Structural and functional definition of the specificity of a novel caspase-3 inhibitor, Ac-DNLD-CHO.
Structural and functional definition of the specificity of a novel caspase-3 inhibitor, Ac-DNLD-CHO.
复制标题
DOI：
10.1186/1471-2210-7-8
复制
发表时间：
2007-06-27
期刊：
影响因子：
--
通讯作者：
Tanuma S
中科院分区：
文献类型：
--
作者：
Yoshimori A;Sakai J;Sunaga S;Kobayashi T;Takahashi S;Okita N;Takasawa R;Tanuma S
使用其X射线晶体学的胱天蛋白酶家族成员的基于肽的特异性抑制剂的合理设计是用于化学敲除以定义每种酶在细胞凋亡和炎症中的关键作用的重要策略。最近，我们设计了一种新的有效的肽抑制剂，Ac-DNLD-CHO，用于半胱天冬酶-3，使用一种新的计算筛选系统命名为氨基酸位置适合度（APF）方法（BMC Pharmacol. 2004，4：7）。在这里，我们报告的特异性DNLD序列对caspase-3的其他主要的caspase家族成员参与细胞凋亡的计算对接和定点诱变研究。Ac-DNLD-CHO抑制半胱天冬酶-3、-7、-8和-9活性，Kiapp值分别为0.68、55.7、>200和>200 nM。相比之下，众所周知的半胱天冬酶-3抑制剂Ac-DEVD-CHO以相似的Kiapp值抑制所有这些半胱天冬酶。通过使用荧光甲基香豆素酰胺（MCA）融合肽底物的底物偏好研究证实了caspase-3对DNLD序列的选择性识别。在计算对接研究中，分别基于底物Asn（N）和胱天蛋白酶-3残基S3亚位点中Ser 209之间的显著相互作用以及底物Leu（L）和胱天蛋白酶-3疏水S2亚位点之间的紧密相互作用评估其选择性和效力的基础。预期地，用丙氨酸取代Ser 209导致Ac-DNLD-MCA上的切割活性的丧失，并且对切割Ac-DEVD-MCA几乎没有影响。这些结果表明，N和L残基的Ac-DNLD-CHO的选择性和有效的抑制活性对caspase-3的决定因素。根据我们的结果，我们得出结论，Ac-DNLD-CHO是一种可靠的，有效的和选择性的caspase-3抑制剂。对caspase-3的特异性抑制作用表明，该抑制剂可能成为研究caspase-3生物学功能的重要工具。此外，Ac-DNLD-CHO可能是一个有吸引力的先导化合物，以产生新的有效的非肽类药物的半胱天冬酶介导的凋亡疾病，如神经退行性疾病和病毒感染性疾病。
The rational design of peptide-based specific inhibitors of the caspase family members using their X-ray crystallographies is an important strategy for chemical knockdown to define the critical role of each enzyme in apoptosis and inflammation. Recently, we designed a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 using a new computational screening system named the Amino acid Positional Fitness (APF) method (BMC Pharmacol. 2004, 4:7). Here, we report the specificity of the DNLD sequence against caspase-3 over other major caspase family members that participate in apoptosis by computational docking and site-directed mutagenesis studies. Ac-DNLD-CHO inhibits caspases-3, -7, -8, and -9 activities with Kiapp values of 0.68, 55.7, >200, and >200 nM, respectively. In contrast, a well-known caspase-3 inhibitor, Ac-DEVD-CHO, inhibits all these caspases with similar Kiapp values. The selective recognition of a DNLD sequence by caspase-3 was confirmed by substrate preference studies using fluorometric methylcoumarin-amide (MCA)-fused peptide substrates. The bases for its selectivity and potency were assessed on a notable interaction between the substrate Asn (N) and the caspase-3 residue Ser209 in the S3 subsite and the tight interaction between the substrate Leu (L) and the caspase-3 hydrophobic S2 subsite, respectively, in computational docking studies. Expectedly, the substitution of Ser209 with alanine resulted in loss of the cleavage activity on Ac-DNLD-MCA and had virtually no effect on cleaving Ac-DEVD-MCA. These findings suggest that N and L residues in Ac-DNLD-CHO are the determinants for the selective and potent inhibitory activity against caspase-3. On the basis of our results, we conclude that Ac-DNLD-CHO is a reliable, potent and selective inhibitor of caspase-3. The specific inhibitory effect on caspase-3 suggests that this inhibitor could become an important tool for investigations of the biological function of caspase-3. Furthermore, Ac-DNLD-CHO may be an attractive lead compound to generate novel effective non-peptidic pharmaceuticals for caspase-mediated apoptosis diseases, such as neurodegenerative disorders and viral infection diseases.