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
基本信息
- 批准号:9141039
- 负责人:
- 金额:$ 34.78万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressBindingBiological AssayBiological ModelsCategoriesCharacteristicsComplexCrystallizationDiseaseDrug DesignEnzyme ActivatorsEnzyme Inhibitor DrugsEnzyme InhibitorsEnzymesGuidelinesLeadLibrariesLigand BindingLigandsLiteratureMeasuresMedicalMethodsModelingMolecularMolecular ConformationNerve DegenerationNon-Insulin-Dependent Diabetes MellitusParkinson DiseasePatternPharmaceutical ChemistryPharmaceutical PreparationsPhaseProbabilityProcessProductionProtein ConformationProteinsRoentgen RaysSiteStructureTemperatureTherapeuticTherapeutic AgentsUbiquitinationbasecancer typedrug discoveryenv Gene Productsfeedingflexibilityhigh throughput screeninghuman genome sequencinginhibitor/antagonistinsightinterestmeltingparkin gene/proteinpreventprogramsprotein protein interactionprotein structurepublic health relevancescreeningsmall molecule libraries
项目摘要
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(由申请人提供):与酶抑制剂相比,酶激活剂作为治疗剂的代表性明显不足。科学文献中只有大约十几个激活剂发现的例子。激活剂治疗方法的缺乏并不是因为各种未满足的医学目标。神经退行性疾病、癌症和 2 型糖尿病等疾病都可以从激活剂治疗中受益。该应用引入了一个新平台 HOT-ROXS,用于发现治疗方法。此类“非成药”靶标解决了激活剂发现中的三个常见问题:富含激活剂的库、针对激活剂识别和激活剂结构表征的通用测定法,以驱动药物化学优化。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,概率计算表明复杂分子与特定分子结合的可能性要小得多。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,这会扰乱药物化学优化,并可能导致 SAR 平坦。被定义为直接结合目标靶点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熔解的正向转变来稳定蛋白质活性构象的化合物。在无法筛选活性构象的情况下,将筛选非活性构象,并在程序早期通过平行活性筛选来表征激活剂对解链温度的影响,首先使用广角 X 在溶液中进行测量。射线散射(WAXS)为蛋白质-配体复合物提供了分子包膜,并且对构象变化非常敏感,可以通过该方法检测到小到环变化的变化。单晶 X 射线衍射的迭代过程。初始 X 射线结构(可能是 apo 或配体复合物)适合 WAXS 模式,并且可根据识别的激活剂结合进行改变。 WAXS 对构象均匀性也非常敏感,这也是蛋白质结晶的一个关键特征,而激活剂被认为可以增加蛋白质的灵活性,从而使复合物的结晶更具挑战性。模式还可以识别高通量共结晶研究的复合物并对其进行优先级排序,WAXS 还可以识别不同的蛋白质构象类别,这可以简化共结晶过程或有可能将活化剂浸泡到已使用的 HOT-ROXS 中。以确定帕金森病高优先级目标的激活剂,并且将使用该模型系统进一步开发和完善该方法。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Vicki Nienaber其他文献
Vicki Nienaber的其他文献
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Discovery of inhibitors of the lipopolysaccharide synthesis pathway enzymes LpxA
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- 资助金额:
$ 34.78万 - 项目类别:
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$ 34.78万 - 项目类别:
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