New Strategies and Screening Methods for Metalloproteinase Inhibition

描述（由申请人提供）：本提案的主要目的是：a）制备多种锌（II）金属蛋白的双齿磺酰胺抑制剂，和B）开发一种基于细胞的筛选方法，用于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（MMP）在炎症性疾病、中风、关节炎、癌症和心脏病的进展中起核心作用。所有MMP都利用锌（II）离子来进行结缔组织和其他生物基质的催化水解。该项目的总体目标是开发利用锌（II）特异性磺酰胺螯合剂作为新型锌结合基团（ZBG）的改良基质金属蛋白酶抑制剂（MMPi）。螯合磺酰胺将增强与锌（II）金属蛋白活性位点的结合，同时保留其他金属酶（例如铁依赖性酶）。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MMP的选择性。本提案的目标将通过以下具体目标实现：1.简单的螯合磺酰胺ZBG的合成和表征。结合生物无机模型复合物，再现MMP活性位点，磺胺ZBG将提供这些配体的锌（II）离子的结合模式和亲和力的基本信息。2.新型MMPi的设计与合成。使用我们实验室的组合生物无机计算方法，将基于新的磺酰胺ZBG设计有效和选择性的MMPi。3.使用荧光和比色测定法评估新型MMPi。将针对多种重组MMP评价新MMP i。4.在炎症的巨噬细胞模型中针对几种金属酶筛选MMPi。将在一种新的基于巨噬细胞的测定中检查MMP i，该测定将确定MMP抑制剂相对于其他锌（II）依赖性金属酶、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，我们将鉴定出比当前MMPi具有更大亲和力、选择性和生物相容性的新MMPi，从而鉴定出将在临床上成功的抑制剂。该项目的成功将产生至少两个极具创新性和影响力的结果：a）首次使用锌（II）-选择性螯合磺酰胺，其经常在锌（II）离子分子传感器中发现，将用于锌（II）-依赖性金属蛋白抑制，和B）开发用于金属蛋白抑制剂的基于细胞的筛选方法，该方法评估化合物对一组广泛的金属-在生物环境中依赖酶。开发能够选择性抑制金属依赖酶的药物对于治疗中风、关节炎、癌症和心脏病等疾病至关重要。本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：开发能够选择性抑制金属依赖酶的药物对于治疗中风、关节炎、癌症和心脏病等疾病至关重要。本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.