背景:组织蛋白酶K,参与骨吸收,是治疗骨质疏松症的好目标,但是尚未开发出临床批准的药物。最近,变构抑制剂具有高特异性和很少的副作用,在新药中的使用引起了人们的注意。
方法:使用组织蛋白酶K抑制活性辅助多步色谱法从Chamaecristanomame(Leguminosae)的甲醇提取物中分离组织蛋白酶K抑制剂。当使用分离的抑制剂及其衍生物时,采用标准动力学分析来检查组织蛋白酶K抑制的机制。使用AutoDockvina的对接研究支持了这些组织蛋白酶K抑制剂的变构结合。通过组织蛋白酶K抑制测定法检查了预期与不同变构位点结合的变构组织蛋白酶K抑制剂的组合。
结果:在Chamaecristanomame的甲醇提取物中鉴定出两种类型的组织蛋白酶K抑制剂。一种类型包括仙人掌B和Torachrysone8-β-龙胆苷,并以相似的抑制潜力抑制组织蛋白酶K和B,而另一种类型的抑制剂由脱镁叶绿素a组成,抑制组织蛋白酶K但不抑制组织蛋白酶B,表明脱茶素a与组织蛋白酶K的变构位点结合。抑制活性的动力学分析表明脱茶素a及其衍生物,菲奥普B,对组织蛋白酶K的对接研究支持了这种可能性。通过将其与变构抑制剂NSC13345和NSC94914结合到组织蛋白酶K上的其他变构位点,可以增强脱茶素a和脱茶酚b的组织蛋白酶K抑制活性。
IONS:组合结合到不同位点的不同变构抑制剂,如这项研究所示,可用于设计具有高特异性和副作用少的新型变构抑制药物。
BACKGROUND: Cathepsin K, which is involved in bone resorption, is a good target for treating osteoporosis, but no clinically approved medicine has been developed. Recently, allosteric inhibitors with high specificity and few side effects have been attracting attention for use in new medicines.
METHODS: Cathepsin K inhibitors were isolated from the methanol extract of Chamaecrista nomame (Leguminosae) using cathepsin K inhibition activity-assisted multi-step chromatography. Standard kinetic analysis was employed to examine the mechanism of cathepsin K inhibition when an isolated inhibitor and its derivative were used. The allosteric binding of these cathepsin K inhibitors was supported by a docking study using AutoDock vina. Combinations of allosteric cathepsin K inhibitors expected to bind to different allosteric sites were examined by means of cathepsin K inhibition assay.
RESULTS: Two types of cathepsin K inhibitors were identified in the methanol extract of Chamaecrista nomame. One type consisted of cassiaoccidentalin B and torachrysone 8-β-gentiobioside, and inhibited both cathepsin K and B with similar inhibitory potential, while the other type of inhibitor consisted of pheophytin a, and inhibited cathepsin K but not cathepsin B, suggesting that pheophytin a binds to an allosteric site of cathepsin K. Kinetic analysis of inhibitory activity suggested that pheophytin a and its derivative, pheophorbide b, bind allosterically to cathepsin K. This possibility was supported by a docking study on cathepsin K. The cathepsin K inhibitory activity of pheophytin a and pheophorbide b was enhanced by combining them with the allosteric inhibitors NSC 13345 and NSC94914, which bind to other allosteric sites on cathepsin K.
CONCLUSIONS: Different allosteric inhibitors that bind to different sites in combination, as shown in this study, may be useful for designing new allosteric inhibitory drugs with high specificity and few side effects.