Substrate Activity Screening: A New Approach to Inhibitor Discovery

底物活性筛选：抑制剂发现的新方法

• 批准号: 7869641
• 负责人: JONATHAN A ELLMAN
• 金额: $ 7万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7869641
• 关键词: Acquired Immunodeficiency Syndrome; African Trypanosomiasis; Angiotensin-Converting Enzyme Inhibitors; Binding; Biological Assay; Cardiovascular Diseases; Chagas Disease; Collection; Cysteine; Cysteine Protease; Development; Dipeptidyl Peptidases; Disease; Drug Delivery Systems; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Evaluation; Fluorescence; HIV Protease; Lead; Libraries; Life; Ligands; Malaria; Methods; Molecular Weight; Mycobacterium tuberculosis; Peptide Hydrolases; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Plasmodium falciparum; Preparation; Protein Tyrosine Phosphatase; RNA-Directed DNA Polymerase; Renin; Research; Reverse Transcriptase Inhibitors; Screening procedure; Serine Protease; Signal Transduction; Therapeutic Agents; Trypanosoma brucei brucei; Tuberculosis; acyl group; analog; base; cruzain; drug development; drug market; high throughput screening; in vivo; inhibitor/antagonist; inorganic phosphate; mimetics; neglect; new therapeutic target; non-drug; novel; novel strategies; pharmacophore; phosphatase inhibitor; public health relevance; small molecule; tuberculosis treatment

DESCRIPTION (provided by applicant): Enzymes represent a significant percentage of drug targets for the treatment of life threatening disease as exemplified by the effective management of AIDS with cocktails of HIV protease and reverse transcriptase inhibitors and the widespread treatment of cardiovascular disease with statins, angiotensin converting enzyme inhibitors, and most recently, renin inhibitors. Indeed, an analysis of the top 20 marketed drugs established that over one third were enzyme inhibitors. The two primary strategies for the development of small molecule enzyme inhibitors are (1) high throughput screening (HTS) of large compound collections, and (2) conversion of natural substrates to mechanism-based inhibitors. While both strategies have successfully been applied to a number of enzyme targets, for several classes of enzymes, neither approach has proven to be particularly effective. For these enzyme classes HTS has resulted in low hit rates and/or a high rate of false positives (compounds that are incorrectly identified as inhibitors) and natural substrate-based methods have resulted in non-drug like inhibitors with poor efficacy in vivo. A powerful new method for small molecule inhibitor discovery called Substrate Activity Screening (SAS) is proposed. The SAS method, which is the first substrate-based method for fragment discovery and optimization, consists of three steps: (1) a diverse library of low molecular weight substrates is screened against the enzyme target to identify hit fragments, (2) the identified fragments are rapidly optimized by subsequent rounds of analogue synthesis and evaluation, and (3) the optimized substrates are converted to inhibitors by direct incorporation of mechanism-based inhibitor pharmacophores. Screening for substrate as opposed to ligand fragments has two significant advantages. Because the assay requires productive substrate binding and turnover, false positives often seen in traditional high-throughput inhibitor screens are eliminated. Secondly, catalytic substrate turnover results in signal amplification enabling the identification of very weakly active lead fragments. The SAS method will be developed for cysteine proteases and tyrosine protein phosphatases, two large enzyme classes that encompass many important targets for the treatment of life-threatening disease and for which inhibitor development by traditional methods has been challenging. The SAS method will be demonstrated by the development of potent small molecule inhibitors to: (1) the cysteine protease cruzain, which is a validated target for the treatment of Chagas' disease, (2) the two essential cysteine proteases encoded by Trypanosoma brucei for the treatment of African sleeping sickness, (3) the dipeptidyl peptidase DPAP3 encoded by Plasmodium falciparum that recently was established as an extremely promising target for the treatment of malaria, and (4) the two phosphatases encoded by the Mycobacterium tuberculosis, PtpA and PtpB, both implicated as important new therapeutic targets for the treatment of tuberculosis. PUBLIC HEALTH RELEVANCE: Inhibitors of enzyme represent greater than 30% of all therapeutic agents. A new method will be developed that will enable the rapid identification of new, potent and drug like enzyme inhibitors. The method will be demonstrated by developing enzyme inhibitors that could serve as leads for the development of drugs to treat the life-threatening neglected diseases malaria, tuberculosis, Chagas' disease and African sleeping sickness.

描述（由申请人提供）：酶在治疗危及生命的疾病的药物靶点中占很大比例，例如用HIV蛋白酶和逆转录酶抑制剂的鸡尾酒有效管理艾滋病，以及用他汀类药物、血管紧张素转换酶抑制剂和最近的肾素抑制剂广泛治疗心血管疾病。事实上，对前20种上市药物的分析表明，超过三分之一是酶抑制剂。开发小分子酶抑制剂的两个主要策略是：(1)大化合物集合的高通量筛选（HTS）和(2)将天然底物转化为基于机制的抑制剂。虽然这两种策略已经成功地应用于许多酶目标，但对于几种酶，这两种方法都没有被证明是特别有效的。对于这些酶类，HTS导致低命中率和/或高假阳性率（化合物被错误地识别为抑制剂），而基于天然底物的方法导致体内疗效较差的非药物类抑制剂。提出了一种强大的小分子抑制剂发现新方法——底物活性筛选（SAS）。SAS方法是第一个基于底物的片段发现和优化方法，包括三个步骤：(1)针对酶目标筛选多种低分子量底物库以识别命中的片段；(2)通过后续的类似物合成和评估快速优化已识别的片段；(3)通过直接结合基于机制的抑制剂药效团将优化的底物转化为抑制剂。筛选底物与筛选配体片段有两个显著的优点。由于该分析需要有效的底物结合和周转，因此消除了传统高通量抑制剂筛选中常见的假阳性。其次，催化底物翻转导致信号放大，从而能够识别非常弱活性的铅碎片。SAS方法将用于半胱氨酸蛋白酶和酪氨酸蛋白磷酸酶，这两大酶类包含许多重要靶点，用于治疗危及生命的疾病，并且通过传统方法开发抑制剂一直具有挑战性。SAS方法将通过开发有效的小分子抑制剂来证明：(1)半胱氨酸蛋白酶cruzain，这是治疗恰加斯病的有效靶点；(2)布鲁氏锥虫编码的两种必需半胱氨酸蛋白酶，用于治疗非洲昏睡病；(3)恶性疟原虫编码的二肽基肽酶DPAP3，最近被确定为治疗疟疾的极有希望的靶点；(4)结核分枝杆菌编码的两种磷酸酶PtpA和PtpB。两者都是结核病治疗的重要新靶点。