Phe caused no change in binding affinity at CGRP receptors. All other substitutions for Phe resulted in substantial reductions in binding affinity. Indeed, no binding was observed for analogues 7, 9, and 11, all of which contained a d-amino acid residue in the C-terminal position, and the binding affinities of the remaining analogues were >10-fold lower than that of h-alpha-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-alpha-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) produced analogues 12-14 and 16-18, respectively. A byproduct was isolated by RP-HPLC from the resin-cleaved crude product of each benzylated analogue, which was characterized as the dibenzylated derivative of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and (1)H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His(10). Radioligand binding experiments showed that derivatizing the N-termini substantially increased binding affinities at CGRP receptors. The benzoylated and dibenzylated derivatives had the highest affinities, which were approximately 50-fold greater than those of h-alpha-CGRP(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-alpha-CGRP(8-37) are antagonists that are more potent than h-alpha-CGRP(8-37). In conclusion, these studies underscore the importance of Phe(37) of h-alpha-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His(10) as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.