Macrocyclization enhances affinity of chemokine-binding peptoids.

Peptoids that bind to protein targets can be selected from one-bead-one-compound libraries. Macrocyclization has been often used to increase conformational rigidity and binding affinity in both peptides and peptoids. Here we describe a combined strategy to label and cyclize hexameric peptoid sequences previously identified in a screen against the inflammatory chemokine interleukin-8/CXCL8 that is involved in a number of inflammatory diseases. Cyclization can be performed on-bead in the presence of side-chain protecting groups so that this strategy can be applied to a large variety of sequences. The affinity of the resulting tetramethylrhodamine-labeled macrocyclic peptomers to CXCL8 is increased by at least 1 order of magnitude compared to the original linear sequences.

Cell-based actin polymerization assay to analyze chemokine inhibitors.

Chemokines play an important role in various diseases as signaling molecules for immune cells. Therefore, the inhibition of the chemokine-receptor interaction and the characterization of potential inhibitors are important steps in the development of new therapies. Here, we present a new cell-based assay for chemokine-receptor interaction, using chemokine-dependent actin polymerization as a readout. We used interleukin-8 (IL-8, CXCL8) as a model chemokine and measured the IL-8-dependent actin polymerization with Atto565-phalloidin by monitoring the fluorescence intensity in the cell layer after activation with IL-8. This assay needs no transfection, is easy to perform and requires only a few working steps. It can be used to confirm receptor activation and to characterize the effect of chemokine receptor antagonists. Experiments with the well-known CXCR1/2 inhibitor reparixin confirmed that the observed increase in fluorescence intensity is a result of chemokine receptor activation and can be inhibited in a dose-dependent manner. With optimized parameters, the difference between positive and negative control was highly significant and statistical Z´-factors of 0.4 were determined on average.

Immobilisation of CXCL8 gradients in microfluidic devices for migration experiments.

The release of inflammatory chemokines leads to the formation of chemokine gradients that result in the directed migration of immune cells to the site of injury. In this process, cells respond to soluble gradients (chemotaxis) as well as to immobilised gradients (haptotaxis). Surface-bound chemokine gradients are mostly presented by endothelial cells and supported by glycosaminoglycans (GAGs), such as heparan sulfate, involving the GAG binding site of chemokines. Microfluidic devices have been used to analyse cell migration along soluble chemokine gradients, as these devices allow the generation of stable gradients with resolutions in the range of microns. To immobilise well-controlled soluble gradients of interleukin-8 (CXCL8), an inflammatory chemokine, we developed a simple procedure using a heparin-coated PDMS-microfluidic device. We used these immobilised gradients for migration experiments with CXCL8-responsive THP-1 cells and confirmed directed cell migration. This setup might be useful for the examination of factors that may alter chemotaxis and haptotaxis as well as synergistic and antagonistic effects of other soluble and immobilised chemokines.

Virtual Screening Identifies Irreversible FMS-like Tyrosine Kinase 3 Inhibitors with Activity toward Resistance-Conferring Mutations.

The use of covalent irreversible binding inhibitors is an established concept for drug development. Usually, the discovery of new irreversible kinase inhibitors occurs serendipitously, showing that efficient rational approaches for the rapid discovery of new drugs are needed. Herein, we report a virtual screening strategy that led to the discovery of irreversible inhibitors of FMS-like tyrosine kinase 3 (FLT3) involved in the pathogenesis of acute myeloid leukemia. A virtual screening library was designed to target the highly conserved Cys828 residue preceding the DFG motif by modification of reported reversible inhibitors with chemically reactive groups. Prospective covalent docking allowed the identification of two lead series, resulting in a massive increase in inhibition of kinase activity and cell viability by irreversible inhibitors compared to the corresponding reversible scaffolds. Lead compound 4b (BSc5371) displays superior cytotoxicity in FLT3-dependent cell lines to compounds in recent clinical trials and overcomes drug-resistant mutations.