CCR1 plays a critical role in modulating pain through hematopoietic and non-hematopoietic cells.

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1-/- animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freund's adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1-/- mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.

Circulating monocytes are reduced by sphingosine-1-phosphate receptor modulators independently of S1P3.

Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and TNF-α production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720 correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes and offer additional insights on the mechanism of action of S1P receptor modulators in disease.

Pathogenic mechanisms and experimental models of multiple sclerosis.

Multiple sclerosis (MS) is a devastating autoimmune disease that affects more than 1 million people worldwide and severely compromises motor and sensory function through demyelination and axonal loss. This review covers current therapies, lessons learned from failed clinical trials, genetic susceptibility, key cell types involved, animal models, gene expression, and biomarker information. The current first-line therapies for MS include the type I interferons (IFN-I) and glatiramer acetate (GA) but because of their limited effectiveness new therapeutic modalities are required. Tysabri is an anti very late antigen-4 antibody that antagonizes the migration of multiple cell types and appears more efficacious as compared to the IFNs or GA. Tysabri blocks the transmigration of T cells and monocytes, which indicates that blocking multiple cell types may increase the effectiveness of the therapy. However, this therapy may increase the risk of progressive multifocal leukoencephalopathy. The major cell types hypothesized to be pathogenic include T cells and antigen-presenting cells, including B cells. The correlation of the animal model experimental autoimmune encephalomyelitis (EAE) of MS and its predictive value to determine efficacy in the clinic appears limited. However, all current therapies do demonstrate efficacy in EAE models. There are also examples of mechanisms that have worked in EAE but have failed in the clinic, such as the TNFα antagonists and anti-p40 (a subunit of IL-12 and IL-23). The MS field would benefit if clinical biomarkers were available to monitor clinical efficacy. The etiology of MS remains elusive but additional understanding of mechanisms involved in the pathogenesis of MS may guide us to more effective treatment and management of this autoimmune disease.

Identification of a novel class of succinyl-nitrile-based Cathepsin S inhibitors.

The synthesis and in vitro activities of a series of succinyl-nitrile-based inhibitors of Cathepsin S are described. Several members of this class show nanomolar inhibition of the target enzyme as well as cellular potency. The inhibitors displaying the greatest potency contain N-alkyl substituted piperidine and pyrrolidine rings spiro-fused to the alpha-carbon of the P1 residue.

Discovery of potent and selective PKC-theta inhibitors.

An uHTS campaign was performed to identify selective inhibitors of PKC-theta. Initial triaging of the hit set based on selectivity and historical analysis led to the identification of 2,4-diamino-5-nitropyrimidines as potent and selective PKC-theta inhibitors. A homology model and initial SAR is presented demonstrating that a 2-arylalkylamino substituent in conjunction with suitable 4-diamino substituent are essential for achieving selectivity over many kinases. Additional hit to lead profiling is presented on selected compounds.

An orally active reversible inhibitor of cathepsin S inhibits human trans vivo delayed-type hypersensitivity.

Cathepsin S is a major histocompatibility complex (MHC) class II associated invariant chain (Ii) degrading enzyme expressed in antigen presenting cells such as B cells and dendritic cells. This enzyme is essential for MHC class II associated antigen processing and presentation to CD4(+) T cells. Compound I, a selective, reversible and orally bioavailable, inhibitor of cathepsin S, with molecular IC(50)=9 nM, has been recently described. We have tested the effects of compound I in a trans vivo model of delayed-type hypersensitivity. Human peripheral blood mononuclear cells (7-10 x 10(6)) from tetanus-sensitized donors were co-injected with tetanus toxoid (0.25 Lf) into C57Bl/6 mouse footpads. At 24 h, significant footpad swelling (+0.024+/-0.001 cm) characterized by an influx of mouse neutrophils and monocytes was observed. Injection of peripheral blood mononuclear cells alone caused negligible swelling (0.002+/-0.0002 cm). Anti-human MHC class II (HLA-DR, DP, DQ) antibody (5 mg/kg, i.p.) inhibited the swelling 91+/-7%, thus demonstrating a role of human antigen presenting cells in this model. Compound I (10, 30, and 100 mg/kg, p.o.) inhibited the response with an ED50 of approximately 18 mg/kg. Compound III, a less active analogue (molecular IC50>20 microM) had no effect. Furthermore, pretreatment of peripheral blood mononuclear cells with 10 nM compound II, an irreversible inhibitor (molecular IC50=11 nM) inhibited swelling 87+/-4%. These findings support the role of cathepsin S in human delayed-type hypersensitivity. Inhibition of cathepsin S with compound I may be useful in the treatment of human autoimmune diseases like rheumatoid arthritis and multiple sclerosis.

The design of potent hydrazones and disulfides as cathepsin S inhibitors.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.

Design and synthesis of dipeptide nitriles as reversible and potent Cathepsin S inhibitors.

The specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.