Nicholas B. Lydon

Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia

BACKGROUND BCR-ABL is a constitutively activated tyrosine kinase that causes chronic myeloid leukemia (CML). Since tyrosine kinase activity is essential to the transforming function of BCR-ABL, an inhibitor of the kinase could be an effective treatment for CML. METHODS We conducted a phase 1, dose-escalating trial of STI571 (formerly known as CGP 57148B), a specific inhibitor of the BCR-ABL tyrosine kinase. STI571 was administered orally to 83 patients with CML in the chronic phase in whom treatment with interferon alfa had failed. Patients were successively assigned to 1 of 14 doses ranging from 25 to 1000 mg per day. RESULTS Adverse effects of STI571 were minimal; the most common were nausea, myalgias, edema, and diarrhea. A maximal tolerated dose was not identified. Complete hematologic responses were observed in 53 of 54 patients treated with daily doses of 300 mg or more and typically occurred in the first four weeks of therapy. Of the 54 patients treated with doses of 300 mg or more, cytogenetic responses occurred in 29, including 17 (31 percent of the 54 patients who received this dose) with major responses (0 to 35 percent of cells in metaphase positive for the Philadelphia chromosome); 7 of these patients had complete cytogenetic remissions. CONCLUSIONS STI571 is well tolerated and has significant antileukemic activity in patients with CML in whom treatment with interferon alfa had failed. Our results provide evidence of the essential role of BCR-ABL tyrosine kinase activity in CML and demonstrate the potential for the development of anticancer drugs based on the specific molecular abnormality present in a human cancer.

Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr–Abl positive cells

The bcr–abl oncogene, present in 95% of patients with chronic myelogenous leukemia (CML), has been implicated as the cause of this disease. A compound, designed to inhibit the Abl protein tyrosine kinase, was evaluated for its effects on cells containing the Bcr–Abl fusion protein. Cellular proliferation and tumor formation by Bcr–Abl–expressing cells were specifically inhibited by this compound. In colony–forming assays of peripheral blood or bone marrow from patients with CML, there was a 92–98% decrease in the number of bcr–abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr–abl–positive leukemias.

Lessons learned from the development of an Abl tyrosine kinase inhibitor for chronic myelogenous leukemia

Protein kinases are a large family of homologous proteins comprising 2 major subfamilies, the protein serine/threonine kinases and protein tyrosine kinases (PTKs). Protein kinases function as components of signal transduction pathways, playing a central role in diverse biological processes such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases with abnormalities in these signaling pathways is considered a promising approach for drug development. Because of their deregulation in human cancers, Bcr-Abl, EGFR, HER2, and protein kinase C (PKC), were among the first protein kinases considered as targets for the development of selective inhibitors. Subsequently, as protein kinases have been implicated in more human cancers (1), drug-discovery efforts have been extended and several first-generation small-molecule inhibitors are now in various stages of development. A selection of these agents is shown in Table ​Table11. Table 1 Selected small-molecule ATP-competitive protein kinase inhibitors in development Based on its clear disease association, we saw the Bcr-Abl tyrosine kinase as an ideal target for validating the clinical utility of protein kinase inhibitors. Here, we discuss our experience in the preclinical and clinical development of a Bcr-Abl inhibitor as a therapeutic agent for chronic myelogenous leukemia (CML), and we consider how this experience and other recent advances in the field could contribute to drug development for other diseases.

Potent and selective inhibitors of the Abl-kinase : Phenylamino-pyrimidine (PAP) derivatives

Abstract Due to its relatively clear etiology, Chronic myelogenous leukemia (CML) represents an ideal disease target for a therapy using a selective inhibitor of the Bcr-Abl tyrosine protein kinase. Extensive optimization of the class of phenylamino-pyrimidines yielded highly potent and selective Bcr-Abl kinase inhibitors. Compound 1 shows high potency (IC50 = 38 nM) and selectivity for the Abl tyrosine protein kinase at the in vitro level.

The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase

The signalling mechanism and cellular targets of the AT2 receptor are still unknown. We report that angiotensin II (Ang II) inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase (pGC) activity through AT2 receptors in rat adrenal glomerulosa and PC12W cells. This inhibition is blocked by the phosphotyrosine phosphatase (PTPase) inhibitor orthovanadate but not by the Ser/Thr phosphatase inhibitor okadaic acid, suggesting the involvement of a PTPase in this process. Moreover, Ang II induces a rapid, transient and orthovanadate sensitive dephosphorylation of phosphotyrosine containing proteins in PC12W cells. Our findings suggest that AT2 receptors signal through stimulation of a PTPase and that this mechanism is implicated in the regulation of pGC activity. This observation is also the first example of hormonal inhibition of basal pGC activity.

PHENYLAMINO-PYRIMIDINE (PAP)-DERIVATIVES : A NEW CLASS OF POTENT AND HIGHLY SELECTIVE PDGF-RECEPTOR AUTOPHOSPHORYLATION INHIBITORS

Abstract Phenylamino-pyrimidines represent a novel class of inhibitors of the PDGF-receptor autophosphorylation with a high degree of selectivity versus other tyrosine and serine/threonine kinases. Optimum activity of ca 10 nM (IC50) was observed when the phenylamino-group which is attached to the pyrimidine carries a benzamide-moiety with a lipophilic substituent in 4-position.

Modulation of the SH2 Binding Specificity and Kinase Activity of Src by Tyrosine Phosphorylation within Its SH2 Domain

The Src family of kinases are held in an inactive state by interaction of their SH2 domain with a C-terminal phosphotyrosine. Dephosphorylation of this site can reactivate Src; however, recent evidence suggests that activation can also occur without dephosphorylation. In this study, platelet-derived growth factor receptor phosphorylation of Src on Tyr-213 specifically blocked binding of its SH2 domain to a phosphopeptide corresponding to the C-terminal regulatory sequence, while binding to other sequences, such as the platelet-derived growth factor receptor or a peptide from the epidermal growth factor receptor, was unaffected. Consequently, Src was activated over 50-fold. This is the first demonstration of regulation of a SH2 domain specificity by post-translational modification and is likely to be a general mechanism for regulation of all Src-like kinases.

Modelling study of protein kinase inhibitors: Binding mode of staurosporine and origin of the selectivity of CGP 52411

SummaryA model for the binding mode of the potent protein kinase inhibitor staurosporine is proposed. Using the information provided by the crystal structure of the cyclic-AMP-dependent protein kinase, it is suggested that staurosporine, despite a seemingly unrelated chemical structure, exploits the same key hydrogen-bond interactions as ATP, the cofactor of the protein kinases, in its binding mode. The structure-activity relationships of the inhibitor and a docking analysis give strong support to this protein tyrosine kinase is rationalized on the basis of the model. It is proposed that this selectivity originates in the occupancy, by one of the anilino moieties of the inhibitor, of the region of the enzyme cleft that normally binds the ribose ring of ATP, which appears to possess a marked lipophilic character in this kinase.

Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.

A potent protein-tyrosine kinase inhibitor which selectively blocks proliferation of epidermal growth factor receptor-expressing tumor cells in vitro and in vivo.

A calculated 3‐D model of the kinase domain of the epidermal growth factor receptor (EGF‐R) protein‐tyrosine kinase (PTK) was used to develop a pharmacophore model for ATP‐competitive inhibitors and, subsequently, a new class of selective EGF‐R kinase inhibitors. CGP 59326A, a highly selective and potent inhibitor of the EGF‐R in vitro, inhibited the proliferation of EGF‐R‐expressing epithelial lines, while having little anti‐proliferative activity against EGF‐R‐negative lines. In contrast to previously described inhibitors, CGP 59326A had potent and selective in vivo anti‐tumor activity at well‐tolerated doses against EGF‐R‐expressing tumors (e.g., ED50 of 0.78 to 1.5 mg/kg for inhibition of A431 tumor growth). CGP 59326A inhibited growth of human tumor xenografts expressing the EGF‐R but showed little activity against EGF‐R‐negative xenografts. Combination of CGP 59326A with cytotoxic agents resulted in tumor regression and cures. The high selectivity and attractive biological profile of CGP 59326A suggest that it could have therapeutic value in the treatment of proliferative diseases which involve mitogenic signaling from the EGF‐R. Int. J. Cancer 76:154–163, 1998.© 1998 Wiley‐Liss, Inc.