In vitro testing of P-glycoprotein inhibitors identified from in silico database

从计算机数据库中鉴定出的 P-糖蛋白抑制剂的体外测试

• 批准号: 8100678
• 负责人: Pia D. Vogel
• 金额: $ 32.41万
• 依托单位: SOUTHERN METHODIST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-05 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8100678
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Affect; Affinity; Antibiotic Resistance; Antineoplastic Agents; Antiviral Agents; Binding; Binding Proteins; Binding Sites; Biochemical; Catalysis; Cells; Characteristics; Chemicals; Clinical; Computer Simulation; Databases; Development; Disease; Docking; Drug Binding Site; Drug Design; Drug Industry; Drug resistance; Effectiveness; Enzymes; Family; Goals; HIV; HIV Infections; Hand; Harvest; Homology Modeling; Human; Malignant Neoplasms; Measures; Molecular; Molecular Chaperones; Molecular Conformation; Multi-Drug Resistance; Nucleotides; P-Glycoprotein; P-Glycoproteins; Patients; Performance; Pharmaceutical Preparations; Phosphotransferases; Plant Roots; Procedures; Property; Protein Family; Proteins; Public Health; Publishing; Pump; Reporting; Resistance; Resolution; Roentgen Rays; Role; Screening procedure; Site; Specificity; Spin Labels; Staging; Structural Models; Structure; Testing; Therapeutic; Translating; Work; analog; base; chemotherapy; conformational conversion; drug candidate; drug mechanism; drug structure; effective therapy; enzyme pathway; human disease; in vitro testing; inhibitor/antagonist; interest; member; microorganism; research study; small molecule

DESCRIPTION (provided by applicant): Multidrug resistances pose a major obstacle to the effective treatment of many severe human diseases and remain an enormous public health problem because they are responsible for the loss of effectiveness of many anticancer and antiviral agents. High resolution structures of bacterial ABC transporters that are homologous to multidrug resistance proteins have been published. Recently the first X-ray structure of a eukaryotic multidrug resistance protein, P-glycoprotein (P-gp), that is thought to confer drug resistance in cancers and HIV infection, has been reported. Our group has used these structural models as well as homology models based on conserved structures to investigate the mechanism of drug export. Computational drug docking studies have revealed putative drug binding sites on both the human and Sav1866 pumps that correlate well with previously reported biochemical results. Most of the drug binding sites were observed in the transmembrane helical part of the protein, potentially making these sites part of the ATP-driven drug expulsion pathway of the enzyme. We hypothesize that inhibitors of P-gp that would chemosensitize drug resistant cells will be most efficient if they interact with the nucleotide binding sites of the enzyme and inhibit the energy harvesting steps of catalysis. In Aim 1 of this proposed study we will screen at least 9.5 million members of a drug-like chemical database for molecules that bind with high calculated affinity to the nucleotide binding domains, but that bind weakly at drug binding domains of P-gp. Candidates with these properties are hypothesized to inhibit P-gp, while not being good transport substrates. In Aim 2 we will test the small molecules identified in Aim 1 for the ability to specifically inhibit ATP- hydrolysis of purified, soluble P-glycoprotein as well as drug-stimulated ATP-hydrolysis of P-gp without negatively affecting other important ATP-utilizing enzymes. We will determine if binding of the inhibitors changes the overall affinity of P-gp for ATP in ESR studies using spin-labeled ATP. We will also determine whether the inhibitor molecules change the way that the enzyme hydrolyzes ATP by comparing transition state characteristics in the absence or presence of drug with and without normal transport substrate present. These studies are expected to provide us with a set of drug-like molecule leads that may translate into drugs to be co-administered with chemotherapeutics for effective chemotherapy. At the very least the identified molecules will serve as good candidates for rational drug design for these much needed inhibitors of multidrug resistance proteins. PUBLIC HEALTH RELEVANCE: Multidrug resistance phenomena remain an enormous public health problem and a major obstacle to the effective treatment of many severe human diseases. Up to ~40% of all human cancers develop multidrug resistance. These phenomena are responsible for the loss of effectiveness of many anticancer and antiviral agents and are root cause of many of the emerging antibiotic resistances of microorganisms. Even though considerable effort has been expended in the elucidation of the structure and enzymatic mechanism of the family of proteins responsible for multidrug resistance, there remain large gaps in our understanding of these important enzymes. The discovery and development of effective drugs that inhibit these enzymes and allow effective treatment of often intractable diseases like cancer or HIV infection will be of great importance. In this project we propose to in-silico screen large chemical data bases for small molecules that specifically and tightly interact with the energy harvesting parts of the multidrug resistance P-glycoprotein. Good candidates will then be tested in the lab as to their inhibitory effect on ATP hydrolysis and ATP binding to the enzyme, the steps thought to be needed for effective drug export. Drug candidates that are shown in the lab to inhibit these steps will be tested to extrapolate their potential effects on other important nucleotide binding proteins. Such small molecules that strongly inhibit P-gp activity while not or weakly interacting with other classes of enzymes may then be further developed for co-therapeutics in therapy of drug resistant cancers or HIV patients.

描述（由申请人提供）：多药耐药性对许多严重的人类疾病的有效治疗构成了主要障碍，并且仍然是一个巨大的公共卫生问题，因为它们导致许多抗癌和抗病毒药物失去有效性。与多药耐药蛋白同源的细菌 ABC 转运蛋白的高分辨率结构已发表。最近报道了真核多药耐药蛋白 P-糖蛋白 (P-gp) 的第一个 X 射线结构，该蛋白被认为在癌症和 HIV 感染中具有耐药性。我们课题组利用这些结构模型以及基于保守结构的同源模型来研究药物输出机制。计算药物对接研究揭示了人类泵和 Sav1866 泵上的推定药物结合位点，与之前报道的生化结果密切相关。大多数药物结合位点在蛋白质的跨膜螺旋部分中观察到，可能使这些位点成为酶的 ATP 驱动的药物排出途径的一部分。我们假设，如果 P-gp 抑制剂与酶的核苷酸结合位点相互作用并抑制催化的能量收集步骤，那么它们将使耐药细胞化学敏化，将是最有效的。在这项拟议研究的目标 1 中，我们将筛选类药物化学数据库中至少 950 万个成员，寻找与核苷酸结合域以高计算亲和力结合、但与 P-gp 药物结合域结合较弱的分子。假设具有这些特性的候选物能够抑制 P-gp，但不是良好的转运底物。在目标 2 中，我们将测试目标 1 中确定的小分子特异性抑制纯化的可溶性 P-糖蛋白的 ATP 水解以及药物刺激的 P-gp ATP 水解的能力，而不会对其他重要的 ATP 利用产生负面影响酶。我们将在使用自旋标记 ATP 的 ESR 研究中确定抑制剂的结合是否会改变 P-gp 对 ATP 的总体亲和力。我们还将通过比较不存在或存在药物以及存在和不存在正常转运底物的过渡态特征来确定抑制剂分子是否改变酶水解 ATP 的方式。这些研究有望为我们提供一组类药物分子先导化合物，这些分子可能转化为与化疗药物联合给药的药物，以实现有效的化疗。至少，所鉴定的分子将作为这些急需的多药耐药蛋白抑制剂的合理药物设计的良好候选者。 公共卫生相关性：多重耐药现象仍然是一个巨大的公共卫生问题，也是有效治疗许多严重人类疾病的主要障碍。高达约 40% 的人类癌症会产生多重耐药性。这些现象导致许多抗癌和抗病毒药物失去有效性，并且是许多新出现的微生物抗生素耐药性的根本原因。尽管在阐明负责多药耐药性的蛋白质家族的结构和酶促机制方面已经付出了相当大的努力，但我们对这些重要酶的理解仍然存在很大差距。发现和开发有效的药物来抑制这些酶并有效治疗癌症或艾滋病毒感染等顽固性疾病将具有非常重要的意义。在这个项目中，我们建议通过计算机筛选大型化学数据库来寻找与多药耐药性 P-糖蛋白的能量收集部分特异性且紧密相互作用的小分子。然后，好的候选药物将在实验室中测试其对 ATP 水解和 ATP 与酶结合的抑制作用，这些步骤被认为是有效药物输出所需的步骤。实验室中显示可抑制这些步骤的候选药物将进行测试，以推断它们对其他重要核苷酸结合蛋白的潜在影响。这种强烈抑制 P-gp 活性，同时不与其他类别的酶相互作用或微弱相互作用的小分子可以进一步开发用于治疗耐药癌症或 HIV 患者的联合治疗。