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创造创新的平台产品，改善蛋白质和药物的结晶。 在潜在参与药物反应的104700种人类膜蛋白中，有1094%尚未被发现。 由于结晶困难而具有结构特征。第二阶段的目标是调整DeNovX的 表面科学不可知的方法，以改善与膜蛋白一起使用的结晶， 以结构为基础的理解物质使用障碍（SUD），以造福公共卫生。DeNovX改进 基于来自双功能自组装的化学相互作用的使用可调衬底的结晶 单分子层（SAM）;来自工程化成核特征（ENF）的表面能修饰;和混合物 使用化学和能源改性ENF（CENF）的战略。高置信度的POC是使用 膜蛋白醌：延胡索酸还原酶（QFR）和琥珀酸：醌氧化还原酶（SQR）。衍射 在双功能SAM上形成优质QFR晶体，而在对照上没有晶体形成，并且选择ENF 与对照相比，QFR晶体产生高达19倍的增加。假设是双功能自组装膜， 与膜蛋白或其去污剂包膜相互作用的ENF或杂合CENF可以促进 预组织和晶体成核从过饱和溶液。具体目标1-行为受到控制， 使用β-原型双功能SAM对膜蛋白结晶结果进行重复（n ≥ 6）研究， ENF和CENF，以确定最有利地影响QFR晶体成核的那些特征， SQR基准膜蛋白由Co-I Iverson在范德比尔特的子合同下提供。这些严格 和定量结晶研究将补充同步加速器X射线衍射，以确保 通过在斯坦福大学的Co-I Cohen的子奖项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征 在≥ 12 μ m原型上影响膜蛋白结晶为双功能SAM、ENF和CENF𝛾 24/96/384孔HTS结晶板。优化含洗涤剂系统中的结晶， 先进的六个成核表面的开发，显示出可再现的（n ≥ 6）结晶 命中率增加≥ 15%、起效时间减少≥ 20%或 产生的晶体与对照相比。具体目标3-阐明NIH的SUD相关目标指导， 领域专家通过Co-Is的易处理性评估， 成核表面至≥ 8个不同类别的膜蛋白，这些膜蛋白将最受益于近原子 解析结构数据在SSRL为≥ 2个目标生成晶体进行研究。具体目标4-展示 为膜蛋白结晶筛选部署表面科学产品套件 与制药公司，NIH和学术研究人员合作，以促进治疗靶点的识别， SUD。DeNovX将在4.5亿至6.5亿美元的市场上销售其专利的高通量结晶板。