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（由申请方提供）：与酶抑制剂相比，酶激活剂作为治疗剂的代表性明显不足。在科学文献中只有大约十几个激活剂发现的例子。活化剂疗法的缺乏并不是因为缺乏各种未满足的医疗需求的靶点。神经变性、癌症和2型糖尿病等疾病都可以从激活剂治疗中受益。该应用程序引入了一个新的平台，HOT-ROXS，用于发现这类“非药物”靶点的治疗方法。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，针对激活剂鉴定和激活剂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，大多数激活剂的发现都是通过高通量筛选（HTS）进行的。HTS文库通常由大的复杂分子组成。概率计算表明，复杂的分子比更小的简单化合物（药物片段）更不可能与靶标结合。此外，HTS命中的配体结合效率（每个原子的结合能）通常比片段命中低得多。这混淆了药物化学优化，并可能导致SAR平坦。在这里，活化剂被定义为直接结合目标靶标并将其稳定在活性构象的化合物。在HOT-ROXS中，潜在的片段激活剂理想地被鉴定为通过蛋白质解链温度的正移来稳定蛋白质的活性构象的化合物。在活性构象无法筛选的情况下，筛选非活性构象，并通过平行活性筛选在程序早期表征活化剂对解链温度的影响。激活剂的蛋白质结构最初使用广角X射线散射（WAXS）在溶液中测量。WAXS为蛋白质-配体复合物提供分子包膜，并且对构象变化非常敏感。这种方法可以检测到小到环路移位的变化。WAXS用作单晶X射线衍射迭代过程的一部分。初始X射线结构（可能是载脂蛋白或配体复合物）与WAXS图谱相吻合，并在识别激活剂结合后发生变化。X射线结构可以被重塑以适应新的WAXS模式。WAXS对构象均匀性也非常敏感，这也是蛋白质结晶的关键特征。活化剂被认为增加了蛋白质的灵活性，这将使复合物的结晶更具挑战性。因此，WAXS模式还可以识别和优先考虑用于高通量共结晶研究的复合物。WAXS还可以鉴定不同的蛋白质构象类别，其可以简化共结晶过程或潜在地提供活化剂浸泡到预形成的晶体中。HOT-ROXS已被用于识别高优先级帕金森病靶点的激活剂，该方法将使用该模型系统进一步开发和完善。