Joseph R. Woska

Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells

Abstract The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.

A small-molecule antagonist of LFA-1 blocks a conformational change important for LFA-1 function

Lymphocyte function‐associated antigen(LFA)‐1/intercellular adhesion molecule (ICAM)‐1interactions mediate several important steps in the evolution of animmune response. LFA‐1 is normally expressed in a quiescent state onthe surface of leukocytes and interacts weakly with its ligands ICAM‐1,‐2, and ‐3. LFA‐1 activity may be regulated by receptor clustering andby increasing the affinity of LFA‐1 for its ligands. Affinitymodulation of LFA‐1 has been shown to occur via a conformational changein the LFA‐1 heterodimer that can be detected by using monoclonalantibody 24 (mAb24). We have recently described a small‐moleculeantagonist of LFA‐1, BIRT 377, that demonstrates selective in vitro andin vivo inhibition of LFA‐1/ICAM‐1‐mediated binding events. We nowdemonstrate that BIRT 377 blocks the induction of the mAb24 reporterepitope on LFA‐1 on the surface of SKW‐3 cells treated with variousagonists known to induce high‐affinity LFA‐1. These data imply thatBIRT 377 exerts its inhibitory effects by preventing up‐regulation ofLFA‐1 to its high‐affinity conformation.

5-Aminomethylbenzimidazoles as potent ITK antagonists

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.

Discovery, SAR and X-ray structure of 1H-benzimidazole-5-carboxylic acid cyclohexyl-methyl-amides as inhibitors of inducible T-cell kinase (Itk)

A series of novel potent benzimidazole based inhibitors of interleukin-2 T-cell kinase (Itk) were prepared. In this report, we discuss the structure-activity relationship (SAR), selectivity, and cell-based activity for the series. We also discuss the SAR associated with an X-ray structure of one of the small-molecule inhibitors bound to ITK.

2-Aminobenzimidazoles as potent ITK antagonists: trans-stilbene-like moieties targeting the kinase specificity pocket.

Based on the information from molecular modeling and X-ray crystal structures, the kinase specificity pocket of ITK could be occupied upon extension of the right-hand-side of the 2-benzimidazole core of the inhibitors. 2-Aminobenzimidazoles with a trans-stilbene-like extension were designed and synthesized as novel ITK antagonists. Significant improvement on binding affinity and cellular activity were obtained through the trans-stilbene-like antagonists. Several compounds showed inhibitory activity in an IL-2 functional assay.

5-Aminomethyl-1H-benzimidazoles as orally active inhibitors of inducible T-cell kinase (Itk).

A series of novel 5-aminomethyl-1H-benzimidazole based inhibitors of Itk were prepared. Structure-activity relationships, selectivity and cell activity are reported for this series. Compound 2, a potent and selective antagonist of Itk, inhibited anti-CD3 antibody induced IL-2 production in vivo in mice.

Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor

Background: Signaling by the IL-36 receptor is poorly characterized. Results: Activation of IL-36R signaling is coupled with its endocytosis to lysosomes. Tollip mediates IL-1 receptor turnover and increases the accumulation of IL-36R. Conclusion: IL-36R signaling has differences in signaling from the IL-1R. Significance: This work defines the requirements for IL-36R signaling and trafficking. Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.

Overview of Approaches to the Identification of Inhibitors of Cytokine Action

Cytokines are key regulatory molecules that serve as critical mediators of cell‐to‐cell communication. They play a central role in immune function and are important molecular targets for drug discovery because of their dysregulation in immune disease. Initial attempts to develop agents that block the actions of cytokines focused on identifying small‐molecule antagonists that would directly compete with these cytokines for their cognate receptors. These efforts were, for the most part, unsuccessful. Outlined in this unit are strategies for developing new approaches. The first section describes strategies for how one might build a conceptual framework to approach this problem. The second section focuses on the technical approaches that can be utilized to accomplish the strategies outlined in the first section. These concepts are illustrated using interleukin‐2 (IL‐2) as a prototype, since there is a substantial body of knowledge regarding this cytokine, including information on its functions, signaling pathways, and physiological effects in normal and diseased tissue.