Here, we have studied the activity of a novel proteintyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colonystimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

When challenged with extracellular ATP, leukocytes respond and activate processes attributed to the P2X7 receptor (P2X7R), an unusual ligand-gated ion channel. To prove P2X7R involvement, blood samples from P2X7R-deficient mice were characterized. Monocytes and lymphocytes associated with wild-type blood responded to ATP and underwent volume/shape changes and shed L-selectin. In contrast, leukocytes from P2X7R-deficient animals demonstrated no change in physical properties or L-selectin expression following ATP challenge. Blood stimulated with LPS or ATP individually generated minimal quantities of the leaderless polypeptide IL-1β, but sequential treatment of wild-type, but not P2X7R-deficient, blood with LPS and ATP yielded large amounts of cell-free cytokine. Based on these differences, wild-type and P2X7R-deficient animals were compared following induction of monoclonal anti-collagen-induced arthritis. Ab-treated wild-type animals subsequently challenged with LPS developed inflamed, swollen paws; their joint cartilage demonstrated lesions, loss of proteoglycan content, and the presence of collagen degradation products. P2X7R-deficient animals subjected to the same challenge were markedly less affected; both the incidence and severity of disease were reduced. These data indicate that ATP does act via the P2X7R to affect leukocyte function and that the P2X7R can serve as an important component of an in vivo inflammatory response.

Cathepsins have been implicated in the degradation of proteins destined for the MHC class II processing pathway and in the proteolytic removal of invariant chain (Ii), a critical regulator of MHC class II function. Mice lacking the lysosomal cysteine proteinase cathepsin S (catS) demonstrated a profound inhibition of Ii degradation in professional APC in vivo. A marked variation in the generation of MHC class II-bound Ii fragments and presentation of exogenous proteins was observed between B cells, dendritic cells, and macrophages lacking catS. CatS-deficient mice showed diminished susceptibility to collagen-induced arthritis, suggesting a potential therapeutic target for regulation of immune responsiveness.

There is a critical need for safer and more convenient treatments for organ transplant rejection and autoimmune disorders such as rheumatoid arthritis. Janus tyrosine kinases (JAK1, JAK3) are expressed in lymphoid cells and are involved in the signaling of multiple cytokines important for various T cell functions. Blockade of the JAK1/JAK3-STAT pathway with a small molecule was anticipated to provide therapeutic immunosuppression/immunomodulation. The Pfizer compound library was screened against the catalytic domain of JAK3 resulting in the identification of a pyrrolopyrimidine-based series of inhibitors represented by CP-352,664 (2a). Synthetic analogues of 2a were screened against the JAK enzymes and evaluated in an IL-2 induced T cell blast proliferation assay. Select compounds were evaluated in rodent efficacy models of allograft rejection and destructive inflammatory arthritis. Optimization within this chemical series led to identification of CP-690,550 1, a potential first-in-class JAK inhibitor for treatment of autoimmune diseases and organ transplant rejection.

We report how rotational variations in transmembrane (TM) helix interactions participate in the activity states of the thrombopoietin receptor (TpoR), a type 1 cytokine receptor that controls the production of blood platelets. We also explore the mechanism of small-molecule agonists that do not mimic the natural ligand. We show, by a combination of cysteine cross-linking, alanine-scanning mutagenesis, and computational simulations, that the TpoR TM dimerizes strongly and can adopt 3 different stable, rotationally related conformations, which may correspond to specific states of the full-length receptor (active, inactive, and partially active). Thus, our data suggest that signaling and inactive states of the receptor are related by receptor subunit rotations, rather than a simple monomer-dimer transition. Moreover, results from experiments with and without agonists in vitro and in cells allow us to propose a novel allosteric mechanism of action for a class of small molecules, in which they activate TpoR by binding to the TM region and by exploiting the rotational states of the dimeric receptor. Overall, our results support the emerging view of the participation of mutual rotations of the TM domains in cytokine receptor activation.

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Objective-Lysosomal proteinases have been implicated in a number of pathologies associated with extracellular matrix breakdown. Therefore, we investigated the possibility that the lysosomal proteinase cathepsin S may be involved in atherosclerotic plaque destabilization. Methods and Results-Atherosclerotic plaques in the brachiocephalic arteries of fat-fed apolipoprotein E/cathepsin S double knockout mice had 73% fewer acute plaque ruptures (Pϭ0.026) and were 46% smaller (Pϭ0.025) than those in age-, strain-, and sex-matched apolipoprotein E single knockout controls. When the incidence of acute plaque rupture was normalized for plaque size, the reduction in the double knockouts was 72% (Pϭ0.039). The number of buried fibrous layers, indicative of an unstable plaque phenotype, was reduced by 67% in the double knockouts (Pϭ0.008). The cysteine proteinase inhibitor, egg white cystatin, was biotinylated and used as an active-site-directed probe for cathepsins. Biotinylated cystatin selectively detected cathepsin S in extracts of human carotid atherosclerotic plaque. Active cathepsin S was detectable in extracts of human atherosclerotic plaque but not in nondiseased carotid arteries. Active cathepsins were especially prominent in macrophages in the shoulder regions of plaques, areas considered to be vulnerable to rupture. Cathepsin S protein colocalized with regions of elastin degradation in human coronary plaques. Conclusion-These data provide direct evidence that an endogenous proteinase, cathepsin S, plays an important role in atherosclerotic plaque destabilization and rupture. Key Words: atherosclerosis Ⅲ plaque Ⅲ pathology Ⅲ cathepsin T he highly thrombogenic gruel in the core of an atherosclerotic plaque is luminally covered by a fibrous cap, weakening of which leads to plaque rupture and thrombus formation. Macrophages and T cells accumulate at sites of plaque disruption. 1 Degradation of collagen by macrophagederived matrix metalloproteinases has been reported, 2 but less is known of other classes of proteinase, which may be released by activated macrophages.The lysosomal cathepsins have been implicated in the development and progression of atherosclerosis. Increased levels of cathepsins F, K, and S are present in atherosclerotic lesions, 3,4 whereas levels of the major extracellular inhibitor of cysteine proteinases, cystatin C, are decreased. 5 In humans, an association has been shown between a genetically determined decrease in cystatin C levels and the severity of coronary artery disease. 6 Cathepsin S/low-density lipoprotein (LDL) receptor double knockout mice have impaired atherogenesis when compared with LDL receptor single knockout controls. 7 Grading of atherosclerosis in the aortic arch showed that there was a delay in lesion progression in the double knockouts. For example, 12 weeks of feeding atherogenic diet to the double knockouts resulted in an average lesion severity similar to that seen in single knockout controls after just 8 weeks of feeding, and 26 weeks of atherogenic diet feeding in the ...