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）：酶是药物靶标的很大一部分用于治疗生命威胁疾病的靶标，这是用HIV蛋白酶和逆转录酶抑制剂的有效治疗辅助药物的有效管理，以及用statins毒素，血管素转化enzyme抑制剂的毒素的广泛治疗。确实，对前20种药物的分析确定，三分之一以上是酶抑制剂。开发小分子酶抑制剂的两种主要策略是（1）大型化合物集合的高吞吐量筛选（HTS），以及（2）将天然底物转化为基于机制的抑制剂。尽管这两种策略已成功地应用于多种酶靶标，但对于几类酶，两种方法都没有被证明尤其有效。对于这些酶类，HTS导致较低的命中率和/或较高的假阳性率（错误地识别为抑制剂的化合物），基于天然底物的方法导致非药物抑制剂，例如抑制剂，在体内效力较差。提出了一种称为底物活性筛选（SAS）的小分子抑制剂发现的强大新方法。 SAS方法是第一种基于碎片发现和优化的基于底物的方法，由三个步骤组成：（1）筛选低分子量底物的多样化库与酶靶标筛选以识别命中片段，（2）确定的片段迅速优化了通过对随后的综合和（3）的效力，以及（3）的效法，以及（3）综合效力，以及（3），以及（3），（3）均值融合了（3）3）抑制剂药理。与配体碎片相反的底物筛选具有两个重要优势。由于该测定需要生产性的底物结合和营业额，因此消除了传统高通量抑制剂屏幕中经常看到的假阳性。其次，催化底物的离心额导致信号扩增，从而鉴定出非常弱的活性铅片段。 SAS方法将针对半胱氨酸蛋白酶和酪氨酸蛋白磷酸酶开发，这是两个大型酶类别，包括许多用于治疗威胁生命的疾病的重要目标，并且通过传统方法开发抑制剂是具有挑战性的。 SAS方法将通过开发有效的小分子抑制剂的发展来证明：（1）半胱氨酸蛋白酶cruzain，这是治疗Chagas疾病的经过验证的靶标，（2）由Brucei用锥类锥虫编码的两个必需的半胱氨酸蛋白酶通过非洲的dipass smote dipspose（3）（3）最近被确定为治疗疟疾的极有前途的恶性菌，以及（4）由结核分枝杆菌，PTPA和PTPB编码的两种磷酸酶，两种磷酸酶对结核病治疗的重要新治疗靶标均牵涉到重要的新治疗靶标。 公共卫生相关性：酶抑制剂占所有治疗剂的30％以上。将开发一种新方法，将能够快速鉴定新，有效和药物（如酶抑制剂）。该方法将通过开发酶抑制剂来证明，这些酶抑制剂可以作为开发药物来治疗威胁生命的被忽视疾病的疟疾，结核病，chagas疾病和非洲睡眠疾病的铅。