Identification of chemical compounds to inhibit the caseinolytic protease ClpXP complex and evaluate their biological activity

在寻找针对多重耐药病原体的新药时，我们采用了一种与经典抗生素相反的策略，该策略不会杀死细菌，但会解除其致病性（毒力）。抗毒力方法的优点是缺乏选择压力，导致减少的耐药性发展和更持久的药物。细菌毒力的重要调节剂是酪蛋白溶解蛋白酶ClpP和相关的伴侣蛋白ClpX。虽然我们能够特异性抑制ClpP和相应的毒力在S。金黄色葡萄球菌与β-内酯，其有限的稳定性代表了药理学应用的主要挑战。本研究计划旨在鉴定新型ClpP和ClpX毒力抑制剂，其具有适合体内应用的药理学特性。在最初的实验中，我们已经通过高通量筛选（HTS）鉴定了可逆的ClpP抑制剂。将通过突变研究详细研究它们与ClpP的结合模式。这些实验的结果将用于合成优化的衍生物，其将用于测试改进的酶抑制和抗毒力特性。由于ClpX代表了抗毒力方法的另一个重要靶标，我们将在柏林的FMP进行HTS，以鉴定第一个ClpP/ClpX特异性抑制剂。所有筛选命中将再次优化，并且最有希望的化合物进一步用炔柄以及氮丙啶光连接剂修饰，以分析其靶选择性。最后，将检测这些分子的药理学特性，并在小鼠脓肿模型中检测适当参数的有效性。

In the search for new drugs against multiresistant pathogens we utilize a strategy which contrary to classical antibiotics does not kill bacteria but disarms their pathogenicity (virulence). The advantage of the anti-virulence approach is a lack of selective pressure leading to reduced resistance development and longer lasting drugs.Important regulators of bacterial virulence are the caseinolytic protease ClpP and is associated chaperone ClpX. While we were able to specifically inhibit ClpP and correspondingly virulence in S. aureus with beta-Lactones, their limited stability represents a major challenge for pharmacological application. This research proposal aims to identify novel ClpP and ClpX based virulence inhibitors with suitable pharmacological properties for in vivo application. In initial experiments we already identified reversible ClpP inhibitors via a high-throughput-screen (HTS). Their binding mode into ClpP will be investigated in detail by mutational studies. The outcome of these experiments will be utilized for the synthesis of optimized derivatives which will be tested for improved enzyme inhibition and anti-virulence properties. As ClpX represents an additional important target for an anti-virulence approach we will perform an HTS at the FMP in Berlin to identify the first ClpP/ClpX specific inhibitors. All screening hits will be again optimized and the most promising compounds further modified with an alkyne handle as well as with an aziridine photolinker in order to analyze their target selectivity. Finally, these molecules will be tested for their pharmacological properties and in case of suitable parameters tested for efficacy in a mouse abscess model.