Targeting the first barrier of immune response: Rational design of small molecule Toll-like receptor modulators

Toll样受体（TLR）形成先天免疫应答中的第一屏障并触发炎症应答，其对于疾病如败血症、1型糖尿病或癌症的出现和进展至关重要。尽管TLR的重要性已被认识到，但迄今为止仅开发了很少的小分子调节剂，这主要是由于缺乏关于结合位点的合理信息。在我们的初步工作中，我们设法建立了一种虚拟筛选方法，该方法允许鉴定新型小分子TLR配体并表征其与受体的结合。通过最近获得的晶体结构，提供TLR二聚化的见解，合理和有针对性的设计新的小分子TLR调节剂变得可行。这些配体具有平衡炎症反应的潜力，因此成为对抗上述疾病的有价值的治疗剂。我们的研究目标是通过分子建模，合成和化学优化，了解其作用模式和开发新的虚拟筛选策略来识别小分子TLR调节剂。应对已鉴定的化合物进行生物学试验，并对其结合模式进行机械研究。首先，最近可用的TLR8和TLR2的晶体结构应通过分子相互作用场的计算、对接和分子动力学模拟在已知小分子调节剂的背景下表征，这导致第一组合理的结合模型。所得的静态和动态3D药效团模型代表激动或拮抗活性和受体特异性所必需的关键特征，并用于针对可用的商业化合物文库（约100 μ g/ml）进行TLR调节剂的虚拟筛选。600万种化合物），然后对最有希望的虚拟命中进行生物测试。应在各种特定的基于细胞的测定中测试选定的虚拟命中物调节TLR8和TLR2的促炎反应的能力，同时使用荧光各向异性滴定研究特异性受体结合。关于化合物活性和机制的信息被整合到预测模型中，随后用于实现进一步的筛选和合成回合，从而产生一组化学优化的TLR调节剂。新开发的抑制剂潜在地将代表用于治疗上述疾病的新的先导结构，并提供对Toll样受体功能的新见解。

Toll-like receptors (TLRs) form the first barrier in innate immune response and trigger inflammatory responses, which are central to the emergence and progression of diseases such as sepsis, type 1 diabetes or cancer. Although the importance of TLRs is recognized, only few small molecule modulators have been developed so far, mainly due to the lack of plausible information about binding sites. In our preliminary work we managed to establish a virtual screening method that allows identifying novel classes of small molecule TLR ligands and characterizing their binding to the receptor. Through the recent availability of crystal structures that provide insights into TLR dimerization, the rational and targeted design of novel small molecule TLR modulators becomes feasible. Such ligands bear the potential to balance inflammatory responses and thus become valuable therapeutics to combat the above-mentioned diseases. Our research aim is to identify small molecule TLR modulators by molecular modeling, their synthesis and chemical optimization, the understanding of their mode of action and the development of new virtual screening strategies. Identified compounds shall be biologically tested and their binding mode mechanistically investigated. At first, recently available crystal structures of TLR8 and TLR2 shall be characterized in the context of known small molecule modulators through calculation of molecular interaction fields, docking and molecular dynamics simulations, which leads to a first set of plausible binding models. Resulting static and dynamic 3D pharmacophore models represent key features necessary for agonistic or antagonistic activity and receptor specificity and are employed to perform virtual screening for TLR modulators against available commercial compound libraries (approx. 6 million compounds), followed by biological testing of most promising virtual hits. Selected virtual hits shall be tested for their ability to modulate the pro-inflammatory response of TLR8 and TLR2 in a variety of specific cell-based assays, while specific receptor binding is investigated using fluorescence anisotropy titration. Information on compound activity and mechanism is integrated into predictive models, which are subsequently used to realize a further screening and synthesis round leading to a set of chemically optimized TLR modulators. The newly developed inhibitors potentially will represent new lead structures for the treatment of the above- mentioned diseases and provide new insights into the function of Toll-like receptors.