HOT-ROXS: An integrated platform for identifying activators of "non-druggable" targets using biophysical screening, x-ray solution scattering and high-throughput co-crystallization

HOT-ROXS：使用生物物理筛选、X 射线溶液散射和高通量共结晶来识别“非成药”靶点激活剂的集成平台

• 批准号: 9141039
• 负责人: Vicki Nienaber
• 金额: $ 34.78万
• 依托单位: ZENOBIA THERAPEUTICS, INC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9141039
• 关键词: Address; Binding; Biological Assay; Biological Models; Categories; Characteristics; Complex; Crystallization; Disease; Drug Design; Enzyme Activators; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Guidelines; Lead; Libraries; Ligand Binding; Ligands; Literature; Measures; Medical; Methods; Modeling; Molecular; Molecular Conformation; Nerve Degeneration; Non-Insulin-Dependent Diabetes Mellitus; Parkinson Disease; Pattern; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Probability; Process; Production; Protein Conformation; Proteins; Roentgen Rays; Site; Structure; Temperature; Therapeutic; Therapeutic Agents; Ubiquitination; base; cancer type; drug discovery; env Gene Products; feeding; flexibility; high throughput screening; human genome sequencing; inhibitor/antagonist; insight; interest; melting; parkin gene/protein; prevent; programs; protein protein interaction; protein structure; public health relevance; screening; small molecule libraries

 DESCRIPTION (provided by applicant): Enzyme activators are significantly underrepresented as therapeutic agents versus enzyme inhibitors. There are only about a dozen examples of activator discovery in the scientific literature. The lack of activator therapeutics is not from a ack of targets for diverse unmet medical needs. Diseases such as neurodegeneration, cancer and type 2 diabetes could all benefit from an activator therapeutic. This application introduces a new platform, HOT-ROXS, for discovery of therapeutics for this class of "non-druggable" targets. HOT-ROXS addresses three of the common issues in activator discovery: a library rich in activators, a generally applicable assay directed at activator identification and structural characterization of the activators to drive medicinal chemistry optimization of the hits. To date, most activator discovery has been through high-throughput screening (HTS). HTS libraries are typically composed of large complex molecules. Probability calculations indicate that complex molecules are much less likely to bind to a target than a smaller simpler compound (fragments of drugs). Furthermore, the ligand binding efficiency (binding energy per atom) is typically much lower for HTS hits versus fragment hits. This confounds medicinal chemistry optimization and can lead to flat SAR. Here, activators are defined as compounds that bind directly to the target of interest and stabilize it in the active conformation. In HOT-ROXS, potential fragment activators are ideally identified as compounds that stabilize the active conformation of the protein by a positive shift in protein melting temperature. In cases where the active conformation cannot be screened, the inactive conformation is screened and the effect of activators on the melting temperature characterized early in the program by parallel activity screens. Protein structure for the activators is initially measured in solution using Wide Angle X-ray Scattering (WAXS). WAXS provides the molecular envelope for the protein-ligand complex and is very sensitive to conformational shifts. Changes as small as loop shifts can be detected by this method. WAXS is used as part of an iterative process with single crystal x-ray diffraction. The initial x-ray structure (maybe apo or a ligand complex) is fit to the WAXS pattern and changes upon activator binding identified. The x-ray structure may be remodeled to fit the new WAXS pattern. WAXS is also very sensitive to conformational uniformity which is also a key characteristic for protein crystallization. Activators are thought to increase the flexibility of proteins which would make crystallization of the complex more challenging. So, the WAXS pattern also identifies and prioritizes complexes for high-throughput co-crystallization studies. WAXS may also identify different protein conformation classes which may streamline the co-crystallization process or potentially provide for soaking of activators into pre-formed crystals. HOT-ROXS has been used to identify activators for a high priority Parkinson's disease target and the method will be further developed and refined using this model system.

 描述（通过应用程序提供）：酶激活剂与酶抑制剂相比治疗剂的代理显着不足。科学文献中只有大约十几个激活剂发现的例子。缺乏激活剂疗法不是来自未满足医疗需求的潜水员目标的AC。神经退行性，癌症和2型糖尿病等疾病都可以从激活剂治疗中受益。该应用程序介绍了一个新的平台Hot-Roxs，以发现此类“不可驱动”目标的理论。 Hot-Roxs解决了激活剂发现中的三个常见问题：富含激活剂的库，这是一种针对激活剂识别和激活剂的结构表征的普遍适用的测定，以驱动命中的药物化学优化。迄今为止，大多数激活剂发现已经通过高通量筛选（HTS）。 HTS文库通常由大型复合分子组成。概率计算表明，与较小的简单化合物（药物片段）相比，复杂分子与靶标的结合的可能性要小得多。此外，HTS命中与片段命中的配体结合效率（每个原子的结合能）通常要低得多。这会混淆药物化学优化，并可能导致扁平的SAR。在这里，激活剂定义为直接与感兴趣的目标结合并在活动会议中稳定它的化合物。在热糖中，理想地将潜在的片段激活剂鉴定为通过蛋白质熔化温度的正变化来稳定蛋白质的活性会议的化合物。如果无法筛选主动会议，则筛选了非活动会议，并且激活剂对通过并行活动筛选在程序早期表征的熔化温度的影响。最初，使用广角X射线散射（蜡）在溶液中测量激活剂的蛋白质结构。蜡为蛋白质配体复合物提供了分子包膜，并且对会议转移非常敏感。该方法可以检测到尽可能小的循环变化。蜡用作具有单晶X射线衍射的迭代过程的一部分。最初的X射线结构（也许是APO或配体配合物）适合蜡模式，并在鉴定的激活剂结合后变化。 X射线结构可以进行重塑以适合新的蜡模式。蜡也对宪法均匀性非常敏感，这也是蛋白质结晶的关键特征。人们认为激活剂会增加蛋白质的柔韧性，从而使复杂的结晶更具挑战性。因此，蜡模式还确定并确定了高通量共结晶研究的复合物。蜡还可以鉴定出不同的蛋白质会议类别，这些类别可能会简化共结晶过程，或者有可能将激活剂浸泡到预成型的晶体中。 Hot-Roxs已用于鉴定高优先级帕金森氏病目标的激活剂，并将使用此模型系统进一步开发和完善该方法。