Substrate Activity Screening: A New Approach to Inhibitor Discovery

描述(申请人提供)：酶在治疗危及生命的疾病的药物靶标中占相当大的比例，例如用HIV蛋白酶和逆转录酶抑制剂的鸡尾酒有效地治疗艾滋病，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，一项对前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)对大的化合物集合进行高通量筛选(HTS)；(2)将天然底物转化为基于机理的抑制剂。虽然这两种策略都成功地应用于一些酶靶标，但对于几类酶，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(被错误地识别为抑制剂的化合物)，而基于自然底物的方法导致非药物样抑制剂在体内效果不佳。提出了一种发现小分子抑制剂的新方法--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)针对酶靶标筛选不同的低分子底物文库以识别命中片段；(2)通过随后的几轮类似物合成和评估快速优化识别的片段；(3)通过直接掺入基于机理的抑制剂药效载体将优化的底物转化为抑制剂。筛选底物而不是配体片段有两个显著的优点。由于该分析需要高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经常出现的假阳性。其次，催化底物周转导致信号放大，从而能够识别非常弱的活性铅片段。将为半胱氨酸蛋白酶和酪氨酸蛋白磷酸酶开发SAS方法，这两大类酶包括许多重要的靶点，用于治疗危及生命的疾病，传统方法开发抑制剂具有挑战性。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，它是治疗恰加斯病的有效靶点；(2)布氏锥虫编码的两种用于治疗非洲昏睡病的基本半胱氨酸蛋白酶；(3)恶性疟原虫编码的二肽基肽酶DPAP3，它最近被确定为治疗疟疾的非常有希望的靶点；以及(4)结核分枝杆菌编码的两种磷酸酶，PTPA和PtpB，两者都被认为是治疗结核病的重要新的治疗靶点。 与公共卫生相关：酶抑制剂占所有治疗剂的30%以上。将开发一种新的方法，使快速识别新的、有效的和类药物的酶抑制剂成为可能。这种方法将通过开发酶抑制剂进行演示，这些酶抑制剂可以作为开发治疗疟疾、结核病、恰加斯病和非洲昏睡病等威胁生命的被忽视疾病的药物的先导。

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.