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.

项目概要 DeNovX 创造了可改善蛋白质和药物结晶的创新平台产品。 在大约 4700 种可能参与药物反应的人类膜蛋白中，大约 94% 尚未得到证实 由于结晶困难而在结构上进行了表征 第二阶段的目标是适应 DeNovX 的。 与表面科学无关的方法来改善结晶，与膜蛋白一起使用，以促进 改善对物质使用障碍 (SUD) 的基于结构的理解，以造福公共健康。 使用基于双功能自组装化学相互作用的可调基质进行结晶 单层（SAM）；工程成核特征（ENF）和混合层的表面能修饰； 使用化学和能量修饰的 ENF (CENF) 策略使用高置信度的 POC 来实现。 膜蛋白喹啉：富马酸还原酶（QFR）和琥珀酸：醌氧化还原酶（SQR）。 在双功能 SAM 上形成优质 QFR 晶体，而在对照上没有形成晶体，并选择 ENF 与对照相比，QFR 晶体的产量增加了 19 倍。假设双功能 SAM， ENF 或混合 CENF 与膜蛋白或其去污剂包膜相互作用可以促进 过饱和溶液的预组织和晶体成核具体目标 1 - 行为受控， 使用 β 原型双功能 SAM 重复 (n ≥ 6) 膜蛋白结晶结果研究， ENF 和 CENF 来识别那些最有利于影响 QFR 和晶体成核的特征 SQR 基准膜蛋白由 Co-I Iverson 在范德比尔特大学的资助下提供。 同步加速器 X 射线衍射将补充定量结晶研究，以确保 通过向斯坦福大学的 Co-I Cohen 提供子奖项来支持结合和构象分析的决议 同步辐射光源 (SSRL)。具体目标 2 - 最有利地结合表面特性。 影响膜蛋白在 ≥ 12 𝛾 原型上结晶成双功能 SAM、ENF 和 CENF 24/96/384 孔 HTS 结晶板优化含有洗涤剂的系统和系统中的结晶。 先进的前六个成核表面显示出可重复的 (n ≥ 6) 结晶 命中率增加 ≥ 15%，起效时间减少 ≥ 20%，或数量增加 ≥ 25% 生成的晶体与对照的具体目标 3 - 纳入 NIH 和 SUD 的相关目标指导。 领域专家通过 Co-Is 进行易处理性评估，通过优化扩大结晶筛选 成核表面≥ 8个不同类别的膜蛋白，这些膜蛋白最受益于近原子 生成 ≥ 2 个目标的晶体以在 SSRL 进行研究 具体目标 4 - 演示 切实的好处并部署表面科学产品套件进行膜蛋白结晶筛选 与制药公司、NIH 和学术研究人员合作，促进治疗靶点的识别 DeNovX 将在 4.5 亿至 6.5 亿美元的市场上销售其专利高通量结晶板。