PRR antagonists and agonists – Screening for new immunmodulators

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-inflammatory 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 therapeutic approach for immunotherapy by using them as immunomodulators. 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.

TLR9 ODN complex.
© Photo Theoretical Biology and Medical Modelling 2013, 10:1. doi:10.1186/1742-4682-10-18
Structure of the human TLR9 when bound to the antagonist ODN (receptor-antagonist complex) [6].

Molecular simulation of screening for drug candidates

The search for TLR antagonists/agonists is conventionally carried 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 University 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 properties, statistical evaluation and research algorithms.

Reporter gene assay.
Schematic representation of the cell-based reporter gene assay.

Verifying the TLR-modulating effect in a reporter gene assay

The compound library predicted by the simulation of specific 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-kB. This, in turn, induces the expression of the reporter gene, e.g. a secreted alkaline phosphatase (SEAP) (Fig. 2) [4, 5].

There is direct and quantitative evidence of the effect of substances, antagonists as well as agonists, on the expression of the reporter gene. The PRR-specific cell-based assay is therefore a quick and flexible tool to identify lead compounds for drug development.

Cell-based reporter gene assay.
Cell-based reporter gene assay in cell-culture plates.

First hits identified

Initially, antagonists for TLR9, based on the algorithms of our cooperators  at the Hebrew University were identified. The initially predicted compound libraries for specific TLR9 binding molecules were synthesized and examined in the cell-based reporter assay established at Fraunhofer 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 referred to as IC50. These promising hits will be optimized by the experts at the Hebrew University, using the data gained by Fraunhofer IGB, and finally tested again at Fraunhofer IGB.


The molecules identified with the aid of the cell-based assay at Fraunhofer IGB represent potential drug candidates for the prevention and therapy of immunological diseases. The complementing expertise of the partners, the unique, patented procedure for molecular simulation by the Hebrew University and the cell-based TLR screening assay developed at Fraunhofer IGB, have provided significant added value regarding the chances of this challenging aim: to find new TLR-based immunomodulators for the therapy and prevention of various medical indications.


  1. Akira, S.; Takeda, K. (2004) Toll-like receptor signalling, Nat Rev Immunol 4: 499–511
  2. Akira, S.; Uematsu, S.; Takeuchi, O. (2006) Pathogen recognition and innate immunity, Cell 124: 783–801
  3. Vasilakos, J. P.; Tomai, M. A. (2013) The use of Toll-like receptor 7/8 agonists as vaccine adjuvants, Expert Rev. Vaccines 12: 809–19
  4. Burger-Kentischer, A.; Abele, I. S.; Finkelmeier, D.; Wiesmüller, K. H.; Rupp, S. (2010) A new cell-based innate immune receptor assay for the examination of receptor activity, ligand specificity, signalling pathways and the detection of pyrogens, J Immunol. Methods 358: 93–103
  5. Zellbasiertes Testsystem zur Identifizierung und Differenzierung von Keimspektren (2009) Patent DE 10 2006 031 483; EP 2 041 172
  6. Zhou, W. et al. (2013) Toll-like receptor 9 interaction with CpG ODN – an in silico analysis approach, Theor. Biol. Med. Model. 10: 18

Project information

Project title

Discovery and delivery of PRR antagonists and agonists to regulate innate immune reaction


Project duration

November 2012 – October 2016


Project partner

Hebrew University of Jerusalem, Institute for Drug Research (IDR), Jerusalem, Israel


Project coordination

Fraunhofer IGB


We would like to thank the Fraunhofer-Gesellschaft for funding the project "Discovery and delivery of PRR antagonists and agonists to regulate innate immune reaction" within the ICON program.