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

描述(由申请人提供)：尽管肝素和硫酸肝素在生长和形态、凝血、血管生成、免疫反应和病毒感染方面发挥了重要作用，但它们与除抗凝血酶外的几乎所有蛋白质的相互作用仍然不清楚。2008年年中，美国药用级肝素中的一种污染物导致81人死亡的事件进一步证明了了解肝素对蛋白质识别的关键重要性。肝素/硫酸肝素构效关系不明确的一个主要原因是它们惊人的结构多样性。肝素和硫酸肝素都是复杂的、高度阴离子的多糖，含有数百万个结构，只有几个序列被认为是优先识别目标蛋白的。鉴定这些结构不仅对设计可能调节肝素/硫酸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化原理也很重要，例如识别的特异性、复合体的组装机制和顺序等。尽管生物物理技术取得了重大进展，但还没有技术来研究肝素/硫酸肝素呈现的数百万种不同的结构。在这种背景下，计算对接方法代表了一种强大的手段，既可以推断相互作用的基本生化原理，也可以识别关键序列(S)或大海捞针(S)。我们开发了一种稳健的计算方法，称为组合虚拟文库筛选(CVLS)方法，它从一个包含近7,000种不同硫化程度的六糖的文库中预测与抗凝血酶结合的高亲和力和高特异性的肝素/硫酸肝素序列。CVLS在了解肝素/硫酸肝素与蛋白质相互作用方面取得了重大进展，并具有破译和解开肝素/硫酸肝素相互作用多样性的重要能力。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模拟分子方面，我们建议：1.进一步发展研究H/HS与蛋白质结合的CVLS技术。这一目标的工作假设是，将CVLS应用于i)蛇类(抗凝血酶和肝素辅助因子II)；ii)蛋白酶(凝血级联的IIa、Xa和IXa因子)；以及iii)病毒糖蛋白(HSV-1的gD)将增强对相互作用的基本机制的理解，并识别可能作为调节目标的“特定”相互作用。2.设计了一个芳香、非糖H/HS模拟物的组合虚拟文库，以确定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一类分子，而不仅仅是硫酸盐化的H/HS寡糖。我们之前已经发现，双环-单环支架可以有效识别抗凝血酶。这些硫化有机结构的文库将以组合的方式建立，并使用双过滤器方法进行评估，以鉴定先进的H/HS模拟物。我们假设我们的策略可以推广到其他H/HS-蛋白质系统。 与公共卫生相关：尽管肝素和硫酸肝素在生长和形态、凝血、血管生成、免疫反应和病毒感染方面发挥了重要作用，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，美国药用级肝素中的一种污染物导致81人死亡的事件进一步证明了了解肝素对蛋白质识别的关键重要性。拟议中的研究旨在开发一种强大的技术来了解肝素和肝素硫酸盐如何与蛋白质结合，目的是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.