PRR antagonists and agonists – Screening for new immunmodulators

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.

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.

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.

Outlook

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

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.