Exploration of cathepsin S inhibitors characterized by a triazole P1-P2 amide replacement.

This paper details exploration of a class of triazole-based cathepsin S inhibitors originally reported by Ellman and co-workers. SAR studies involving modifications across the whole inhibitor provide a perspective on the strengths and weaknesses of this class of inhibitors. In addition, we put the unique characteristics of this class of compounds into perspective with other classes of cathepsin S inhibitors.

Fully human antibodies against the Protease-Activated Receptor-2 (PAR-2) with anti-inflammatory activity.

PAR-2 belongs to a family of G-protein coupled Protease-Activated Receptors (PAR) which are activated by specific proteolytic cleavage in the extracellular N-terminal region. PAR-2 is activated by proteases such as trypsin, tryptase, proteinase 3, factor VIIa, factor Xa and is thought to be a mediator of inflammation and tissue injury, where elevated levels of proteases are found. Utilizing the HuCAL GOLD® phage display library we generated fully human antibodies specifically blocking the protease cleavage site in the N-terminal domain. In vitro affinity optimization resulted in antibodies with up to 1000-fold improved affinities relative to the original parental antibodies with dissociation constants as low as 100 pM. Corresponding increases in potency were observed in a mechanistic protease cleavage assay. The antibodies effectively inhibited PAR-2 mediated intracellular calcium release and cytokine secretion in various cell types stimulated with trypsin. In addition, the antibodies demonstrated potent inhibition of trypsin induced relaxation of isolated rat aortic rings ex vivo. In a short term mouse model of inflammation, the trans vivo DTH model, anti-PAR-2 antibodies showed inhibition of the inflammatory swelling response. In summary, potent inhibitors of PAR-2 were generated which allow further assessment of the role of this receptor in inflammation and evaluation of their potential as therapeutic agents.

Inhibition of pro-inflammatory cytokine production by the dual p38/JNK2 inhibitor BIRB796 correlates with the inhibition of p38 signaling.

The characterization of the potent p38 inhibitor BIRB796 as a dual inhibitor of p38/Jun N-terminal kinases (JNK) mitogen-activated protein kinases (EC 2.7.11.24) has complicated the interpretation of its reported anti-inflammatory activity. To better understand the contribution of JNK2 inhibition to the anti-inflammatory activities of BIRB796, we explored the relationship between the effects of BIRB796 and analogues on cytokine production and on cellular p38 and JNK signaling. We determined the binding affinity for BIRB796 and structural analogues to p38alpha and JNK2 and characterized compound 2 as a p38 inhibitor that binds to p38alpha with an affinity equivalent to BIRB796 but does not bind to any of the JNK isoforms. High-content imaging enabled us to show that the inhibition of p38 signaling by BIRB796 and analogues correlates with the ability of these compounds to inhibit the lipopolysaccharide (LPS)-induced TNF-alpha production in THP-1 monocytes. This finding was extended to cytokine release by disease-relevant human primary cells: to the production of TNF-alpha by peripheral blood mononuclear cells, and of IL-8 by neutrophils. Furthermore, BIRB796 and compound 2 inhibited the production of TNF-alpha in THP-1 monocytes and the IL-12/IL-18-induced production of interferon-gamma in human T-cells with similar potencies. In contrast, cellular JNK signaling in response to cytokines or stress stimuli was only weakly inhibited by BIRB796 and analogues and not affected by compound 2. In summary, our data suggest that p38 inhibition alone is sufficient to completely suppress cytokine production and that the added inhibition of JNK2 does not significantly contribute to the effects of BIRB796 on cytokine production.