Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological eects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our ®ndings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.

Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Despite the progress achieved in its treatment, 20% of cases relapse and no longer respond to chemotherapy. The most common phenotype of ALL cells share surface antigens with very early precursors of B cells and are therefore believed to originate from this lineage. Characterization of the growth requirement of ALL cells indicated that they were dependent on various cytokines, suggesting paracrine and/or autocrine growth regulation. Because many cytokines induce tyrosine phosphorylation in lymphoid progenitor cells, and constitutive tyrosine phosphorylation is commonly observed in B-lineage leukaemias, attempts have been made to develop protein tyrosine kinase (PTK) blockers of leukaemia cell growth. Here we show that leukaemic cells from patients in relapse have constitutively activated Jak-2 PTK. Inhibition of Jak-2 activity by a specific tyrosine kinase blocker, AG-490, selectively blocks leukaemic cell growth in vitro and in vivo by inducing programmed cell death, with no deleterious effect on normal haematopoiesis.

The crystal structure of the autoinhibited form of Hck has been determined at 2.0 A resolution, in complex with a specific pyrazolo pyrimidine-type inhibitor, PP1. The activation segment, a key regulatory component of the catalytic domain, is unphosphorylated and is visualized in its entirety. Tyr-416, the site of activating autophosphorylation in the Src family kinases, is positioned such that access to the catalytic machinery is blocked. PP1 is bound at the ATP-binding site of the kinase, and a methylphenyl group on PP1 is inserted into an adjacent hydrophobic pocket. The enlargement of this pocket in autoinhibited Src kinases suggests a route toward the development of inhibitors that are specific for the inactive forms of these proteins.

A novel class of low molecular weight protein tyrosine kinase inhibitors is described. These compounds constitute a systematic series of molecules with a progressive increase in affinity toward the substrate site of the EGF receptor kinase domain. These competitive inhibitors also effectively block the EGF-dependent autophosphorylation of the receptor. The potent EGF receptor kinase blockers examined were found to competitively inhibit the homologous insulin receptor kinase at 10(2)-10(3) higher inhibitor concentrations in spite of the significant homology between these protein tyrosine kinases. These results demonstrate the ability to synthesize selective tyrosine kinase inhibitors. The most potent EGF receptor kinase inhibitors also inhibit the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on EGF independent cell growth. These results demonstrate the potential use of protein tyrosine kinase inhibitors as selective antiproliferative agents for proliferative diseases caused by the hyperactivity of protein tyrosine kinases. We have suggested the name "tyrphostins" for this class of antiproliferative compounds which act as protein tyrosine kinase blockers.