Advanced Nucleation Technologies for Membrane Protein Crystallization to Accelerate Structure-Based Drug Design for Substance Use Disorders

先进的膜蛋白结晶成核技术可加速针对药物滥用疾病的基于结构的药物设计

• 批准号: 10546186
• 负责人: Andrew H. Bond
• 金额: $ 177.56万
• 依托单位: DENOVX, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10546186
• 关键词: Advanced Development; Benchmarking; Binding; Binding Sites; Biological Process; Characteristics; Chemicals; Complement; Crystallization; Data; Detergents; Disease; Drops; Drug Design; Engineering; Ensure; Goals; Health Benefit; Human; Hybrids; Hydrophobicity; Island; Knowledge acquisition; Legal patent; Location; Membrane Proteins; Methods; Modification; Molecular Conformation; National Institute of Drug Abuse; Outcome; Peripheral; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Productivity; Proteins; Proteome; Public Health; Reproducibility; Research Personnel; Resolution; Science; Signal Transduction; Solubility; Source; Structure; Substance Use Disorder; Succinate dehydrogenase (ubiquinone); Surface; Synchrotrons; Systems Development; Technology; Temperature; Time; United States National Institutes of Health; Universities; X ray diffraction analysis; base; commercial application; drug development; flexibility; high throughput screening; hydrophilicity; improved; innovation; monolayer; prototype; public health research; quinol fumarate reductase; response; screening; structural biology; surfactant; synchrotron radiation; technological innovation; therapeutic target

PROJECT SUMMARY DeNovX creates innovative platform products that improve the crystallization of proteins and pharmaceuticals. Of the ≈ 4700 human membrane proteins potentially involved in drug responses, ≈ 94% have yet to be structurally characterized owing to difficulties in crystallization. The goal of Phase II is to adapt DeNovX’s surface science agnostic approach to improving crystallization for use with membrane proteins to advance the structure-based understanding of substance use disorders (SUDs) to benefit Public Health. DeNovX improves crystallization using tunable substrates based on chemical interactions from bifunctional self-assembled monolayers (SAMs); surface energy modifications from engineered nucleation features (ENFs); and a hybrid strategy using chemically and energy modified ENFs (CENFs). A high confidence POC was achieved using the membrane proteins quinol:fumarate reductase (QFR) and succinate:quinone oxidoreductase (SQR). Diffraction quality QFR crystals were formed on a bifunctional SAM while no crystals formed on controls, and select ENFs produced up to a 19-fold increase in QFR crystals vs. controls. The hypothesis is that bifunctional SAMs, ENFs, or hybrid CENFs interacting with a membrane protein or its detergent envelope can facilitate preorganization and crystal nucleation from supersaturated solutions. Specific Aim 1 - Conduct controlled, replicate (n ≥ 6) studies of membrane protein crystallization outcomes using β-prototype bifunctional SAMs, ENFs, and CENFs to identify those characteristics most favorably impacting crystal nucleation of the QFR and SQR benchmark membrane proteins provided by Co-I Iverson under a subaward to Vanderbilt. These rigorous and quantitative crystallization studies will be complemented with synchrotron X-ray diffraction to ensure resolution that supports binding and conformational analyses through a subaward to Co-I Cohen at Stanford’s Synchrotron Radiation Lightsource (SSRL). Specific Aim 2 - Incorporate surface characteristics most favorably impacting membrane protein crystallization into bifunctional SAM, ENF, and CENFs on ≥ 12 𝛾-prototype 24/96/384 well HTS crystallization plates. Optimize for crystallization in detergent containing systems and advance development of the top six nucleation surfaces showing reproducible (n ≥ 6) crystallization improvements of ≥ 15% increase in hits, ≥ 20% reduction in onset times, or ≥ 25% increase in the quantity of crystals generated vs. controls. Specific Aim 3 - Incorporating SUD relevant target guidance from NIH and domain experts with a tractability assessment by the Co-Is, expand crystallization screening with optimized nucleation surfaces to ≥ 8 membrane proteins of varying class that would most benefit from near atomic resolution structural data. Generate crystals for ≥ 2 targets for study at SSRL. Specific Aim 4 - Demonstrate the tangible benefits and deploy the surface science product suite for membrane protein crystallization screening with pharmaceutical company, NIH, and academic researchers to facilitate therapeutic target identification for SUDs. DeNovX will sell its patented high throughput crystallization plates in a $450M-650M market.

项目摘要 Denovx创建了创新的平台产品，可改善蛋白质和药物的结晶。 在潜在参与药物反应的≈4700个人膜蛋白中，≈94％尚未是 由于结晶的难度而在结构上表征。第二阶段的目的是适应Denovx的 表面科学不可知的方法，用于改善与膜蛋白一起使用的结晶以促进 基于结构的对物质使用障碍（SUD）的理解以使公共卫生受益。 DENOVX改进 基于双功能自组装的化学相互作用的可调底物结晶 单层（SAMS）；工程成核特征（ENF）的表面能量修饰；和混合动力 使用化学和能量改性ENF（CENF）的策略。使用 膜蛋白喹酚：富马酸盐还原（QFR）和琥珀酸酯：喹酮氧化氧化激酶（SQR）。衍射 优质QFR晶体在双功能的SAM上形成，而在对照组上没有晶体，然后选择ENF 与对照组相比，QFR晶体的产生高达19倍。假设是双功能SAM， ENFS或与膜蛋白相互作用或其敏感性包膜相互作用的混合CENF可以促进 过饱和溶液的组织前和晶体成核。特定目标1-受控的行为， 复制（n≥6）研究膜蛋白结晶结果的研究，使用β-功能型双功能SAM， ENF和CENF确定这些特征最有利影响QFR的晶体成核和 Co-I Iverson在范德比尔特的亚武器下提供的SQR基准膜蛋白。这些严格 定量结晶研究将使用同步加速器X射线衍射完成，以确保 通过子宣告与斯坦福大学的Co-i Cohen进行约束和构象分析的决议 同步子辐射光线（SSRL）。特定目标2-最有利的表面特征 在≥12𝛾-蛋白质上，影响膜蛋白结晶成双功能的SAM，ENF和CENFS 24/96/384井HTS结晶板。确定包含系统和 提前开发前六个核表面，显示可再现（n≥6）结晶 命中率增加≥15％，发作时间减少20％，或≥25％的增长数量增加 晶体生成与控件。特定目的3-合并NIH的SUD相关目标指导 通过共同评估易于评估的领域专家，扩展结晶筛选，并优化 成核表面至≥8种不同类别的膜蛋白 分辨率结构数据。在SSRL上生成≥2个目标的晶体。特定目标4-证明 切实的好处并部署表面科学产品套件以膜蛋白结晶筛选 与制药公司，NIH和学术研究人员一起促进治疗目标识别 泡沫。 Denovx将在4.5亿至6.5亿美元的市场中出售其专利的高通量结晶板。