Correction: AMN107 (nilotinib): a novel and selective inhibitor of BCR-ABL.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Beta interferon subtype 1 induction by tumor necrosis factor.

Tumor necrosis factor (TNF) induces an antiviral state in various cell lines. This antiviral state is quite similar to that established by interferon (IFN), e.g., TNF treatment of HEp-2 cells induces 2',5'-oligoadenylate synthetase activity. Both antiviral activity and synthetase induction are greatly reduced when TNF treatment occurs in the presence of a beta interferon subtype 1 (IFN-beta 1)-neutralizing antiserum. However, no one has yet directly demonstrated IFN-beta 1 induction, either as an antiviral activity in supernatants from TNF-treated cells or as IFN-specific mRNA by Northern (RNA) blot analysis. We have adopted a recently described in vitro DNA amplification protocol for the detection of specific RNAs. By applying this method to RNA from HEp-2 cells, we could demonstrate increased levels of IFN-beta 1-specific transcripts after TNF treatment. Dose response and kinetics of IFN-beta 1 induction coincided with the TNF-induced antiviral state. Nuclear run-on analysis showed enhanced transcriptional activity of the IFN-beta 1 gene in TNF-treated cells. Our data substantiate a role of IFN-beta 1 as mediator of the biological activity of TNF in HEp-2 cells.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Inhibitors of protein kinases: CGP 41251, a protein kinase inhibitor with potential as an anticancer agent.

CGP 41251 was originally identified as an inhibitor of protein kinase C (PKC), inhibiting mainly the conventional PKC subtypes, and subsequently shown to inhibit the vascular endothelial growth factor (VEGF) receptor kinase insert domain-containing receptor, which is involved in angiogenesis. CGP 41251 inhibits reversibly intracellular PKC activity, induction of c-fos and the corresponding activation of the mitogen-activated protein kinase induced by either tumor promoting phorbol esters, platelet-derived growth factor, or basic fibroblast growth factor, but not by the epidermal growth factor. CGP 41251 inhibited the ligand-induced autophosphorylation of the receptors for platelet-derived growth factor, stem cell factor, and VEGF (kinase insert domain-containing receptor) that correlated with the inhibition of the mitogen-activated protein kinase activation, but did not affect the ligand-induced autophosphorylation of the receptors for insulin, insulin-like growth factor-I, or epidermal growth factor. CGP 41251 showed broad antiproliferative activity against various tumor and normal cell lines in vitro, and is able to reverse the p-glycoprotein-mediated multidrug resistance of tumor cells in vitro. CGP 41251 showed in vivo antitumor activity as single agent and inhibited angiogenesis in vivo. Thus, CGP 41251 may suppress tumor growth by inhibiting tumor angiogenesis (via its effects on the VEGF receptor tyrosine kinases) in addition to directly inhibiting tumor cell proliferation (via its effects on PKCs).

Induction of intracellular and plasma 2',5'-oligoadenylate synthetase by pentostatin.

Similar to interferon alpha, pentostatin is highly effective in hairy cell leukemia and moderately active in other chronic lymphoid malignancies. In ten patients with hairy cell leukemia (HCL) and seven patients with other B-cell chronic leukemias (BCL), we have studied the intracellular 2',5'-oligoadenylate synthetase (2,5OAS) activity of the mononuclear cells before, 4 h, 24 h, and 48 h after pentostatin administration. In patients with HCL the median level of intracellular 2,5OAS increased 4.6-fold at 4 h and 11.5-fold at 24 h compared to the pretreatment value. Among the other seven patients, the median intracellular 2,5OAS remained unchanged in three patients and rose slightly by 2 to 14 times in four patients. Eleven patients (eight with HCL and two with BCL) responded to pentostatin. The median increase in 2,5OAS among the responders was 13.0-fold (range 4.8-30.0) whereas that among non-responders was 2.2-fold (range 0.2-6.3). The difference was highly significant (p less than 0.0001). In five of the total seventeen patients, the plasma levels of 2,5OAS activity were also determined and changes in plasma levels paralleled those measured intracellularly. To determine if the elevation of 2,5OAS is mediated by induction of interferon alpha, the expressions of mRNA for interferon alpha and beta were investigated by means of reverse transcription and polymerase chain reaction using the corresponding sense primers. In none of the five patients thus studied could we find an induction of mRNA for interferon alpha or beta in the leukemic cells during treatment with pentostatin. Thus, response to pentostatin correlates with induction of 2,5OAS directly and the 2',5'-oligoadenylate system seems to be involved in cytotoxicity.

Aza-peptide analogs as potent human immunodeficiency virus type-1 protease inhibitors with oral bioavailability.

A series of aza-peptide analogs with a (hydroxyethyl)hydrazine isostere has been synthesized as HIV-1 protease inhibitors using a simple synthetic scheme. Structure-activity studies based on the X-ray of a previously described inhibitor-enzyme complex led to potent inhibitors with antiviral activity in the low-nanomolar range. The S-configuration of the transition-state hydroxyl group was preferred in this series. Small modifications of the P2P3 and P2'P3' substituents had little effect on enzyme inhibition but greatly influenced the pharmacokinetic profile. As a result of these studies, the symmetrically acylated compound 8a and its close analog 24a bearing a methyl carbamate in P3 and an ethyl carbamate in P3' position were identified as potent inhibitors with plasma concentrations exceeding antiviral ED50 values 150-fold following oral application in mice.

New aza-dipeptide analogues as potent and orally absorbed HIV-1 protease inhibitors: candidates for clinical development.

On the basis of previously described X-ray studies of an enzyme/aza-dipeptide complex,8 aza-dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetrically acylated aza-dipeptides can be synthesized. This approach led to the discovery of very potent inhibitors with antiviral activities (ED50) in the subnanomolar range. Acylation of the (hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine derivatives. The bis(L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP 75176 combine excellent antiviral activity with high blood concentration after oral administration. Furthermore, they show no cross-resistance with saquinavir-resistant strains and maintain activity against indinavir-resistant ones. Consequently they qualify for further profiling as potential clinical candidates.

New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Preactivation of macrophages in mice acutely infected with Schistosoma mansoni.

This paper shows that peritoneal murine macrophages become preactivated in vivo during the course of a Schistosoma mansoni infection. Thus, less macrophage-activating factor (MAF) was required to induce in vitro tumoricidal and schistosomulicidal activity in macrophages from S. mansoni-infected mice than in macrophages from uninfected control animals. Moreover, the respiratory burst activity, as measured by chemiluminescence, was enhanced in macrophages from S. mansoni-infected mice as compared to controls, whether or not lymphokine (LK) was present in the macrophage cultures. This response appeared at 3 weeks and persisted at least until 12 weeks after infection. Interferon-gamma (IFN-gamma) is most likely involved in the mechanisms leading to such an increased cytolytic and oxidative activity, since in vitro experiments showed: 1) that less IFN-gamma was required to induce tumoricidal activity in macrophages from infected as compared to macrophages from uninfected animals, 2) that the activity of (2'-5')-adenylate synthetase (2'-5' A-synthetase), an enzyme strongly induced by IFN, was elevated in cells from livers of S. mansoni-infected mice.

[Measurement of mean pulmonary pressures and right systolic ventricular pressure by radiocardiography]. / Mesure des pressions pulmonaires moyennes et de la pression ventriculaire droite systolique par radiocardiographie

Computer exploitation of some parameters of the radiocardiogram and of the pressures measured by catheterization in a series of 678 subjects, studied both in Paris and in Prague, has made it possible to establish regression equations providing the rates of mean pulmonary arterial and wedge pressures together with the systolic right ventricular pressure on the basis of the radiocardiogram data. The latter was obtained by the conventional technique of the isotope dilution curves or by gamma-angiocardiography. Easy repetition of radiocardiography makes it an interesting investigation for the haemodynamic follow-up of the patients with heart disease.

AMN107 (nilotinib): a novel and selective inhibitor of BCR-ABL.

Chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukaemia (ALL) are caused by the BCR-ABL oncogene. Imatinib inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. However, accelerated or blast-crisis phase CML patients and Ph+ ALL patients often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase domain. This has stimulated the development of new kinase inhibitors that are able to over-ride resistance to imatinib. The novel, selective BCR-ABL inhibitor, AMN107, was designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib. In addition to being more potent than imatinib (IC50< 30 nM) against wild-type BCR-ABL, AMN107 is also significantly active against 32/33 imatinib-resistant BCR-ABL mutants. In preclinical studies, AMN107 demonstrated activity in vitro and in vivo against wild-type and imatinib-resistant BCR-ABL-expressing cells. In phase I/II clinical trials, AMN107 has produced haematological and cytogenetic responses in CML patients, who either did not initially respond to imatinib or developed imatinib resistance. Dasatinib (BMS-354825), which inhibits Abl and Src family kinases, is another promising new clinical candidate for CML that has shown good efficacy in CML patients. In this review, the early characterisation and development of AMN107 is discussed, as is the current status of AMN107 in clinical trials for imatinib-resistant CML and Ph+ ALL. Future trends investigating prediction of mechanisms of resistance to AMN107, and how and where AMN107 is expected to fit into the overall picture for treatment of early-phase CML and imatinib-refractory and late-stage disease are discussed.

Antiviral activity of tumour necrosis factor. Synergism with interferons and induction of oligo-2',5'-adenylate synthetase.

Tumour necrosis factor (TNF) induces antiviral activity in HEp-2 cells. Virus yield reduction assays with vesicular stomatitis virus as challenging virus demonstrated that the antiviral state was more pronounced in confluent cultures under low serum conditions. A significant enhancement of the antiviral state was obtained by combining TNF with low concentrations of either interferon (IFN)-beta 1 or IFN-gamma. The reduction in virus yield was significantly higher than that expected from summation of the independent antiviral activities of either substance alone, i.e. TNF and IFN acted synergistically as antiviral agents. Synergism of TNF with IFN-beta or IFN-gamma appeared to be mediated by different pathways, since different requirements for pretreatment and different effects on oligo-2',5'-adenylate synthetase (2-5AS) induction were observed. Induction of 2-5AS by TNF could be shown to be an indirect event that was sensitive to an antiserum against natural IFN-beta 1.

Heat-treated myosin does not bind ATPase--inhibiting antibodies.

Polyclonal antibodies to native chicken pectoral fast-twitch myosin are directed to all subfragments of the molecule (S1, S2 and LMM), as seen in the ELISA and Western blotting techniques. The antibodies inhibit the Ca(2+)-activated myosin ATPase. Absorption of the antibodies with native myosin abolishes these reactions. Heat treatment of myosin for 2h at 40 degrees C will inactivate myosin ATPase and alter its antibody binding pattern: the binding of antibodies to the rod fractions is reduced, that to the globular head (S1) completely abolished. Thus, these antibodies are useful as sensitive probes for the structural integrity of the myosin head.

Structural properties of myosin from the particulate fraction of human blood platelets.

Fractionation of human blood platelets has revealed that myosin, a contractile and mechanochemical protein, is present in both the soluble and particulate fraction. The aim of this study was to elucidate whether platelets contain more than one myosin isoform, especially in view of the fact that in other cellular systems (cardiac muscle, amoeba) several myosin isoenzymes were found. The particulate fraction was solubilized by Triton X-100, and the myosin was purified by the same procedure used for the cytoplasmic myosin. The final preparation contained, in addition to myosin, a 130-kDa polypeptide, which was observed also in myosin preparations obtained from the soluble fraction. The electrophoretic mobilities of the two myosins were identical under both dissociating and nondissociating conditions. Comparison of the molecular structure of the heavy chain of the two myosins by limited proteolysis with Staphylococcus aureus V8 protease showed that the proteolytic fragments of the two myosins were rather similar, with only minor alterations in the quantitative distribution of the products. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the map of the particulate myosin, as compared to its soluble counterpart. According to the two-dimensional peptide map, the 130-kDa polypeptide seems to be a proteolytic fragment of the myosin heavy chain and most probably the rod portion of the molecule. The observed minor variations in the structure of myosins isolated from the soluble and the fractions of human platelets may reflect differences in their respective physiological functions.

SH3GLB, a new endophilin-related protein family featuring an SH3 domain.

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.