New Strategies and Screening Methods for Metalloproteinase Inhibition

描述(由申请人提供)：这项建议的主要目标是：a)制备多种锌(II)金属蛋白的双齿磺胺类抑制剂，以及b)发展一种基于细胞的筛选方法，以同时检测这些抑制剂对几种金属酶的影响。基质金属蛋白酶(MMPs)在炎症性疾病、中风、关节炎、癌症和心脏病的进展中起着核心作用。所有MMPs都利用锌(II)离子进行结缔组织和其他生物底物的催化水解。这个项目的总体目标是开发改进的基质金属蛋白酶抑制剂(MMPI)，它利用锌(II)特异性的磺胺螯合剂作为新的锌结合基团(ZBGs)。螯合磺胺类化合物将增强与锌(II)金属蛋白活性部位的结合，同时保留其他金属酶(例如铁依赖的酶)。为了验证这一假设，将使用一种新的基于巨噬细胞的筛选方法来评估这些抑制剂对MMPs的选择性。这项建议的目标将通过以下具体目标来实现：1.简单、螯合磺胺ZBGs的合成和表征。与再现基质金属蛋白酶活性部位的生物无机模型络合物相结合，磺胺ZBGs将提供有关这些配体与锌(II)离子结合模式和亲和力的重要信息。2.新型MMPI的设计与合成。利用我们实验室的生物无机-计算相结合的方法，将基于新的磺胺ZBGs设计出有效的和选择性的MMPI。3.荧光和比色法对新型MMPI的评价。新的MMPI将与各种重组MMPs进行比较。4.在巨噬细胞炎症模型中筛选抗几种金属酶的MMPI。MMPI将在一种新的基于巨噬细胞的测试中进行检测，该测试将确定MMPs抑制剂相对于其他依赖锌(II)的金属酶、血红素-铁金属酶和非血红素-铁金属酶的选择性。通过使用这种方法并实现这些目标，我们将发现新的MMPI比现有的MMPI具有更高的亲和力、选择性和生物相容性，从而确定将在临床上成功的抑制剂。该项目的成功将至少产生两项极具创新性和高影响力的发现：a)首次将锌(II)离子分子传感器中经常发现的选择性锌(II)螯合磺胺用于锌(II)依赖的金属蛋白抑制，以及b)开发一种基于细胞的金属蛋白抑制剂筛选方法，该方法在生物环境中评估一种化合物对广泛的金属依赖酶的效果。能够选择性抑制金属依赖酶的药物的开发对中风、关节炎、癌症和心脏病等疾病的治疗至关重要。该项目旨在发现选择性金属蛋白抑制剂，并开发一种简便的方法，通过该方法可以快速评估这些抑制剂的选择性。公共卫生相关性：能够选择性抑制金属依赖酶的药物的开发对中风、关节炎、癌症和心脏病等疾病的治疗至关重要。该项目旨在发现选择性金属蛋白抑制剂，并开发一种简便的方法，通过该方法可以快速评估这些抑制剂的选择性。

DESCRIPTION (provided by applicant): The major objectives of this proposal are: a) to prepare a variety of bidentate sulfonamide inhibitors of zinc(II) metalloproteins, and b) develop a cell-based screening method for examining the effect of these inhibitors on several metalloenzymes simultaneously. Matrix metalloproteinases (MMPs) play a central role in the progression of inflammatory disease, stroke, arthritis, cancer, and heart disease. All MMPs utilize a zinc(II) ion to perform the catalytic hydrolysis of connective tissue and other biological substrates. The overall aim of this project is to develop improved matrix metalloproteinase inhibitors (MMPi) that utilize zinc(II)-specific sulfonamide chelators as novel zinc-binding groups (ZBGs). Chelating sulfonamides will enhance binding to the zinc(II) metalloprotein active site, while sparing other metalloenzymes (e.g. iron- dependent enzymes). To validate this hypothesis, the selectivity of these inhibitors for MMPs will be assessed using a novel macrophage-based screening methodology. The goals of this proposal will be achieved via the following specific aims: 1. The synthesis and characterization of simple, chelating sulfonamide ZBGs. Combined with bioinorganic model complexes that reproduce the MMP active site, sulfonamide ZBGs will provide essential information on the binding mode and affinity of these ligands for the zinc(II) ion. 2. The design and synthesis of novel MMPi. Using a combined bioinorganic-computational approach from our laboratory, potent and selective MMPi will be devised based on new sulfonamide ZBGs. 3. The evaluation of novel MMPi using fluorescence and colorimetric assays. New MMPi will be evaluated against a variety of recombinant MMPs. 4. The screening of MMPi against several metalloenzymes in a macrophage cell model of inflammation. MMPi will be examined in a novel macrophage cell-based assay that will determine the selectivity of the inhibitors for MMPs versus other zinc(II)-dependent metalloenzymes, heme- iron metalloenzymes, and non-heme-iron metalloenzymes. By using this approach and achieving these aims, we will identify new MMPi with greater affinity, selectivity, and biocompatibility than current MMPi, and thereby identify inhibitors that will be clinically successful. The success of this project will generate at least two highly innovative and high-impact findings: a) the first time that the use of zinc(II)-selective chelating sulfonamides, which are frequently found in zinc(II) ion molecular sensors, will be utilized for zinc(II)-dependent metalloprotein inhibition, and b) the development of a cell-based screening method for metalloprotein inhibitors that evaluates the effect of a compound against a broad set of metal-dependent enzymes in a biological setting. The development of drugs that can selectively inhibit metal-dependent enzymes is critical to the treatment of illnesses such as stroke, arthritis, cancer, and heart disease. This project seeks to discover selective metalloprotein inhibitors and develop a facile method by which the selectivity of these inhibitors can be rapidly assessed. PUBLIC HEALTH RELEVANCE: The development of drugs that can selectively inhibit metal-dependent enzymes is critical to the treatment of illnesses such as stroke, arthritis, cancer, and heart disease. This project seeks to discover selective metalloprotein inhibitors and develop a facile method by which the selectivity of these inhibitors can be rapidly assessed.