Substrate Activity Screening: A New Approach to Inhibitor Discovery

描述(申请人提供)：小分子酶抑制剂作为基本药物用于治疗广泛的普遍和危及生命的疾病。开发这些抑制剂所需的起始化合物通常要么通过高通量筛选(HTS)鉴定，要么由天然底物提供。然而，对于几类酶，如蛋白酶和磷酸酶，这两种方法都不是非常有效的。底物活性筛选(SAS)是发现小分子抑制剂的一种强有力的新方法，它具有加速酶抑制剂开发的巨大潜力。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HIT片段；(2)优化识别的底物片段；(3)通过直接掺入抑制剂药效团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关键的优点。首先，在传统的高通量抑制剂筛选中经常看到的假阳性被消除，因为该分析需要有效的底物结合和周转。其次，催化底物周转导致信号放大，从而能够识别弱活性的铅片段。本应用程序的总体目标是开发并将SAS方法应用于三种重要的酶类--半胱氨酸蛋白酶、磷酸酶和精氨酸脱亚胺酶。我们的中心假设是，SAS方法可以用于可靠和有效地发现小分子抑制剂，这将在化学生物学、药理学和药物发现应用中特别有用。已经开发或提出的抑制剂将带来一些非常重要的进展和积极影响，包括：(A)天冬氨酸S显像剂的进展，用于临床用于体外肿瘤成像，在手术中实时识别肿瘤边缘。(B)发展新的、有效的和口服可用的蛋白水解酶抑制剂，这种抑制剂已被证明可以消除动物模型中急性恰加斯病的症状。(C)在亨廷顿病动物模型中应用半胱氨酸氨基转移酶抑制剂的进展，这种动物模型已被证明可使亨廷顿病体外模型中的神经元免于细胞死亡，并可跨越血脑屏障。(D)将有效和选择性的磷酸酶抑制剂用于阿尔茨海默病的细胞和动物模型。(E)小分子抑制剂的进展，这将有助于确定蛋白质精氨酸脱亚胺酶的生物学作用，该酶催化POST 翻译修饰与类风湿性关节炎等疾病有关。将SAS方法应用于上述酶的实施和应用将产生许多创新。预期的结果将是开发非常有用的小分子抑制剂的强大和有效的战略。考虑到目标特定酶的巨大生物学和治疗重要性，预计也会产生相当大的积极影响。

DESCRIPTION (provided by applicant): Small molecule enzyme inhibitors serve as essential drugs for the treatment of a wide range of pervasive and life threatening diseases. The starting compounds needed for the development of these inhibitors are most often either identified by high throughput screening (HTS) or are provided by natural substrates. However, for several classes of enzymes such as proteases and phosphatases neither approach is very effective. A powerful new method for small molecule inhibitor discovery called Substrate Activity Screening (SAS) has enormous potential to accelerate the development of enzyme inhibitors. 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 substrate fragments are optimized, and (3) the optimized substrates are converted to inhibitors by direct incorporation of inhibitor pharmacophores. Screening for substrate as opposed to ligand fragments has two key advantages. First, false positives often seen in traditional high throughput inhibitor screens are eliminated because the assay requires productive substrate binding and turnover. Secondly, catalytic substrate turnover results in signal amplification enabling the identification of weakly active lead fragments. The overall objective of this application is to develop and apply the SAS method to three important enzyme classes - cysteine proteases, phosphatases and arginine deiminases. Our central hypothesis is that the SAS method can be used for the reliable and efficient discovery of small molecule inhibitors that will be exceptionally useful for chemical biology, pharmacology and drug discovery applications. A number of highly significant advances and positive impacts will result from inhibitors that have already been developed or are proposed, including: (A) Advancement of cathespin S imaging agents for clinical use in ex vivo tumor imaging for real time identification of tumor margins during surgery. (B) Advancement of novel, potent and orally available inhibitors of the protease cruzain that already have been shown to eliminate symptoms of acute Chagas' disease in animal models. (C) Advancement of caspase inhibitors to Huntington's disease animal models that have already been shown to rescue neurons from cell death in Huntington's disease ex vivo models and that cross the blood-brain barrier. (D) Advancement of potent and selective inhibitors of the phosphatase STEP to cell and animal models of Alzheimer's disease. (E) Advancement of small molecule inhibitors that will help to define the biological roles of protein arginine deiminases, which catalyze a post translational modification implicated in diseases such as rheumatoid arthritis. Many innovations will result from the implementation and application of the SAS method to the aforementioned enzymes. The expected outcomes will be robust and efficient strategies for the development of exceedingly useful small molecule inhibitors. Considerable positive impacts are also anticipated given the enormous biological and therapeutic importance of the specific enzymes being targeted.