David W. Fry

Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2.

ABSTRACT Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130Cas, were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKβ/RAFTK/CadTK.

Inhibition of the epidermal growth factor receptor family of tyrosine kinases as an approach to cancer chemotherapy: progression from reversible to irreversible inhibitors.

The rationale to inhibit the epidermal growth factor receptor (EGFR) tyrosine kinase family as an approach to cancer chemotherapy has continued to grow stronger over the last 10 years. Both preclinical and clinical data strongly support the involvement of these receptors in the formation and progression of human cancers, as well as establish a high correlation in cancer patients between receptor/ ligand expression and poor prognosis. During the past 4 years, significant progress has been made in the area of EGFR tyrosine kinase inhibitors, and new structural classes have emerged that exhibit enormous improvements with regard to potency, specificity, and in vitro and in vivo activity. Very recently, further advancements in this field have been made whereby very specific, irreversible inhibitors of the EGFR family have been synthesized that provide unique pharmacological properties and exceptional efficacy. The in vivo performance of these modern kinase inhibitors has improved to the point where several compounds are either in clinical trials or very near to that point in their development. This review will briefly address the justification for targeting the EGFR family for cancer therapeutics, and then will highlight some of the more promising kinase inhibitors that are in development.

Inhibition of type II topoisomerase by fostriecin

Fostriecin is a new antitumor antibiotic which is being developed further as an anticancer agent based on its marked activity in murine leukemias. Its mechanism of action, however, has thus far remained unknown. The present study demonstrates that fostriecin inhibits the catalytic activity of partially purified type II topoisomerase from Ehrlich ascites carcinoma. Under the experimental conditions employed, fostriecin completely inhibited the enzyme at 100 microM. A general kinetic analysis showed that fostriecin inhibited topoisomerase in an uncompetitive manner with a Ki,app of 110 microM and produced kinetics that were distinctly different from those of VM-26 which exhibited noncompetitive inhibition. Fostriecin did not cause DNA strand breaks in L1210 cells, suggesting that it did not stabilize a cleavable complex as do other known inhibitors of this enzyme. Fostriecin, however, did partially inhibit DNA strand breaks produced by amsacrine. An analysis by flow cytometry showed that L1210 cells exposed to 5 microM fostriecin for 12 hr caused a block in the G2 phase of the cell cycle. These studies thus suggest that the mechanism by which fostriecin produces its antitumor effects may be through inhibition of topoisomerase II and that the type of inhibition is markedly different from existing antitumor agents which inhibit this enzyme.

Biochemical pharmacology of the lipophilic antifolate, trimetrexate

Trimetrexate is a novel lipophilic folate antagonist that causes growth inhibition, inhibition of nucleic acid biosynthesis, and cytotoxicity at nanomolar concentrations in tissue cultures. The potency of trimetrexate cytotoxicity against most cell lines is greater than that of methotrexate. Trimetrexate has antitumor activity in vivo in several murine leukemia and solid tumor systems, including tumors in which methotrexate is inactive. Antitumor activity was seen following oral, intravenous, or intraperitoneal administration. Trimetrexate causes a pronounced and early depression in incorporation of deoxyuridine into DNA. In tumor cell lines resistant to methotrexate because of a drug transport defect, trimetrexate retains activity. In many such cases the methotrexate-resistant tumors show collateral sensitivity to trimetrexate. In methotrexate-resistant cells with impaired drug transport, trimetrexate sensitivity was even more pronounced when cells were grown in folate-free medium supplemented with physiological levels of tetrahydrofolate cofactor. In the human tumor stem cell colony assay, trimetrexate, at concentrations achievable in vivo, gave activity against many human tumors, including samples that were unresponsive to methotrexate. Trimetrexate crosses the blood-brain barrier, and at very high doses may cause neurotoxicity. At conventional doses the primary toxic effects in mice are gastrointestinal. This toxicity is reversible at therapeutic doses. Unlike earlier lipophilic antifolates, trimetrexate has rapid plasma clearance (t1/2 in mice of 45 minutes). Trimetrexate is a tight-binding competitive inhibitor of dihydrofolate reductase. The Ki,slope for inhibition of the human enzyme was 4 X 10(-11) M. A dose-dependent decrease in cellular purine ribonucleotide pools is given by trimetrexate. Pyrimidine ribonucleotide pools tend to increase in treated cells. Trimetrexate caused a marked depression of cellular pools of dTTP and dGTP, and a lesser depression in dATP. Cytotoxicity of trimetrexate in vitro was prevented by leucovorin. Leucovorin also protected mice from trimetrexate toxicity. Thymidine protected cells from lethal effects of low concentrations of trimetrexate, but not from high concentrations. The combination of thymidine and hypoxanthine completely protected cells from low and high concentrations of trimetrexate. A new, stable and highly water-soluble formulation of trimetrexate has been developed. Because of the interesting biochemical and pharmacological properties of trimetrexate, and its experimental antitumor activity, clinical trials are planned.

discovery and structure-activity studies of a novel series of pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors

Abstract The inhibition of tyrosine kinase-mediated signal transduction pathways represents a therapeutic approach to the intervention of proliferative diseases such as cancer, atherosclerosis, and restenosis. A novel series of pyrido[2,3-d]pyrimidine inhibitors of the PDGFr, bFGFr, and c-Src tyrosine kinases was developed from compound library screening and lead optimization.1 In addition, highly selective inhibitors of the FGFr tyrosine kinase were also discovered and developed from this novel series of pyrido[2,3-d]pyrimidines. The syntheses, biological evaluation, and structure-activity relationships of this series are reported.

Biochemical and antiproliferative properties of 4-[Ar(alk)ylamino]pyridopyrimidines, a new chemical class of potent and specific epidermal growth factor receptor tyrosine kinase inhibitor

The tyrosine kinase inhibitors PD 69896, 153717, and 158780, which belong to the chemical class 4-[ar(alk)ylamino]pyridopyrimidines, have been characterized with respect to enzymology, target specificity, and antiproliferative effects in tumor cells. These compounds were competitive inhibitors with respect to ATP against purified epidermal growth factor (EGF) receptor tyrosine kinase and inhibited EGF receptor autophosphorylation in A431 human epidermoid carcinoma with IC50 values of 2085, 110, and 13 nM, respectively. Onset of inhibition was immediate once cells were exposed to these compounds, whereas recovery of receptor autophosphorylation activity after the cells were washed free of the compound was dependent on inhibitory potency. Thus, full activity returned immediately after removal of PD 69896 but required 8 hr after exposure to PD 158780. PD 158780 was highly specific for the EGF receptor in Swiss 3T3 fibroblasts, inhibiting EGF-dependent receptor autophosphorylation and thymidine incorporation at low nanomolar concentrations while requiring micromolar levels for platelet-derived growth factor- and basic fibroblast growth factor-dependent processes. PD 158780 inhibited heregulin-stimulated phosphorylation in the SK-BR-3 and MDA-MB-453 breast carcinomas with IC50 values of 49 and 52 nM, respectively, suggesting that the compound was active against other members of the EGF receptor family. The antiproliferative effects of this series of compounds against A431 cells correlated precisely with the inhibitory potency against EGF receptor autophosphorylation. PD 158780 reduced clone formation in soft agar of fibroblasts transformed by EGF, EGF receptor, or the neu oncogene but not ras or raf, further demonstrating its high degree of specificity. Finally, this compound was active against clone formation in several breast tumors having different expression patterns of the erbB family, indicating an anticancer utility in tumors expressing these receptors.

Protein tyrosine kinases as therapeutic targets in cancer chemotherapy and recent advances in the development of new inhibitors

Future approaches to cancer chemotherapy will inevitably include ways to modulate the function of oncogenes, suppressor genes or components of mitogenic or cell cycle signal transduction. One set of likely targets for therapeutic intervention are tyrosine kinases. Over the past decade, tyrosine phosphorylation/dephosphorylation has irrefutably been shown to be intimately involved in growth regulation, mitogenesis and cell cycle progression. Many of the known tyrosine kinases are encoded for by proto-oncogenes, and transforming mutations or uncontrolled activity of these enzymes have been observed in tumours. Certain growth factors known or suspected to participate in autocrine mechanisms of tumour propagation bind to receptors having tyrosine kinase activity and there now exists a significant amount of clinical data to implicate a role for many of these enzymes in the development or progression of disease in cancer patients. Throughout the last decade many inhibitors of tyrosine kinases have been discover...

Anticancer efficacy of the irreversible EGFr tyrosine kinase inhibitor PD 0169414 against human tumor xenografts.

Purpose: The involvement of the EGF receptor (EGFr) family of receptors in cancers suggests that a selective inhibitor of the tyrosine kinase activity of the EGFr family could have a therapeutic effect. PD 0169414, an anilinoquinazoline, is a potent irreversible inhibitor of the EGFr family tyrosine kinase activity with IC50 values of 0.42 nM against the isolated EGF receptor, and 4.7 nM and 22 nM against EGF- and heregulin-mediated receptor phosphorylation in A431 and MDA-MB-453 cells, respectively. Methods and Results: Oral administration of 260 mg/kg per day PD 0169414 for 15 days to animals bearing advanced-stage A431 epidermoid carcinoma produced a 28.2-day delay in tumor growth and resulted in three complete and three partial tumor regressions in six animals. Toxicity at this dose level was limited to <6% loss of initial body weight. Doses of 160 and 100 mg/kg per day produced tumor growth delays of 29.5 and 20.9 days and two and one complete regressions in six animals, respectively. Subcutaneous, intraperitoneal, and oral routes of administration have also shown in vivo antitumor activity of PD 0169414 in a panel of human tumor xenografts. Responsive tumor lines include A431 (human epidermoid carcinoma), H125 (NSCL carcinoma), MCF-7 and UISO-BCA1 (human breast carcinoma), and SK-OV-03 (human ovarian carcinoma). The therapeutic effect ranged from delayed tumor growth (6.4 days delayed tumor growth for 14 days of treatment) to tumor regressions (32.2 days delayed tumor growth and five partial regressions in six animals) in these model systems. Conclusion: PD 0169414 is a specific, irreversible inhibitor of EGFr family tyrosine kinases with significant in vivo activity against a variety of relevant human tumor xenografts.

In vitro DNA strand scission and inhibition of nucleic acid synthesis in L1210 leukemia cells by a new class of DNA complexers, the anthra[1,9-cd]pyrazol-6(2H)-ones (anthrapyrazoles)

CI-937 and CI-942 belong to a new class of DNA complexers, the anthra[1,9-cd]pyrazol-6(2H)-ones (anthrapyrazoles), and are being further developed as antitumor drugs based on their curative properties against murine solid tumour models. The biochemical effects of these agents were studied in L1210 leukemia in relation to other clinically used intercalators. After a 1-hr exposure, CI-937 and CI-942 reduced the cloning efficiency of L1210 cells by 50% at 3.0 X 10(-8) and 1.5 X 10(-7) M respectively. Based on an ethidium displacement assay, these drugs bound strongly to DNA, reducing the fluorescence of an ethidium-DNA complex by 50% at concentrations of 23 and 33 nM for CI-937 and CI-942 respectively. This was comparable to mitoxantrone at 15 nM, but much more potent than Amsacrine which required over 1.3 microM. A distinct property of the anthrapyrazoles was a much more potent inhibitory effect on whole cell DNA synthesis than on RNA synthesis. After L1210 cells were exposed to drug for 2 hr the concentration needed to inhibit DNA synthesis by 50% was 0.33 and 0.57 microM for CI-937 and CI-942, respectively, whereas 2.0 and 11.3 microM were required to inhibit RNA synthesis by the same extent. This was in contrast to Adriamycin and mitoxantrone which inhibited both activities equally at similar concentrations. It was apparent that the inhibition of these processes was not due to substrate depletion since intracellular ribonucleoside and deoxyribonucleoside triphosphates either remained constant or were elevated after a 2-hr exposure to 1 or 10 microM drug. A similar discriminatory effect was observed on DNA and RNA polymerase in permeabilized cells, and the inhibition of nucleic acid synthesis in this system could be reversed by exogenously added DNA. Since the high incidence of cardiotoxicity associated with the administration of anthracyclines has been related to the formation of reactive oxygen species, the ability of the anthrapyrazoles to augment superoxide dismutase sensitive oxygen consumption was observed in a rat liver microsomal system. CI-937 and CI-942 induced 5- and 10-fold less oxygen consumption than Adriamycin, producing rates of 12.4, 24.2 and 138.9 nmoles/min/mg microsomal protein, respectively, at a drug concentration of 0.5 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Enantioselective inhibition of the epidermal growth factor receptor tyrosine kinase by 4-(α-phenethylamino)quinazolines

4-Benzylaminoquinazolines can be potent reversible inhibitors of the EGFR tyrosine kinase at the ATP binding site. Examination of benzylic methylation reveals that an (R)-methyl group is four- to six-fold activating, with an optimal Ki of 630 pM for compound 11. In sharp contrast, (S)-methylation causes a > 30 to 500-fold loss of inhibitory activity, showing that the ATP-binding site of the receptor has very low tolerance for even moderate out-of-plane bulk in certain directions. It is suggested that the best of these inhibitors can induce a conformation of the kinase not available to poorer inhibitors.