Discovery of small molecules inhibiting Toll-like receptor-mediated inflammation

摘要 Toll样受体（TLR）家族的成员识别各种病原体和宿主组织来源的 分子，并通过炎症过程生理性地启动免疫应答。夸大或 然而，延长的TLR激活导致病理性炎症，如在大量的 病因多样的疾病，如细菌性败血症、自身免疫性疾病和癌症。尽管 显然需要，没有有效的经典TLR信号传导途径的小分子抑制剂可用， 既不是探索TLR生物学的探针，也不是治疗患者的药物。本项目的目标是确定 TLR特异性信号通路的小分子抑制剂。阻碍药物开发的一个因素是 涉及的信号传导机制，即一系列蛋白质相互作用，这些相互作用对于靶向药物来说是固有困难的 发展具体地，TLR通过分级作用蛋白TIRAP、MyD 88、IRAK和TlR信号传导。 TRAF 6，后者定义了共同的TLR非特异性途径的边界。因此，信号事件- TRAF 6的流代表期望的“药物靶窗口”。我们已经开发出一种独特的，细胞高 高通量筛选（HTS）平台，其保留了表型筛选的优点，即， 复杂的反应，但同时，减轻了主要的缺点， 通过消除非特异性化合物。该系统的关键特征是药物诱导（GyrB融合）形式的 TIRAP、MyD 88和TRAF 6，其反应反映了化合物活性的“信号传导水平”。我们有 使用St. Jude生物活性化合物库验证了这种方法，该方法允许连续筛选 步骤容易消除非特异性命中（>99%的命中阻断TRAF 6），并鉴定一种化合物 作为选择性TLR抑制剂。以该化合物为工具分子，建立了命中推进算法 包括SAR分析、定量蛋白质组学、蛋白质相互作用和细胞热位移测定（CETSA）， 共同验证该方法作为TLR拮抗剂的发现工具。在这里，我们建议执行HTS 圣裘德收集（约650，000化合物），以确定一组TLR抑制化合物， 化学型基于TIRAP-GyrB的初步筛选（单一浓度）鉴定了在以下条件下起作用的化合物： TLR通路的任何可能水平。基于TIRAP-，MyD 88-， 而TRAF 6-GyrB细胞定义了信号传导水平。抑制TRAF 6的化合物将作为TLR-非 特定. TIRAP或MyD 88的抑制将定义TLR特异性化合物。TLR特异性化合物将被 通过生理TLR刺激进行验证，并基于化学信息学分析进行优先排序， 化学检验将进行SAR分析，以验证化学型的可处理性， 可用的类似物和探索性化学，以及提到的生物化学算法将用于定义 作用机制和药物靶点。总之，我们希望通过定义， 化学上易于处理的TLR抑制活性作为成功的起点，未来的药物开发。

ABSTRACT Members of the Toll-like receptor (TLR) family recognize various pathogen- and host tissue-derived molecules, and initiate immune responses physiologically via inflammatory processes. Exaggerated or prolonged TLR activation, however, leads to pathological inflammation as observed in a large number of etiologically diverse diseases, such as bacterial sepsis, autoimmune diseases and cancer. Despite the apparent need, no effective small molecule inhibitors of the canonical TLR signaling pathway are available, neither as probes to explore TLR biology nor as drugs to treat patients. The goal of this project is to identify small molecule inhibitors of TLR-specific signaling pathways. One factor impeding drug development is the signaling mechanism involved, i.e. a series of protein interactions that are inherently difficult for targeted drug development. Specifically, TLRs signal via the hierarchically acting proteins TIRAP, MyD88, IRAKs and TRAF6, the latter defining the border towards common, TLR-non-specific pathways. Thus, signaling events up- stream of TRAF6 represent the desirable ‘drug target window’. We have developed a unique, cellular high throughput screening (HTS) platform that retains the advantages of phenotypic screening, i.e. the testing of complex responses in the natural environment of cells but, at the same time, mitigates the major disadvantage by eliminating non-specific compounds. Key feature of this system are drug-inducible (GyrB-fused) forms of TIRAP, MyD88 and TRAF6, whose responses reflect the ‘signaling level’ of compound activity. We have validated this approach using the St. Jude bioactive compound library, which allowed in consecutive screening steps facile elimination of non-specific hits (>99% of hits blocking TRAF6), and identification of one compound as selective TLR inhibitor. Using this compound as tool molecule, we established a hit advancement algorithm including SAR analysis, quantitative proteomics, protein interaction- and cellular thermal shift assays (CETSA), collectively validating this approach as discovery tool for TLR antagonists. Here we propose to perform a HTS of the St. Jude collection (~650,000 compounds) to identify a set of TLR inhibitory compounds with defined chemotype. The primary screen (single concentration) based on TIRAP-GyrB identifies compounds that act at any possible level of the TLR pathway. The secondary screen (dose-response) based on TIRAP-, MyD88- and TRAF6-GyrB cells defines the signaling level. Compounds inhibiting TRAF6 will be discarded as TLR-non- specific. Inhibition of TIRAP or MyD88 will define TLR-specific compounds. TLR-specific compounds will be validated by physiological TLR stimulation and prioritized based on chemoinformatics analysis and chemistry inspection. SAR analysis will be conducted to validate chemotype tractability based on available analogs and exploratory chemistry, and mentioned biochemical algorithm will be used to define mechanism of action and drug target. In summary, we expect to identify diverse chemotypes with defined, chemically tractable TLR-inhibitory activity as starting point for successful, future drug development.