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2013|14 Annual Report Fraunhofer IGB

86 Initial situation The receptors of the innate immune system recognize not only conserved molecular patterns of infectious pathogens, but also isolated chemical structures (PAMPs, pathogen-associated molecular patterns), and are referred to as pattern recognition receptors (PRRs) [1]. Among the PRRs, the toll-like receptors represent the largest and most well-known family. Stimulation of the TLRs leads, via the activation of various signal cascades and transcription factors, to the production of pro-inflam- matory cytokines and thereby plays a significant role in the development of pathological processes in acute and chronic diseases in humans [2]. Agonists and antagonists of TLRs are therefore a new thera- peutic approach for immunotherapy by using them as immu- nomodulators. Agonists stimulate the innate immune system and are frequently used as adjuvant drugs, while antagonists inhibit inflammatory processes [3]. The possible spectrum of indications ranges from allergies, infections and tumors, up to autoimmune diseases. It is the aim of an international project at the IGB to seek out new TLR antagonists/agonists, in order to be able to treat inflammatory reactions and allergies. Molecular simulation of screening for drug candidates The search for TLR antagonists/agonists is conventionally car- ried out using high-throughput screening (HTS). However, this procedure is very time-consuming and expensive. Our project partner, the Institute for Drug Research at the Hebrew Univer- sity of Jerusalem, is able to use computer-assisted simulations to vastly reduce the time and cost of the screening process. The process is greatly simplified through 3D binding models for the relevant receptors. The software used contains a broad range of functions, such as the integration of molecular prop- erties, statistical evaluation and research algorithms. Verifying the TLR-modulating effect in a reporter gene assay The compound library predicted by the simulation of spe- cific TLR agonists/antagonists is synthesized and validated in a cell-based test system established and patented at the Fraunhofer IGB [4, 5]. This test system enables the detection and differentiation of PRR-modulating substances via a simple reporter gene assay and can also be carried out in accordance with GLP (Good Laboratory Practice). In establishing the assay, the relevant human PRR receptor was stably introduced into the NIH3T3 fibroblast cell line, which naturally expresses very low endogenous levels of TLRs. Additionally, a reporter gene, which is induced by PRR activity, was stably integrated into these cells. The induction of the receptor by a specific ligand leads to the activation of the transcription factor NF-κB. This, in turn, induces the expression of the reporter gene, e.g. a se- creted alkaline phosphatase (SEAP) (Fig. 2) [4, 5]. There is direct and quantitative evidence of the effect of sub- stances, antagonists as well as agonists, on the expression of the reporter gene. The PRR-specific cell-based assay is there- fore a quick and flexible tool to identify lead compounds for drug development. First hits identified Initially, antagonists for TLR9, based on the algorithms of our cooperators The Hebrew University were identified. The ini- tially predicted compound libraries for specific TLR9 binding molecules were synthesized and examined in the cell-based reporter assay established at the IGB (Figs. 3 and 4). In doing this we discovered, among other things, the relevant average inhibitory concentration (IC50) of an antagonistic substance. The level at which half the maximum inhibition is seen is re- ferred to as IC50. These promising hits will be optimized by PHARMACY SCREENING FOR NEW IMMUNMODULATORS Angela Mattes B.Sc., Dr. rer. nat. Anke Burger-Kentischer 21 CpG ODN potential antagonist TLR9 SEAP nucleus Endosome TNF-α MIF NO IFN-β IL1.6, 10, 12 phosphatase activity expression MyD 88 IRAK TRAF6 IKK NF-κB NF-κB activation

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