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

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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.