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

项目总结 DeNovX创造了创新的平台产品，改善了蛋白质和药物的结晶。 在可能参与药物反应的≈4700人膜蛋白中，≈94%尚未参与药物反应 由于结晶困难而具有结构特征的。第二阶段的目标是将DeNovX的 表面科学不可知的方法，以改善结晶用于膜蛋白，以促进 基于结构的对物质使用障碍(SUDS)的理解，以造福公众健康。DeNovX有所改善 基于双功能自组装化学相互作用的可调衬底结晶 单分子膜(SAM)；基于工程成核特征(ENF)的表面能修饰；以及混合体 使用化学和能量修饰的ENF(CENF)的策略。使用以下工具实现了高度可信的PoC 膜蛋白喹酚：富马酸还原酶(QFR)和琥珀酸氧化还原酶(SQR)。绕射 在双功能SAM上形成了高质量的QFR晶体，而在对照和部分ENF上没有形成晶体 与对照组相比，QFR晶体的数量增加了19倍。假设是双功能SAM， ENF或杂交CENF与膜蛋白或其洗涤剂包膜相互作用可以促进 过饱和溶液中的预组织和晶体形核。具体目标1--行为受控， 重复(n≥6)使用β原型双功能自组装膜蛋白结晶结果的研究， ENF和CENF，以确定最有利地影响QFR和 SQR基准膜蛋白由Co-I Iverson在授予Vanderbilt的分奖下提供。这些严谨的 定量结晶研究将与同步辐射X射线衍射相辅相成，以确保 通过授予斯坦福大学的Co-I Cohen子奖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征 在≥12𝛾-Prototype上影响膜蛋白结晶为双功能SAM、Enf和CENf 24/96/384孔高温超导结晶板。在含有洗涤剂的系统中优化结晶和 显示可重复(n≥6)结晶的前六个成核面的进展 ≥的改善点击率增加15%，≥的发病时间减少20%，或≥的数量增加25% 产生的晶体与对照的晶体。具体目标3-纳入NIH的SUD相关目标指南和 由Co-IS进行可处理性评估的领域专家，通过优化的 不同类别的≥8膜蛋白的成核面，这将使近原子受益最大 解析结构化数据。为≥2靶产生晶体，以便在上海同步辐射实验室进行研究。具体目标4--展示 实实在在的好处，并部署用于膜蛋白结晶筛选的表面科学产品套件 与制药公司、NIH和学术研究人员合作，促进治疗靶点的确定 肥皂水。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。

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.