新型利尿钠肽药物分子的理性设计及药学分析

Natriuetic Peptide (NP) and its derivatives is a class of pharmaceuticals used in the stage of acute decompensated heart failure, which is tracting more attention recently. The stable blood drug level, among other PK properties, is essential for the sucessful clinic application. The B-type Natriuetic Peptide (BNP), which is already on market, has some drawbacks incluging luck of stability, short halflife, and among others in vivo. Moreover, its functions on natriuresis are still unconfirmed.Therefore its clinical application is limited. This proposal is to use NP as a precursor, a rational design approach for the modifying NP as drug has been proposed. In the present investigation, biochemical modifications on the enzymatic hydrolysis center inside the NPs are suggested and the combining abilities of the functional receptors are identified and compared by using the computational biology technique. Experimentally, the secondary structure of the enzymatic hydrolysis center modified NP with high medical effects are selected by the in vitro refolding technique. The designed NP derivatives are then compared with the experimental determined NPs. These comparisons are adopted to improve the computational biology based rational design. In this way, NPs molecules with more favouable pharmacological properties are expected to be designed and developed. The knowledge of design for macromoleucle drug is expected to be accumulated during this project executing. This knowledge is important for the development of the methodology of rational design for developing peptide based drugs. It also provides theoretical supportive information for the development of peptide based drugs.

利尿钠肽（NP）家族是急性失代偿性心力衰竭阶段治疗药物研发的热点，保持稳态的血药浓度是临床治疗效果的必要条件。已上市的B-型NP（BNP）药物除存在稳定性差、体内半衰期短等缺陷外，其排钠利尿作用尚有疑问。项目以NP为药物前体，计算生物学技术为工具，提出定位改造NP分子内酶解部位，结合功能受体结合差异设计NP成药性分子的理性设计策略。对于合成获得经酶解部位突变的NP，利用体外辅助折叠技术筛选确认优势药效活性的二级结构，与理性设计的候选NP分子进行比较，修正和优化理性设计方法，形成对设计结果的互补验证，获得具有高药学品质的NP药物分子。项目的实施，可积累大分子药物设计的知识，为多肽药物分子的理性设计提供可操作的方法学和相应的理论支持，并可望产生具有自主知识产权的新型NP药物；也提示在多肽药物制备工艺的研究中既要注重序列正确和辅助键形成，更要重视优势药学二级结构的工艺条件建设。

钠尿肽（NP）是一组具有排钠利尿、舒张血管、降低血压和调节电解质平衡等的激素，设计开发活性高、稳定性强的新型NP药物有重要的意义。本研究采用氨基酸虚拟突变和分子动力学模拟技术，以已上市药物BNP为模版，理性设计和实验验证新型的NP多肽分子。主要结果为：（1）同源建模补齐3种天然NP分子序列全长的空间结构，以其为模板构建了人工URO和VNP模型。发现除CNP外，ANP、BNP、URO和VNP的N-和C-末端均呈现立体相交叉模式，推测这种构象更便于它们与受体的输水性口袋识别结合。（2）上述5种NP分子100ns分子动力学模拟计算发现未与NPR受体结合时的ANP、CNP和VNP呈现较为稳定、致密的空间结构，形成的分子内氢键数目相对较少，推测这是它们生物活性较高的原因之一。（3）多肽-蛋白分子对接技术构建了NPRA、NPRB与NP的复合物模型，发现BNP与NPRA的相互作用较强，形成的分子间氢键数目多，相互作用能更低；VNP与NPRB的相互作用强于CNP，复合物的稳定性高。（4）对BNP进行氨基酸虚拟突变设计，借助打分函数和能量优化，确定了理论上最优的10个突变体。分子动力学模拟和轨迹分析确定了两个候选突变体分子BNPmt1 (S20R/S8W/S21R)和BNPmt2 (S19R/G7R/S21R)，其与NPR间的平均成氢键数比野生型BNP分别增加了31.56%和10.59%；BNPmt1与受体间的相互作用能的平均值为-689.789 kcal/mol，BNPmt2为-619.348 kcal/mol，比野生型BNP分别降低了22.78%和8.82%，推测两者具有更高的生理活性。（6）设计和构建了可检测NP活性的NPRA和NPRB受体转染细胞系HEK-293，发现BNPmt1和BNPmt2比野生型BNP具有更强的生物学活性，可作为治疗ADHF的新型候选药物分子。