A series of substituted 2-(aminopyridyl)- and 2-(aminopyrimidinyl)thiazole-5-carboxamides was identified as potent Src/Abl kinase inhibitors with excellent antiproliferative activity against hematological and solid tumor cell lines. Compound 13 was orally active in a K562 xenograft model of chronic myelogenous leukemia (CML), demonstrating complete tumor regressions and low toxicity at multiple dose levels. On the basis of its robust in vivo activity and favorable pharmacokinetic profile, 13 was selected for additional characterization for oncology indications.

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The binding ofantigen to the multicomponent T-cell receptor (TCR) activates several signal transduction pathways via coupling mechanisms that are poorly understood. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC). TCR activation by antigen, lectins, or anti-TCR monoclonal antibody has also been showu to cause increases in tyrosine phosphorylation of TCR-C and other substrates, suggesting stimulation of protein tyrosine kinase (PTK) activity. A critical question is whether these two pathways, PLC and PTK, are independently activated or whether one initiates and/or regulates the other. In the former case, PLC activation could be coupled to the TCR via a GTP-binding protein (G protein).We have reported, however, that tyrosine phosphorylation of intracellular substrates precedes detection of PLC activation and intracellular calcium elevation, suggesting that inositol phospholipid turnover in T cells is initiated by a PTIK pathway.In this study, we test this hypothesis by treating T cells with the drug herbimycin A. We demonstrate that this agent inhibits substrate tyrosine phosphorylation, TCR-mediated inositol phospholipid hydrolysis, and calcium elevation. In contrast, under these conditions G-protein-mediated PLC activity, as tested by addition of aluminum fluoride, remains intact. Furthermore, whereas herbimycin treatment prevents TCRmediated interleukin 2 production and interleukin 2 receptor expression, phorbol ester-induced effects are substantially resistant to herbimycin. The drug thus appears to abrogate TCR-mediated signaling without affecting distal signaling mechanisms.Triggering of the T-cell receptor (TCR) for antigen activates multiple biochemical pathways. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC) (reviewed in refs. 1 and 2) with the generation of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (3). Treatment of T cells with phorbol ester and calcium ionophore, agents whose effects mimic these second messengers, has been shown to reproduce many features of TCR stimulation, leading to the conclusion that the signal transduction pathway mediated by PLC is important for T-cell activation.TCR stimulation has also been shown to cause increases in the tyrosine phosphorylation of several substrates in both murine and human T cells (4-8). Immunoblots using specific anti-phosphotyrosine antibodies have revealed increased tyrosine phosphorylation on multiple proteins, including those of 145, 135, 100, 75, and 40 kDa after ligation of the human TCR with anti-CD3 monoclonal antibody. In a previous report, we demonstrated that tyrosine phosphorylation of these proteins is rapid, with the earliest increase in phosphorylation detectable at 5 sec and with maximal stimulation by 90 sec (7).Herbimycin A is a benzoquinonoid ansamycin antibiotic that was found to reverse oncogenic transformation induced by pp6v-src (9,10). Subsequen...

The p38 kinase plays a central role in inflammation, and it has been the subject of extensive efforts in both basic research and drug discovery. This review summarizes the biology of the p38 kinase with a focus on its role in inflammation. The p38 kinase regulates the production of key inflammatory mediators, including TNFalpha, IL-1beta, and COX-2. In addition, p38 also acts downstream of cytokines such as TNFalpha, mediating some of their effects. The potential efficacy of p38 inhibitors may thus be greater than would be expected from the inhibition of the mediators alone. Inhibitors of p38 kinase are currently in development for the treatment of rheumatoid arthritis. The biological processes regulated by p38 kinase suggest a wide variety of additional potential indications.

2-aminothiazole (1) was discovered as a novel Src family kinase inhibitor template through screening of our internal compound collection. Optimization through successive structure-activity relationship iterations identified analogs 2 (Dasatinib, BMS-354825) and 12m as pan-Src inhibitors with nanomolar to subnanomolar potencies in biochemical and cellular assays. Molecular modeling was used to construct a putative binding model for Lck inhibition by this class of compounds. The framework of key hydrogen-bond interactions proposed by this model was in agreement with the subsequent, published crystal structure of 2 bound to structurally similar Abl kinase. The oral efficacy of this class of inhibitors was demonstrated with 12m in inhibiting the proinflammatory cytokine IL-2 ex vivo in mice (ED50 approximately 5 mg/kg) and in reducing TNF levels in an acute murine model of inflammation (90% inhibition in LPS-induced TNFalpha production when dosed orally at 60 mg/kg, 2 h prior to LPS administration). The oral efficacy of 12m was further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when administered orally at 0.3 and 3 mg/kg twice daily. Dasatinib (2) is currently in clinical trials for the treatment of chronic myelogenous leukemia.

The role of intracellular oxidative stress in the mechanism of action of phosphotyrosine phosphatase (PTP) inhibitors was studied using three vanadiumbased compounds. Sodium orthovanadate (Na 3 VO 4 ), sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) (pV(phen), and bis(maltolato)-oxovanadium(IV) (BMOV) differentially induced oxidative stress in lymphocytes. Treatment with pV(phen), which caused intracellular oxidation, induced strong protein tyrosine phosphorylation compared with Na 3 VO 4 and BMOV. Syk family kinases and the mitogen-activated protein kinase erk2 were rapidly activated by pV(phen) but not by BMOV or Na 3 VO 4 . In contrast, both BMOV and pV(phen) strongly activated NF-B. The antioxidant pyrrolidine dithiocarbamate (PDTC) greatly diminished the intracellular oxidation and protein phosphotyrosine accumulation induced by pV(phen). Pretreatment of cells with PDTC reduced and delayed the activation of Syk kinases and erk2. However, NF-B activation by pV(phen) was markedly enhanced in lymphocytes pretreated with PDTC, and another antioxidant, N-acetylcysteine, did not prevent the activation of NF-B by BMOV. These results indicate a role for oxidative stress in the biological effects of some PTP inhibitors, whereas NF-B activation by PTP inhibitors is mediated by mechanisms independent of intracellular redox status.Lymphocyte signal transduction requires the activation of protein tyrosine kinases (PTKs), 1 with subsequent assembly of signaling complexes, generation of second messengers, activation of transcription factors, and gene expression (1, 2). The balance of protein tyrosine phosphorylation within the cell is controlled by the relative activities of the PTKs and PTPs in the signaling network (3). Besides dephosphorylating a variety of PTK substrates, PTPs have been shown to directly modulate the activities of PTKs (4, 5). Thus PTPs serve a crucial function in lymphocytes by controlling both the initiation and termination of receptor-based signals.The inhibition of PTPs reveals PTK substrates on which phosphotyrosine accumulates in the absence of receptor engagement (6). Some of these substrates are key phosphoproteins in lymphocyte signal transduction pathways, suggesting that PTKs involved in transmission of receptor signals are activated by the absence of PTP regulation (6 -9). However, many of the PTP inhibitors used thus far to explore lymphocyte signal transduction pathways are redox-active compounds. For example, phenylarsine oxide, a thiol-reactive compound, and H 2 O 2 , which generates hydroxyl radicals, both act as potent PTP inhibitors (6, 10). The role of intracellular oxidation in the mechanism of action of PTP inhibitors is unknown, a question this study addresses.Vanadium-based PTP inhibitors, which have been extensively studied as insulin mimetic agents, stimulate glucose uptake and fatty acid synthesis in adipocytes and mimic receptor-based signals in lymphocytes (11-15). The widely used PTP inhibitor pervanadate is a peroxovanadium compound generated by reaction of H 2 O 2 wi...

CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the mu2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The mu1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of mu2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. Mu2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 (165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

Collagen is an important primary stimulus of platelets during the process of hemostasis. As with many other platelet stimuli, collagen signal transduction involves the hydrolysis of inositol phospholipids; however, the mechanisms which underlies this event is not well understood. Neither the collagen receptor nor the isoform of phospholipase C that is activated have been identified. We report that collagen-activation of platelets induces tyrosine phosphorylation of phospholipase C-gamma 2 but not phospholipase C-gamma 1. We also show that the platelet low affinity Fc receptor (Fc gamma RII), which mediates activation of platelets by immune complexes, and wheat germ agglutinin, which binds non-specifically to glycoprotein, stimulate phospholipase C-gamma 2 tyrosine phosphorylation. In contrast, we could not detect phospholipase C-gamma 2 tyrosine phosphorylation in platelets stimulated by either thrombin or a stable thromboxane A2 analogue, U46619.