Chelator Fragment Libraries for Optimizing Metal-Ligand Interactions in Metallopr

描述（由申请人提供）：该提案的重点是开发新的方法和策略来发现金属蛋白抑制剂。金属蛋白是一类重要的药物靶点，与治疗许多疾病相关，包括癌症、关节炎、细菌感染等。大多数抑制金属蛋白的治疗剂使用金属结合基团（MBG）来结合活性位点金属离子。尽管这些MBG的重要性，很少有研究已经开发出的方法来识别，优化，并了解什么MBG是最好的一个给定的金属蛋白的利益。 该提议试图回答关于在金属蛋白抑制剂中使用的MBG的“什么、为什么和如何”，即：a）什么MBG最好地抑制给定的金属蛋白？B）为什么某些MBG对某种金属蛋白的抑制作用最好？c）如何加速MBG的发现、评估和开发，使其从成功走向领先？ 解决这些问题将解决医学和生物无机化学交叉的一个基本问题。我们的研究小组在金属蛋白抑制剂领域经验丰富，我们已经在回答上述问题方面取得了实质性进展。从根本上说，我们的实验室正在寻求一种基于片段的先导发现（FBLD）方法来开发抑制剂，其中第一次将金属螯合剂用作片段，以探索针对结合金属蛋白活性位点的化学空间。在实施这种方法中，我们已经开发了一个螯合剂片段库（CFL），可以很容易地对金属蛋白进行筛选，以确定什么MBGs最好地结合到给定的目标。我们还确定了MBG在金属蛋白活性位点的结构，以了解为什么MBG以高亲和力结合到活性位点。接下来，我们使用了生物无机模型复合物和计算对接方法的组合，以开发改进的方法，以确定如何发现MBG片段可以开发成铅抑制剂。最后，我们结合了所有上述研究来制备螯合剂亚文库，其提供了更高级的片段命中，该片段命中探测金属离子活性位点之外。 在我们正在进行的研究中，我们将针对大量的金属蛋白筛选我们的文库（Aim 1，Aim 4），但将更详细的研究集中在两个特定的Zn 2+依赖系统（Aim 2，Aim 3），基质金属蛋白酶（MMP）和碳酸酐酶（CA）。这些金属蛋白，共享一个共同的金属活性位点基序，但不同的MBG偏好，将作为优秀的系统，对测试我们的想法和比较和对比的MBG结合的细节。我们建议：1.开发和筛选针对各种金属蛋白靶点的螯合片段库（CFL）。使用现有的和新的节能灯，将对十多种不同的金属酶进行筛选，以揭示MBG与各种靶点的最佳结合。 2.对蛋白质进行结构和热力学研究，以了解为什么给定的MBG与给定的金属蛋白活性位点紧密结合。将用MMP-3和hCAI检查CFL的命中，其中：a）共结晶MBG的晶体结构测定将揭示MBG-金属蛋白结合的关键特征，和B）通过等温滴定量热法的热力学研究将提供结合常数和相关能量参数。 3. MMP和CA活性位点中的模型MBG片段与从蛋白质晶体学获得的结构数据进行比较。合成的生物无机模型化合物将被结晶提供MBG结合的无限制模式的结构数据，而计算对接研究将被用来预测的整体构象和第二配位球之间的MBG和金属蛋白的相互作用。通过结合生物无机建模和计算对接，我们将试图准确地概括晶体学数据。 4.将制备含有MBG衍生物的亚文库以使这些支架从命中到引导。对MBG支架的扩增将产生含有化合物的子库，所述化合物可以针对金属蛋白进行筛选以提供更先进的先导结构。这些将被开发用于MMPs和CA，以及各种其他具有药用价值的金属蛋白。 公共卫生相关性：金属蛋白的抑制对于治疗从心脏病到癌症的病理学非常重要。该项目旨在发现新的化合物和新的方法来鉴定金属蛋白抑制剂。将实现对抑制剂与酶活性位点中的金属离子之间的相互作用的更好理解和优化。

DESCRIPTION (provided by applicant): This proposal is focused on developing new approaches and strategies to the discovery of metalloprotein inhibitors. Metalloproteins are an important class of medicinal targets that are relevant to treating numerous diseases including cancer, arthritis, bacterial infections, and many others. Most therapeutics that inhibit metalloproteins use a metal-binding group (MBG) to bind to the active site metal ion. Despite the importance of these MBGs, very few studies have developed methods to identify, optimize, and understand what MBGs are best for a given metalloprotein of interest. This proposal seeks to answer the 'what, why, and how' about the MBGs used in metalloprotein inhibitors, namely: a) WHAT MBGs best inhibit a given metalloprotein? b) WHY some MBGs give the best inhibition against a certain metalloprotein? c) HOW can the discovery, evaluation, and development of MBGs be accelerated from hit-to-lead? Answering these questions will address a fundamental problem at the intersection of medicinal and bioinorganic chemistry. Our research group is experienced in the field of metalloprotein inhibitors, and we have made substantial progress toward answering the aforementioned questions. Fundamentally, our laboratory is pursuing a fragment-based lead discovery (FBLD) approach to inhibitor development, where for the first time, metal chelators are being used as fragments that will explore chemical space targeted to binding metalloprotein active sites. In implementing this approach we have developed a chelator-fragment library (CFL) that can be readily screened against metalloproteins to identify what MBGs bind best to a given target. We have also determined the structure of an MBG in the active site of a metalloprotein to understand why the MBG binds with high affinity to the active site. Next, we have used and combination of bioinorganic model complexes and computational docking approaches to develop improved methods to determine how the discovery of MBG fragments can be developed into lead inhibitors. Finally, we have combined all of the aforementioned studies to prepare chelator sublibraries that provide more advanced fragment hits that probe beyond the metal ion active site. In our ongoing studies we will screen our libraries against a large number of metalloproteins (Aim 1, Aim 4), but will focus our more detailed studies on two specific Zn2+dependent systems (Aim 2, Aim 3), the matrix metalloproteinases (MMPs) and carbonic anhydrases (CAs). These metalloproteins, which share a common metal active site motif, but different MBG preferences, will serve as excellent systems against which to test our ideas and compare and contrast the details of MBG binding. We propose to: 1. Develop and screen chelator fragment libraries (CFLs) against a wide range of metalloprotein targets. Using existing and new CFLs, screens against more than ten different metalloenzymes will be performed to reveal what MBGs bind best to various targets. 2. Perform structural and thermodynamic studies with proteins to obtain a molecular understanding of why a given MBG binds tightly to a given metalloprotein active site. Hits from CFLs will be examined with MMP-3 and hCAI, where: a) crystallographic structure determinations with co-crystallized MBGs will reveal key features of MBG-metalloprotein binding, and b) thermodynamic studies via isothermal titration calorimetry will provide binding constants and relevant energetic parameters. 3. Model MBG fragments in the MMP and CA active site to compare with structural data obtained from protein crystallography. Synthetic bioinorganic model compounds will be crystallized providing structural data on the unrestrained mode of MBG binding, while computational docking studies will be used to predict the overall conformation and second coordination sphere interactions between the MBG and the metalloprotein. By combining bioinorganic modeling and computational docking we will try to accurately recapitulate the crystallographic data. 4. Sublibraries containing MBG derivatives will be prepared to advance these scaffolds from hit- to-lead. Elaboration of MBG scaffolds will produce sublibraries containing compounds that can be screened against metalloproteins to provide more advanced lead structures. These will be developed for the MMPs and CAs, as well as a variety of other metalloproteins of medicinal interest. PUBLIC HEALTH RELEVANCE: The inhibition of metalloproteins is important for the treatment of pathologies ranging from cardiac disease to cancer. This project seeks to discover novel compounds and new approaches to the identification of metalloprotein inhibitors. Improved understanding and optimization of the interactions between the inhibitor and the metal ions in the enzyme active site will be realized.