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1.
J Natl Cancer Inst ; 92(20): 1641-50, 2000 Oct 18.
Article in English | MEDLINE | ID: mdl-11036109

ABSTRACT

BACKGROUND: Chronic myeloid leukemia is caused by a chromosomal translocation that results in an oncogenic fusion protein, Bcr-Abl. Bcr-Abl is a tyrosine kinase whose activity is inhibited by the antineoplastic drug STI571. This drug can cure mice given an injection of human leukemic cells, but treatment ultimately fails in animals that have large tumors when treatment is initiated. We created a mouse model to explore the mechanism of resistance in vivo. METHODS Nude mice were injected with KU812 Bcr-Abl(+) human leukemic cells. After 1 day (no evident tumors), 8 days, or 15 days (tumors >1 g), mice were treated with STI571 (160 mg/kg every 8 hours). Cells recovered from relapsing animals were used for in vitro experiments. Statistical tests were two-sided. RESULTS: Tumors regressed initially in all STI571-treated mice, but all mice treated 15 days after injection of tumor cells eventually relapsed. Relapsed animals did not respond to further STI571 treatment, and their Bcr-Abl kinase activity in vivo was not inhibited by STI571, despite high plasma concentrations of the drug. However, tumor cells from resistant animals were sensitive to STI571 in vitro, suggesting that a molecule in the plasma of relapsed animals may inactivate the drug. The plasma protein alpha1 acid glycoprotein (AGP) bound STI571 at physiologic concentrations in vitro and blocked the ability of STI571 to inhibit Bcr-Abl kinase activity in a dose-dependent manner. Plasma AGP concentrations were strongly associated with tumor load. Erythromycin competed with STI571 for AGP binding. When animals bearing large tumors were treated with STI571 alone or with a combination of STI571 and erythromycin, greater tumor reductions and better long-term tumor-free survival (10 of 12 versus one of 13 at day 180; P:<.001) were observed after the combination treatment. CONCLUSION: AGP in the plasma of relapsed animals binds to STI571, preventing this compound from inhibiting the Bcr/Abl tyrosine kinase. Molecules such as erythromycin that compete with STI571 for binding to AGP may enhance the therapeutic potential of this drug.


Subject(s)
Antineoplastic Agents/pharmacology , Fusion Proteins, bcr-abl/drug effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Orosomucoid/drug effects , Orosomucoid/metabolism , Piperazines/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Animals , Benzamides , Blotting, Western , Drug Resistance, Neoplasm , Drug Synergism , Drug Therapy, Combination , Enzyme Inhibitors/pharmacology , Erythromycin/pharmacology , Female , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Mice , Mice, Nude , Phosphorylation/drug effects , Time Factors , Tumor Cells, Cultured
3.
J Control Release ; 68(2): 291-8, 2000 Aug 10.
Article in English | MEDLINE | ID: mdl-10925137

ABSTRACT

The new chemical entity CGP 70726, a very poorly water-soluble HIV-1 protease inhibitor, was incorporated into pH-sensitive nanoparticles and microparticles made of the poly(methacrylic acid-co-ethylacrylate) copolymer Eudragit((R)) L100-55. The particles were characterized in terms of morphology, size distribution, drug loading, production yield and dispersion state of the drug inside the polymeric matrices. Aqueous dispersions of the particles were administered orally to Beagle dogs against a suspension of free drug (control formulation) all at a dose of 100 mg/kg. Oral administration was conducted in the absence and presence of food. Plasma concentrations and pharmacokinetic parameters were determined within 8 h post-dose. While no measurable absorption of the drug resulted after administration of the control formulation, substantial systemic exposure to the compound was obtained with both kinds of pH-sensitive formulations. The selective release of CGP 70726 in a highly dispersed/amorphous state and creation of high concentrations close to its absorption site was thought to account for this positive result. The largest areas under the plasma concentration-time curve (AUC) were obtained in the fasted state, with slightly better performance of the microparticles over the nanoparticles, in both nutritional states (7.8+/-1.5 versus 5.8+/-0. 8 micromol.h/l in the fasted state; 4.4+/-1.4 versus 2.00+/-0.5 micromol.h/l in the fed state). With these results, the potential of pH-sensitive particles for the oral delivery of HIV-1 protease inhibitors with low water solubility was confirmed.


Subject(s)
Biphenyl Compounds/administration & dosage , HIV Protease Inhibitors/administration & dosage , HIV Protease/drug effects , Administration, Oral , Animals , Biological Availability , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacokinetics , Dogs , Female , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacokinetics , Hydrogen-Ion Concentration , Male , Nutritional Status , Particle Size , Solubility , X-Ray Diffraction
4.
J Med Chem ; 43(12): 2310-23, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-10882357

ABSTRACT

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.


Subject(s)
Angiogenesis Inhibitors/chemical synthesis , Aniline Compounds/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Phthalazines/chemical synthesis , Pyridines , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Growth Factor/antagonists & inhibitors , Administration, Oral , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacokinetics , Angiogenesis Inhibitors/pharmacology , Aniline Compounds/chemistry , Aniline Compounds/pharmacokinetics , Aniline Compounds/pharmacology , Animals , Biological Availability , CHO Cells , Cell Line , Cricetinae , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Humans , Mice , Models, Molecular , Neoplasms/blood supply , Neovascularization, Pathologic , Phosphorylation , Phthalazines/chemistry , Phthalazines/pharmacokinetics , Phthalazines/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor , Structure-Activity Relationship , Transfection , Vascular Endothelial Growth Factor Receptor-1
5.
Cancer Res ; 60(8): 2178-89, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10786682

ABSTRACT

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.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Carcinoma/drug therapy , Carcinoma/pathology , Endothelial Growth Factors/antagonists & inhibitors , Lymphokines/antagonists & inhibitors , Neovascularization, Pathologic/drug therapy , Phthalazines , Pyridines , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Growth Factor/antagonists & inhibitors , Angiogenesis Inhibitors/adverse effects , Angiogenesis Inhibitors/blood , Angiogenesis Inhibitors/therapeutic use , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Carcinoma/blood supply , Cell Division/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Endothelial Growth Factors/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Hematopoiesis/drug effects , Humans , Kidney Neoplasms/blood supply , Kidney Neoplasms/drug therapy , Kidney Neoplasms/pathology , Leukocytes/cytology , Leukocytes/drug effects , Lymphokines/pharmacology , Mice , Mice, Nude , Neoplasm Metastasis/drug therapy , Neoplasm Metastasis/pathology , Neoplasm Transplantation , Neovascularization, Pathologic/pathology , Phosphorylation/drug effects , Platelet-Derived Growth Factor/antagonists & inhibitors , Platelet-Derived Growth Factor/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Wound Healing/drug effects
6.
Int J Cancer ; 81(4): 669, 1999 May 17.
Article in English | MEDLINE | ID: mdl-10336342
7.
J Med Chem ; 41(18): 3387-401, 1998 Aug 27.
Article in English | MEDLINE | ID: mdl-9719591

ABSTRACT

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.


Subject(s)
Anti-HIV Agents , Aza Compounds , Dipeptides , HIV Protease Inhibitors , HIV Protease/metabolism , Administration, Oral , Animals , Anti-HIV Agents/administration & dosage , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Aza Compounds/administration & dosage , Aza Compounds/chemical synthesis , Aza Compounds/pharmacology , Biological Availability , Dipeptides/administration & dosage , Dipeptides/chemical synthesis , Dipeptides/pharmacology , Drug Evaluation, Preclinical , Drug Resistance, Microbial , Female , HIV Protease Inhibitors/administration & dosage , HIV Protease Inhibitors/chemical synthesis , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , HIV-1/enzymology , HIV-1/physiology , Indinavir/pharmacology , Mice , Mice, Inbred BALB C , Saquinavir/pharmacology , Structure-Activity Relationship , Virus Replication/drug effects
8.
Int J Cancer ; 76(1): 154-63, 1998 Mar 30.
Article in English | MEDLINE | ID: mdl-9533776

ABSTRACT

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.


Subject(s)
Neoplasms/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrroles/pharmacology , 3T3 Cells , Animals , Cell Division/drug effects , Enzyme Inhibitors , Epithelial Cells , ErbB Receptors/metabolism , Female , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Neoplasms/pathology , Phosphorylation , Pyrimidines/chemistry , Pyrroles/chemistry , Time Factors
9.
Schweiz Med Wochenschr ; 126(43): 1849-51, 1996 Oct 26.
Article in English | MEDLINE | ID: mdl-8916294

ABSTRACT

CGP 61755 is a novel hydroxyethylene derivative produced by a high yield 10 step chemical synthesis. It is highly specific for HIV-1 protease with an IC50 of 1 nM. The ED90 in MT-2, PBLs and macrophages is infected with laboratory strains of HIV-1 or clinical isolates is 30-100 nM. In chronically infected macrophages the ED90 is 1000 nM (1000 nM for saquinavir and 10 microM for indinavir). When the antiviral activity of CGP 61755 on HIV-1 infected lymphocytes was examined using serum free medium an ED99 of 60 nM was determined, while in the presence of 10% human serum the same activity was achieved with 120 nM. When examined in combination with RT inhibitors or protease inhibitors, either in a co-culture of CEM-SS and chronically infected H9IIIB cells or in a free virus lymphocyte infection, cooperativity of the antiviral activities was observed. Dog pharmacokinetic studies comparing p.o. and i.v. data indicate that CGP 61755 has a bioavailability between 50 and 80%. Following oral administration the area under the concentration curve (AUC) values increased in a dose proportional manner. The plasma levels of the drug at 6 hours after oral administration were above the ED90. Based on these properties we believe that CGP 61755 has an attractive profile that justifies further preclinical evaluation of the drug.


Subject(s)
Anti-HIV Agents/chemical synthesis , Ethylenes/chemical synthesis , HIV Protease Inhibitors/chemical synthesis , Virus Replication/drug effects , Animals , Anti-HIV Agents/pharmacokinetics , Blood Proteins/metabolism , Dogs , Ethylenes/pharmacokinetics , HIV Protease Inhibitors/pharmacokinetics , HIV-1/drug effects , HIV-1/enzymology , Humans , In Vitro Techniques , Mice , Mice, Inbred BALB C , Protein Binding
10.
J Med Chem ; 39(16): 3203-16, 1996 Aug 02.
Article in English | MEDLINE | ID: mdl-8759643

ABSTRACT

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.


Subject(s)
Amino Acids/chemical synthesis , Antiviral Agents/chemical synthesis , HIV Protease Inhibitors/chemical synthesis , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Hydrazines/chemical synthesis , Administration, Oral , Amino Acid Sequence , Amino Acids/administration & dosage , Amino Acids/pharmacokinetics , Amino Acids/pharmacology , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Biological Availability , Cells, Cultured , Female , HIV Protease/metabolism , HIV Protease Inhibitors/administration & dosage , HIV Protease Inhibitors/pharmacokinetics , HIV-1/enzymology , Hydrazines/administration & dosage , Hydrazines/pharmacokinetics , Hydrazines/pharmacology , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Molecular Structure , Structure-Activity Relationship
11.
Arch Pharm (Weinheim) ; 329(6): 273-8, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8767110

ABSTRACT

A series of potent HIV-protease inhibitors has been prepared. Several of the newly synthesized compounds showed high plasma even after oral administration to animals. Based on the overall biological profile, CGP 61755 was chosen for further preclinical evaluation. For this compound, a 10 step synthesis potentially suitable for large scale production was developed.


Subject(s)
HIV Protease Inhibitors/chemical synthesis , Administration, Oral , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , HIV Protease Inhibitors/pharmacology , Molecular Structure , Renin/antagonists & inhibitors , Structure-Activity Relationship
13.
J Pharm Sci ; 84(12): 1387-91, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8748318

ABSTRACT

CGP 57813 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease. This lipophilic compound was successfully entrapped into poly(D,L-lactic acid) (PLA) and pH sensitive methacrylic acid copolymers nanoparticle. The intravenous administration to mice of PLA nanoparticles loaded with CGP 57813 resulted in a 2-fold increase of the area under the plasma concentration-time curve, compared to a control solution. An increase in the elimination half-life (from 13 to 61 min) and in the apparent volume of distribution (1.7-3.6 L/kg) was observed for the nanoparticle incorporated compound vs control solution. Following oral administration, only nanoparticles made of the methacrylic acid copolymer soluble at low pH provided sufficient plasma levels of CGP 57813. In vitro, these nanoparticles dissolved completely within 5 min at pH 5.8. PLA nanoparticles, which are insoluble in the gastrointestinal tract, did not provide significant plasma concentrations of CGP 57813. From these observations, one can conclude that the passage of intact PLA nanoparticles across the gastrointestinal mucosa appears to be very low.


Subject(s)
Carbamates/administration & dosage , Carbamates/pharmacokinetics , Drug Delivery Systems , HIV Protease Inhibitors/administration & dosage , HIV Protease Inhibitors/pharmacokinetics , Lactic Acid , Oligopeptides/administration & dosage , Oligopeptides/pharmacokinetics , Acrylic Resins/chemistry , Administration, Oral , Animals , Biological Availability , Delayed-Action Preparations , Drug Carriers , Half-Life , Injections, Intravenous , Lactates , Mice , Mice, Inbred BALB C , Polyesters , Polymers , Polymethacrylic Acids/chemistry
14.
Antimicrob Agents Chemother ; 37(10): 2087-92, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8257128

ABSTRACT

CGP 53437 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease containing a hydroxyethylene isostere. The compound inhibited recombinant HIV-1 protease with a Ki of 0.2 nM. The inhibition constant versus human cathepsin D and human cathepsin E was 4 nM. Human pepsin and gastricsin were inhibited with Kis of 8 and 500 nM, respectively, and human renin was inhibited with a Ki of 190 microM. The replication of HIV-1/LAV, HIV-1/Z-84, and HIV-1/pLAI was inhibited with a 90% effective dose of 0.1 microM in acutely infected MT-2 cells. The 50% cytotoxic dose was 100 microM. Similar antiviral activity was observed when the compound was added up to 10 h after infection. At the effective concentration, processing of Gag precursor protein p55 was greatly reduced, confirming an action on the late stage of the virus life cycle, as expected. The efficacy of the inhibitor was also demonstrated by using primary human peripheral blood lymphocytes infected with the HIV-1/LAV strain, low-passage clinical isolates obtained from HIV-1-seropositive individuals (including a zidovudine-resistant strain), and HIV-2/ROD. In these cells, CGP 53437 delayed the onset of HIV replication in a dose-dependent fashion (substantial effects with concentrations of > or = 0.1 microM) as long as the inhibitor was maintained in the culture. CGP 53437 was orally bioavailable in mice. Concentrations in plasma 10-fold in excess of the in vitro antiviral 90% effective dose could be sustained for several hours after oral application of 120 mg/kg. Therefore, CGP 53437 has the potential to be a therapeutically useful anti-HIV agent for the treatment of AIDS.


Subject(s)
Antiviral Agents/pharmacology , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Morpholines/pharmacology , Oligopeptides/pharmacology , Acquired Immunodeficiency Syndrome/drug therapy , Acquired Immunodeficiency Syndrome/enzymology , Administration, Oral , Amino Acid Sequence , Animals , Antiviral Agents/pharmacokinetics , Biological Availability , Drug Resistance, Microbial , Female , HIV Protease/drug effects , HIV Protease Inhibitors/pharmacokinetics , HIV-1/enzymology , HIV-1/physiology , HIV-2/drug effects , HIV-2/physiology , Humans , Lymphocytes/microbiology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Morpholines/pharmacokinetics , Oligopeptides/pharmacokinetics , Virus Replication/drug effects , Zidovudine/pharmacology
15.
Eur J Biochem ; 207(1): 265-75, 1992 Jul 01.
Article in English | MEDLINE | ID: mdl-1321046

ABSTRACT

The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor alpha. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 61 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 microM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min-1 mol-1. Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20 degrees C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of Me(2+)-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.


Subject(s)
ErbB Receptors/isolation & purification , Protein-Tyrosine Kinases/isolation & purification , Protein-Tyrosine Kinases/metabolism , Angiotensin II/pharmacology , Animals , Baculoviridae/genetics , Base Sequence , Cell Line , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Gene Expression , Humans , Insecta , Kinetics , Magnesium/pharmacology , Manganese/pharmacology , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides , Protein-Tyrosine Kinases/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Transfection
16.
Antimicrob Agents Chemother ; 33(11): 1864-8, 1989 Nov.
Article in English | MEDLINE | ID: mdl-2482011

ABSTRACT

The murine model of acquired immunodeficiency disease was used to evaluate both the antiretroviral and antiherpetic activities of the acyclic nucleotide analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA). The antiretroviral activity of PMEA was compared with that of azidothymidine (AZT) in mice receiving the drug either immediately after infection or at late times in disease progression. Both AZT (oral, 30 mg/kg) and PMEA (parenteral, 25 and 5 mg/kg) were effective in slowing the development of disease when administered daily beginning on the day of infection. In contrast, neither drug alone was effective in modifying disease outcome when administered several weeks after viral infection. Human recombinant alpha interferon (rhuIFN alpha-B/D at 5 x 10(7) U/kg) was also ineffective when administered late in the course of disease. However, when administered in combination, both alpha interferon and PMEA (25 mg/kg) were able to suppress disease progression even when treatment was initiated as late as 3 weeks postinfection. Mice that were immunocompromised due to LP-BM5 virus infection were highly susceptible to acute (lethal) infection with herpes simplex virus type 1, whereas their immunocompetent littermates were not. PMEA was as effective as acyclovir in the treatment of opportunistic herpes simplex virus type 1 infections in LP-BM5 virus-infected mice. Thus, like AZT, PMEA was effective against retrovirus infection, and, like acyclovir, PMEA was effective against herpes simplex virus type 1 infection. This gives PMEA the unique potential of being useful in the treatment of opportunistic herpes simplex virus infections as well as the underlying retroviral disease.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Adenine/analogs & derivatives , Antiviral Agents/therapeutic use , Herpes Simplex/drug therapy , Opportunistic Infections/drug therapy , Organophosphonates , Acquired Immunodeficiency Syndrome/complications , Adenine/therapeutic use , Animals , Concanavalin A/pharmacology , Herpes Simplex/complications , Interferons/pharmacology , Mice , Mice, Inbred C57BL , Mitogens , Opportunistic Infections/complications , Spleen/cytology , Zidovudine/pharmacology
17.
Antimicrob Agents Chemother ; 33(10): 1819-21, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2574024

ABSTRACT

The carbapenem imipenem and the penem CGP 31608 demonstrated unusually good bactericidal activity against slowly growing bacteria. In contrast to that of penicillin, the rate of killing was independent of growth rate. In logarithmically growing cells, a decrease in growth rate was paralleled by a decrease in the relative activity of only one of four autolysins measured (membrane-bound endopeptidase), suggesting that autolysis induced by penicillin G may be rate limited by this enzyme. Imipenem, on the other hand, appeared to trigger different autolysins in Escherichia coli, as evidenced by differences in the structure of peptidoglycan after imipenem- versus penicillin-induced autolysis.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Imipenem/pharmacology , Lactams , Autolysis , Bacteria/growth & development , Endopeptidases/metabolism , Escherichia coli/drug effects , Escherichia coli/growth & development , Escherichia coli/metabolism , Microbial Sensitivity Tests , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Penicillin G/pharmacology , Peptidoglycan/metabolism
19.
J Bacteriol ; 169(11): 5308-10, 1987 Nov.
Article in English | MEDLINE | ID: mdl-3312172

ABSTRACT

The composition of peptidoglycan of chemostat-grown cultures of Escherichia coli was investigated as a function of growth rate. As the generation time was lengthened from 0.8 to 13.8 h, there was a decrease in the major monomer (disaccharide tetrapeptide) and dimer (bis-disaccharide tetrapeptide), while disaccharide tripeptide moieties increased to greater than 50% of the total wall. The average chain length became much shorter; lipoprotein density tripled, and the number of unusual diaminopimelyl-diaminopimelic acid crossbridges increased fivefold. As cells grew more slowly, amounts of penicillin-binding proteins (PBPs) 1a-1b complex and 4 decreased, while amounts of PBPs 3 and the 5-6 complex increased. We propose that the chemical composition of E. coli cell walls changes with growth rate in a manner consistent with alterations in the activities of PBPs and cell shape.


Subject(s)
Bacterial Proteins , Carrier Proteins/biosynthesis , Escherichia coli/growth & development , Hexosyltransferases , Muramoylpentapeptide Carboxypeptidase/biosynthesis , Peptidoglycan , Peptidyl Transferases , Chromatography, High Pressure Liquid , Escherichia coli/metabolism , Kinetics , Penicillin-Binding Proteins , Peptidoglycan/isolation & purification
20.
J Gen Microbiol ; 132(5): 1297-304, 1986 May.
Article in English | MEDLINE | ID: mdl-3534137

ABSTRACT

Nongrowing bacteria evade the bactericidal activity of beta-lactam antibiotics. We sought to determine if slow growth rate also alters bactericidal activity. The bactericidal activity of two beta-lactams on Escherichia coli grown in glucose limited chemostats was compared for generation times ranging from 0.7 to 12 h. The degree of killing varied with drug structure and with E. coli strain. However, all killing rates were a constant function of the bacterial generation time: slowly growing bacteria became progressively more phenotypically tolerant to beta-lactam antibiotics as the generation time was extended.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins , Escherichia coli/drug effects , Hexosyltransferases , Peptidyl Transferases , Carrier Proteins/analysis , Cefamandole/analogs & derivatives , Cefamandole/pharmacology , Cefonicid , Cell Wall/drug effects , Escherichia coli/growth & development , Muramoylpentapeptide Carboxypeptidase/analysis , Penicillin G/pharmacology , Penicillin Resistance , Penicillin-Binding Proteins
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