Research Group. Towards small-molecule ligands to modulate leukocyte behavior

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1 Research Group Receptor-Ligand-Interactions Towards small-molecule ligands to modulate leukocyte behavior Dr. Katja Schmitz,

2 Chemokines and chemokine receptors Chemotactic cytokines Small, soluble proteins ( aa) about 50 human chemokines known 20 different receptors CCL2 Chemokine (MCP-1) CXCL8 Chemokine (IL-8)

3 Chemokines control leukocyte migration Secretion Glycosaminoglycan binding Gradient formation Recognition by chemokine receptors on leukocytes Leukocyte migration along the gradient Allergies, asthma, chronic inflammation Migration of metastases

4 Blocking chemokine-receptor interactions Receptor-based Chemokine-based

5 Design, test and synthesis of ligands Ligand synthesis Molecular modeling Protein expression Binding assays Cell isolation Bioassays

6 Sources of lead structures

7 Small-molecule microarrays 10,000 immobilized small molecules Fluorescence-based binding assay Potential ligands

8 Simulation of interleukin-8 receptor peptides En nergy Differen nce WT TRP2 PHE4 ASP6 MET8 PRO10 ASP12 ASP14 SER16 Protein structure from PDB Computational Alanine Scan Irene Meliciani Meliciani i i et al. (2009), J. Chem. Phys., in press

9 Simulation of interleukin-8 receptor peptides MWDFDDGMPPADEDYSP MWDFDDGMPFADEDYSP MWDFDDGMPPDDEDYSP Computational full peptide exchange scan Peptide synthesis for experimental verification Irene Meliciani

10 Peptides and peptide mimetics that bind the IL-8 8Nt N-terminus IL-8 N-terminal ELR motif is essential for receptor activation. Combinatorial library Resin N-terminal 10 amino acids labelled with fluoresceine Fluo SAKELRCQCI -D-A-R- G-E-L-K-A -S-Fincubate and screen Elena Heidenreich Colaboration with Ute Schepers and Esther Birthalan

11 Validation of lead structures

12 Expression of recombinant chemokines Cloning of optimized cdna into an expression vector Tag-free expression or expression with bacterial export signal controls 4h-ON-4h-4h-ON-ON (-) (+) (-) IL kda SDS-PAGE for expression analysis Dana Wiese

13 mau Purification of recombinant chemokines Affinity chromatography on Hi-Trap heparin resin Analysis of fractions by SDS-PAGE, Western blot and MALDI Further purification on CIEX cation exchange chromatography kda M A2 A3 A4 A5 X1 A2 A3 A4 A5 L27 A2 L27 A3 L27 A4 L26/27L28 L28 A5 X1 A2 L28 A3 L28 A4 L28 A min X1 Waste A1 A2 A3 A4 A5 Waste Dana Wiese

14 Fluorescence Polarization Polarization of fluorescent light depends on molecular motility Free tracer = high flexibility = low polarization Protein-bound tracer = low flexibility = high polarization Fluorescence Polarization reflects ratio of bound/unbound ligand

15 SAR studies by Fluorescence Polarization If functional groups essential for binding are labelled, binding is prevented All derivatives need to be tested and synthesized

16 FP-Competition assay for lead validation N-terminal binding peptide with fluorescent label as tracer Suitable for comparison of affinities of unlabeled ligands K d of tracer needs to be known Only one binding mode is considered d Colaboration with Bianca Stolzenberger

17 Establishing a Cell Migration Assay Transwell membrane system Chemotaxis medium Neutrophile granulocytes Polycarbonate membran with 3 µm pores Chemotaxis medium with chemokines Nicole Niemeier KIT die Kooperation von Forschungszentrum Karlsruhe GmbH und Universität Karlsruhe (TH)

18 Cell migration evaluated with CASY 1 Cell Counter 1. Quantification of granulocytes migrating along a chemokine gradient 2. Evaluation of the best chemokine concentration for migration 3. Test system of inhibitory peptides and positives from microarrays 100 Differentiated HL-60 cells 80 = neutrophile granulocytes 60 1 million cells seeded Incubation time 1h 20 migrated ce ells [%] 40 Cell migration assay with Il-8 0 Nicole Niemeier KIT die Kooperation von Forschungszentrum Karlsruhe GmbH und Universität Karlsruhe (TH)

19 Acknowledgement Nicole Niemeieri Dana Wiese Irene Meliciani Dorothea Helmer Ebru Diler Elena Heidenreich Katharina Güse Alisha Oster Ilona Schmelcher h IFG: Sonja Berensmeier, Matthias Franzreb, Thomas Schwartz IOC: Stefan Bräse ITG: Ute Schepers INT: Wolfgang g Wenzel IBL: Bianca Stolzenfelder, Jürgen Hubbuch Concept for the future of KIT (German Excellence Initiative)