A Robust Predictive Method for Heparin and Heparan Sulfate Binding to Proteins

描述（由申请方提供）：尽管肝素和硫酸乙酰肝素在生长和形态学、凝血、血管生成、免疫应答和病毒感染中发挥重要作用，但其与几乎所有蛋白质（抗凝血酶除外）的相互作用仍不明确。2008年年中的事件中，一种存在于药物级肝素中的污染物在美国导致81人死亡，这进一步证明了了解肝素对蛋白质的识别的至关重要性。肝素/硫酸乙酰肝素的结构-活性关系不明确的主要原因是它们惊人的结构多样性。肝素和硫酸乙酰肝素都是含有数百万种结构的复杂的高阴离子多糖，预期其中仅选择少数序列优先识别靶蛋白。这些结构的鉴定不仅对于设计可能调节肝素/硫酸乙酰肝素作用的分子很重要，而且对于理解定义这些相互作用的基本生物化学原理也很重要，例如，识别的特异性、复合物的组装机制和顺序等。尽管生物物理技术取得了重大进展，但没有技术可用于研究肝素/硫酸乙酰肝素呈现的数百万种不同结构。在这种情况下，计算对接方法代表了一个强大的手段，推导相互作用的基本生物化学原理，以及确定关键序列或“大海捞针”。我们开发了一种强大的计算方法，称为组合虚拟库筛选（CVLS）方法，预测“高亲和力和高特异性”肝素/硫酸乙酰肝素序列结合抗凝血酶从近7,000个不同水平的硫酸化六糖库。CVLS代表了理解肝素/硫酸乙酰肝素与蛋白质相互作用的重大进展，并具有破译和解开肝素/硫酸乙酰肝素相互作用多样性的主要能力。为了进一步发展我们的CVLS技术在理解生化原理和设计H/HS模拟物，我们建议：1。进一步发展CVLS技术，用于研究H/HS与蛋白质的结合。该目的的工作假设是CVLS应用于i）丝氨酸蛋白酶抑制剂（抗凝血酶和肝素辅因子II）; ii）蛋白酶（凝血级联的因子IIa、Xa和IXa）;和iii）病毒糖蛋白（HSV-1的gD）将增强对相互作用的基本机理的理解，并鉴定可靶向调节的“特异性”相互作用。2.设计芳香族非糖H/HS模拟物的组合虚拟文库以鉴定H/HS -抗凝血酶相互作用的潜在调节剂。工作假设是CVLS技术应该适用于任何类型的分子，而不仅仅是硫酸化H/HS寡糖。我们先前已经发现，双环-单环支架有效地识别抗凝血酶。这些硫酸化的有机结构的库将建立在一个组合的方式，并使用双过滤器的方法进行评估，以确定先进的H/HS模拟。我们假设，我们的策略可以推广，并可用于其他H/HS -蛋白质系统。 公共卫生相关性：尽管肝素和硫酸乙酰肝素在生长和形态学、凝血、血管生成、免疫应答和病毒感染中发挥了重要作用，但它们与除抗凝血酶外的几乎所有蛋白质的相互作用仍不清楚。2008年年中的事件中，一种存在于药物级肝素中的污染物在美国导致81人死亡，这进一步证明了了解肝素对蛋白质的识别的至关重要性。拟议的研究旨在开发一种强大的技术来了解肝素和硫酸乙酰肝素如何与蛋白质结合，旨在改进现有的药物制剂并设计更好的药物。

DESCRIPTION (provided by applicant): Despite major roles played by heparin and heparan sulfate in growth and morphology, coagulation, angiogenesis, immune response and viral infection, their interaction with nearly all proteins, except for antithrombin, remains poorly defined. The events of mid-2008 in which a contaminant present in pharmaceutical grade heparin led to 81 deaths in the US demonstrate further the critical importance of understanding heparin recognition of proteins. A major reason for the ill-defined structure-activity relationships of heparin/heparan sulfate is their phenomenal structural diversity. Both heparin and heparan sulfate are complex, highly anionic polysaccharides containing millions of structures, of which only select few sequences are expected to preferentially recognize a target protein. Identification of these structures is important not only for designing molecules that may modulate the role of heparin/heparan sulfate, but also for understanding fundamental biochemical principles that define these interactions, e.g., specificity of recognition, the mechanism and order of assembly of complexes, etc. Despite the major advances in biophysical techniques, no technology is available to study the millions of diverse structures presented by heparin/heparan sulfate. In this context, computational docking approaches represent a powerful means of deducing both fundamental biochemical principles of interactions as well as identifying key sequence(s) or 'needle(s) in a haystack'. We developed a robust computational approach, called the combinatorial virtual library screening (CVLS) approach, that predicted 'high affinity and high specificity' heparin/heparan sulfate sequences binding to antithrombin from a library of nearly 7,000 hexasaccharides of varying levels of sulfation. CVLS represents a major advance in understanding heparin/heparan sulfate interactions with proteins and possesses major capabilities of deciphering and unraveling the diversity of heparin/heparan sulfate interactions. To further develop our CVLS technology in understanding biochemical principles and designing H/HS mimetics, we propose to: 1. Further develop the CVLS technology for studying H/HS binding to proteins. The working hypothesis in this aim is that application of CVLS to i) serpins (antithrombin and heparin cofactor II); ii) proteases (factors IIa, Xa and IXa of the coagulation cascade); and iii) viral glycoproteins (gD of HSV-1) will enhance fundamental mechanistic understanding the interactions and identify 'specific' interactions that may be targeted for modulation. 2. Design a combinatorial virtual library of aromatic, non-sugar H/HS mimetics to identify potential modulators of H/HS - antithrombin interaction. The working hypothesis is that the CVLS technology should be applicable to any class of molecule, and not just sulfated H/HS oligosaccharides. We have previously discovered that the bicyclic-unicyclic scaffold potently recognizes antithrombin. A library of these sulfated organic structures will be built in a combinatorial manner and assessed using the dual-filter approach for identification of advanced H/HS mimetics. We hypothesize that our strategy can be generalized and may be exploited for other H/HS - protein systems. PUBLIC HEALTH RELEVANCE: Despite major roles played by heparin and heparan sulfate in growth and morphology, coagulation, angiogenesis, immune response and viral infection, their interaction with nearly all proteins, except for antithrombin, remains poorly defined. The events of mid-2008 in which a contaminant present in pharmaceutical grade heparin led to 81 deaths in the US demonstrate further the critical importance of understanding heparin recognition of proteins. The proposed research on developing a robust technology for understanding how heparin and heparan sulfate bind to proteins aims to improve upon current pharmaceutical agents as well as design better drugs.