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1.
Viruses ; 13(12)2021 12 14.
Article in English | MEDLINE | ID: mdl-34960780

ABSTRACT

Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5'-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5'-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.


Subject(s)
Antimetabolites/pharmacology , Antiviral Agents/pharmacology , RNA Viruses/drug effects , Adenosine Triphosphate/metabolism , Amides/pharmacology , Animals , Cell Line , Drug Synergism , Guanosine Triphosphate/metabolism , Humans , Methylthioinosine/pharmacology , Mutation/drug effects , Phosphoribosyl Pyrophosphate/metabolism , Pyrazines/pharmacology , RNA Viruses/classification , RNA Viruses/genetics , RNA, Viral/drug effects , RNA, Viral/genetics , Virus Replication/drug effects
2.
Biomed Pharmacother ; 108: 618-624, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30243096

ABSTRACT

Colon cancer is one of the most common digestive malignant tumors that leads to high mortality worldwide, and metastasis is the primary cause of cancer-related death. It is well accepted that the epithelial-mesenchymal transition (EMT) plays a key role in the process of metastasis. As a cytokine that macrophage secretes, IL-6 is involved in the progression of tumors, including the invasion and metastasis via kinds of signaling pathways. However, the mechanism of interactions between IL-6, macrophage, EMT and colon cancer is not fully understood. Increased CD68+ macrophages and IL-6 level were found in colon tumor as compared to normal colon tissue. Metastatic lymph node showed even more CD68+ macrophages and higher IL-6 level than the primary tumor. These results suggested that macrophages and IL-6 play an important role in EMT of colon cancer. In order to investigate the effect of macrophage and IL-6 on EMT of colon cancer, we cultured human colon carcinoma cell line SW48 with conditioned medium (CM) from PMA-stimulated monocyte THP-1 cells and tested for IL-6 dependent EMT pathways. Wound healing assay and Transwell assay were used to analyze cell migration and invasion. Results showed that CM-treated SW48 cells increased IL-6 production and displayed elevated capacity of migration and invasion compared to untreated cells. Increased expressions of EMT markers (N-cadherin, Vimentin and ß-catenin) and decreased expression of EMT marker(E-cadherin) were found in CM-treated SW48 cells by Western Blot. The addition of an anti-IL-6 antibody significantly inhibited the increase of EMT markers (Vimentin and ß-catenin) as well as cell migration and invasion, suggesting that IL-6 played a critical role in promoting EMT of CM-treated SW48 cells. In addition, we found that the levels of p-STAT3 and p-ERK increased in CM-treated SW48 compared to untreated cells, which can be reversed by AG490, an inhibitor of JAK. In the meantime, the suppression of JAK-associated signaling pathways caused a decrease of ß-catenin. In summary, our study suggested that macrophage-induced IL-6 promotes migration and invasion of colon cancer cell via Wnt/ß-catenin pathway in STAT3/ERK-dependent way.


Subject(s)
Colonic Neoplasms/metabolism , Epithelial-Mesenchymal Transition/physiology , Interleukin-6/metabolism , MAP Kinase Signaling System/physiology , STAT3 Transcription Factor/metabolism , THP-1 Cells/metabolism , Wnt Signaling Pathway/physiology , beta Catenin/metabolism , 6-Aminonicotinamide/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Aspartic Acid/analogs & derivatives , Aspartic Acid/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/physiology , Epithelial-Mesenchymal Transition/drug effects , Humans , MAP Kinase Signaling System/drug effects , Macrophages/drug effects , Macrophages/metabolism , Methylthioinosine/pharmacology , THP-1 Cells/drug effects , Wnt Signaling Pathway/drug effects
3.
Int J Antimicrob Agents ; 50(6): 718-725, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28803932

ABSTRACT

Since the emergence of Zika virus (ZIKV) in Brazil in 2015, 48 countries and territories in the Americas have confirmed autochthonous cases of disease caused by the virus. ZIKV-associated neurological manifestations and congenital defects make the development of safe and effective antivirals against ZIKV of utmost importance. Here we evaluated the antiviral activity of 6-methylmercaptopurine riboside (6MMPr), a thiopurine nucleoside analogue derived from the prodrug azathioprine, against the epidemic ZIKV strain circulating in Brazil. In all of the assays, an epithelial (Vero) and a human neuronal (SH-SY5Y) cell line were used to evaluate the cytotoxicity and effective concentrations of 6MMPr against ZIKV. Levels of ZIKV-RNA, viral infectious titre and the percentage of infected cells in the presence or absence of 6MMPr were used to determine antiviral efficacy. 6MMPr decreased ZIKV production by >99% in both cell lines in a dose- and time-dependent manner. Interestingly, 6MMPr was 1.6 times less toxic to SH-SY5Y cells compared with Vero cells, presenting a 50% cytotoxic concentrations (CC50) of 460.3 µM and 291 µM, respectively. The selectivity index of 6MMPr for Vero and SH-SY5Y cells was 11.9 and 22.7, respectively, highlighting the safety profile of the drug to neuronal cells. Taken together, these results identify, for the first time, the thiopurine nucleoside analogue 6MMPr as a promising antiviral candidate against ZIKV that warrants further in vivo evaluation.


Subject(s)
Antiviral Agents/pharmacology , Methylthioinosine/pharmacology , Virus Replication/drug effects , Zika Virus/drug effects , Animals , Antiviral Agents/toxicity , Brazil , Cell Line , Cell Survival/drug effects , Epithelial Cells/drug effects , Epithelial Cells/physiology , Humans , Methylthioinosine/toxicity , Neurons/drug effects , Neurons/physiology , Zika Virus/isolation & purification , Zika Virus/physiology , Zika Virus Infection/virology
4.
Virol J ; 14(1): 124, 2017 06 26.
Article in English | MEDLINE | ID: mdl-28651549

ABSTRACT

BACKGROUND: Canine distemper (CD) is a widespread infectious disease that can severely impact a variety of species in the order Carnivora, as well as non-carnivore species such as non-human primates. Despite large-scale vaccination campaigns, several fatal outbreaks have been reported in wild and domestic carnivore populations. This, in association with expansion of the disease host range and the development of vaccine-escape strains, has contributed to an increased demand for therapeutic strategies synergizing with vaccine programs for effectively controlling canine distemper. 6-methylmercaptopurine riboside (6MMPr) is a modified thiopurine nucleoside with known antiviral properties against certain RNA viruses. METHODS: We tested the inhibitory effects of 6MMPr against a wild-type CDV strain infection in cell culture. We measured infectious particle production and viral RNA levels in treated and untreated CDV-infected cells. Ribavirin (RIB) was used as a positive control. RESULTS: Here, we report for the first time the antiviral effects of 6MMPr against canine distemper virus (CDV) in vitro. 6MMPr was able to reduce viral RNA levels and to inhibit the production of infectious CDV particles. The therapeutic selectivity of 6MMPr was approximately six times higher than that of ribavirin. CONCLUSION: Our results indicate that 6MMPr has high anti-CDV potential and warrants further testing against other paramyxoviruses, as well as clinical testing of the compound against CDV.


Subject(s)
Antiviral Agents/pharmacology , Distemper Virus, Canine/drug effects , Distemper Virus, Canine/physiology , Methylthioinosine/pharmacology , Microbial Viability/drug effects , Animals , Cell Line , Dogs
5.
Mar Drugs ; 12(2): 999-1012, 2014 Feb 14.
Article in English | MEDLINE | ID: mdl-24534843

ABSTRACT

A new actinomycete strain Micromonospora sp. K310 was isolated from Ghanaian mangrove river sediment. Spectroscopy-guided fractionation led to the isolation of two new compounds from the fermentation culture. One of the compounds is butremycin (2) which is the (3-hydroxyl) derivative of the known Streptomyces metabolite ikarugamycin (1) and the other compound is a protonated aromatic tautomer of 5'-methylthioinosine (MTI) (3). Both new compounds were characterized by 1D, 2D NMR and MS data. Butremycin (2) displayed weak antibacterial activity against Gram-positive S. aureus ATCC 25923, the Gram-negative E. coli ATCC 25922 and a panel of clinical isolates of methicillin-resistant S. aureus (MRSA) strains while 3 did not show any antibacterial activity against these microbes.


Subject(s)
Anti-Bacterial Agents/pharmacology , Lactams, Macrocyclic/pharmacology , Methylthioinosine/analogs & derivatives , Micromonospora/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Escherichia coli/drug effects , Fermentation , Geologic Sediments/microbiology , Ghana , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/isolation & purification , Magnetic Resonance Spectroscopy , Mass Spectrometry , Methicillin-Resistant Staphylococcus aureus/drug effects , Methylthioinosine/chemistry , Methylthioinosine/isolation & purification , Methylthioinosine/pharmacology , Microbial Sensitivity Tests , Rivers/microbiology , Staphylococcus aureus/drug effects
6.
PLoS One ; 6(10): e26697, 2011.
Article in English | MEDLINE | ID: mdl-22039536

ABSTRACT

Many viruses within the Flavivirus genus cause significant disease in humans; however, effective antivirals against these viruses are not currently available. We have previously shown that a thiopurine drug, 6-methylmercaptopurine riboside (6MMPr), inhibits replication of distantly related viruses within the Flaviviridae family in cell culture, including bovine viral diarrhea virus and hepatitis C virus replicon. Here we further examined the potential antiviral effect of 6MMPr on several diverse flaviviruses. In cell culture, 6MMPr inhibited virus production of yellow fever virus, dengue virus-2 (DENV-2) and West Nile virus (WNV) in a dose-dependent manner, and DENV-2 was significantly more sensitive to 6MMPr treatment than WNV. We then explored the use of 6MMPr as an antiviral against WNV in an immunocompetent mouse model. Once a day treatment of mice with 0.5 mg 6MMPr was just below the toxic dose in our mouse model, and this dose was used in subsequent studies. Mice were treated with 6MMPr immediately after subcutaneous inoculation with WNV for eight consecutive days. Treatment with 6MMPr exacerbated weight loss in WNV-inoculated mice and did not significantly affect mortality. We hypothesized that 6MMPr has low bioavailability in the central nervous system (CNS) and examined the effect of pre-treatment with 6MMPr on viral loads in the periphery and CNS. Pre-treatment with 6MMPr had no significant effect on viremia or viral titers in the periphery, but resulted in significantly higher viral loads in the brain, suggesting that the effect of 6MMPr is tissue-dependent. In conclusion, despite being a potent inhibitor of flaviviruses in cell culture, 6MMPr was not effective against West Nile disease in mice; however, further studies are warranted to reduce the toxicity and/or improve the bioavailability of this potential antiviral drug.


Subject(s)
Antiviral Agents/pharmacology , Methylthioinosine/pharmacology , Virus Replication/drug effects , West Nile virus/drug effects , Animals , Cells, Cultured , Chlorocebus aethiops , Cricetinae , Mice , Vero Cells , West Nile virus/physiology
7.
J Gen Virol ; 89(Pt 4): 1000-1009, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18343842

ABSTRACT

The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine (AZA) has in vitro activity against bovine viral diarrhea virus (BVDV). While the mechanism of inhibition is unknown, AZA and related thiopurine nucleoside analogues have been used as immunosuppressants for decades and both AZA metabolites and cellular genes involved in AZA metabolism have been extensively characterized. Here, we show that only certain riboside metabolites have antiviral activity and identify the most potent known antiviral AZA metabolite as 6-methylmercaptopurine riboside (6MMPr). The antiviral activity of 6MMPr is antagonized by adenosine, and is specific to BVDV and not to the related yellow fever virus. An essential step in the conversion of AZA to 6MMPr is the addition of a methyl group onto the sulfur atom attached to position six of the purine ring. Intracellularly, the methyl group is added by thiopurine methyltransferase (TPMT), an S-adenosyl methionine-dependent methyltransferase. Either chemically bypassing or inhibiting TPMT modulates antiviral activity of AZA metabolites. TPMT exists in several variants with varying levels of activity and since 6MMPr is a potent antiviral, the antiviral activity of AZA may be modulated by host genetics.


Subject(s)
Azathioprine/pharmacology , Bovine Virus Diarrhea-Mucosal Disease/drug therapy , Diarrhea Viruses, Bovine Viral/drug effects , Methylthioinosine/pharmacology , Methyltransferases/pharmacology , Adenosine/pharmacology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Azathioprine/metabolism , Bovine Virus Diarrhea-Mucosal Disease/virology , Cattle , Cell Line , Diarrhea Viruses, Bovine Viral/physiology , Methylthioinosine/antagonists & inhibitors , Methylthioinosine/metabolism , Methyltransferases/metabolism , Virus Replication/drug effects
8.
Molecules ; 10(8): 1015-20, 2005 Aug 31.
Article in English | MEDLINE | ID: mdl-18007371

ABSTRACT

6-Methylpurine-beta-D-riboside (beta-D-MPR) has been synthesized by coupling 6-methylpurine and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose using conditions that produce the beta-D-anomer exclusively. The in vitro antitumor effects of beta-D-MPR and 6-methyl-purine-alpha-D-riboside (alpha-D-MPR) in five human tumor cell lines showed that beta-D-MPR was highly active (IC(50) values ranging from 6 to 34 nM). alpha-D-MPR, although less active than beta-D-MPR, also exhibited significant antitumor effects (IC50 values ranging from 1.47 to 4.83 microM).


Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Methylthioinosine/chemical synthesis , Methylthioinosine/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Methylthioinosine/chemistry , Stereoisomerism
9.
Oncol Res ; 14(7-8): 373-9, 2004.
Article in English | MEDLINE | ID: mdl-15301428

ABSTRACT

The aim of this study was to examine the expression of methylthioadenosine phosphorylase (MTAP) in 21 fresh tumor samples from patients with soft tissue sarcomas (STS) and 11 human soft tissue sarcoma cell lines, and to determine if loss of expression of this enzyme was correlated with increased sensitivity to L-alanosine and/or 6-methylmercaptopurine. We used a polyclonal antibody to measure the expression of MTAP in soft tissue sarcoma cell lines and in fresh tumor samples. Transfection of the HT-1080 cell line with a plasmid containing the cDNA for the MTAP gene was also performed to generate cell lines for in vitro and in vivo comparative sensitivity studies. MTAP was not expressed in 8 of 21 fresh STS tumors. The expression of MTAP was also not detectable in 3 of the 11 soft tissue sarcoma cell lines (HT-1080, HS42, and M-9 110). These three cell lines were more sensitive to L-alanosine, a potent inhibitor of de novo AMP synthesis, and to an inhibitor of de novo purine nucleotide synthesis, 6-methylmercaptopurine riboside (MMPR). The IC50 values for L-alanosine and MMPR were >20-fold lower in MTAP-deficient cells than in MTAP-positive cells. Restoration of MTAP into HT-1080 MTAP-deficient cells also led to decreased sensitivity to L-alanosine and MMPR. An in vivo study using HT-1080 cell tumors with and without MTAP expression confirmed that tumors lacking MTAP were more sensitive to L-alanosine than tumors expressing MTAP. These results provide the basis for selective therapy using inhibitors of de novo purine nucleotide synthesis such as L-alanosine or MMPR to treat patients with STS lacking this enzyme.


Subject(s)
Alanine/pharmacology , Antibiotics, Antineoplastic/pharmacology , Gene Expression Regulation, Neoplastic , Methylthioinosine/pharmacology , Purine-Nucleoside Phosphorylase/biosynthesis , Purine-Nucleoside Phosphorylase/pharmacology , Sarcoma/genetics , Sarcoma/physiopathology , Alanine/analogs & derivatives , DNA, Complementary , Drug Screening Assays, Antitumor , Humans , Transfection , Tumor Cells, Cultured
10.
Mol Pharmacol ; 62(6): 1321-31, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12435799

ABSTRACT

Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.


Subject(s)
Mercaptopurine/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Ribosomal Proteins/metabolism , Thioguanine/metabolism , Biological Transport , Cells, Cultured , Chromatography, High Pressure Liquid , Drug Interactions , Humans , Kidney/cytology , Kidney/embryology , Kinetics , Mercaptopurine/pharmacology , Methylthioinosine/pharmacology , Multidrug Resistance-Associated Proteins/biosynthesis , Ribosomal Proteins/biosynthesis , Thioguanine/pharmacology , Transfection
11.
Cancer Res ; 59(10): 2417-24, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10344752

ABSTRACT

Angiogenesis has been identified as an important target for antineoplastic therapy. The use of purine analogue antimetabolites in combination chemotherapy of solid tumors has been proposed. To assess the possibility that selected purine analogues may affect tumor neovascularization, 6-methylmercaptopurine riboside (6-MMPR), 6-methylmercaptopurine, 2-aminopurine, and adenosine were evaluated for the capacity to inhibit angiogenesis in vitro and in vivo. 6-MMPR inhibited fibroblast growth factor-2 (FGF2)-induced proliferation and delayed the repair of mechanically wounded monolayer in endothelial GM 7373 cell cultures. 6-MMPR also inhibited the formation of solid sprouts within fibrin gel by FGF2-treated murine brain microvascular endothelial cells and the formation of capillary-like structures on Matrigel by murine aortic endothelial cells transfected with FGF2 cDNA. 6-MMPR affected FGF2-induced intracellular signaling in murine aortic endothelial cells by inhibiting the phosphorylation of extracellular signal-regulated kinase-2. The other molecules were ineffective in all of the assays. In vivo, 6-MMPR inhibited vascularization in the chick embryo chorioallantoic membrane and prevented blood vessel formation induced by human endometrial adenocarcinoma specimens grafted onto the chorioallantoic membrane. Also, topical administration of 6-MMPR caused the regression of newly formed blood vessels in the rabbit cornea. Thus, 6-MMPR specifically inhibits both the early and the late phases of the angiogenesis process in vitro and exerts a potent anti-angiogenic activity in vivo. These results provide a new rationale for the use of selected purine analogues in combination therapy of solid cancer.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Methylthioinosine/pharmacology , Neovascularization, Physiologic/drug effects , 2-Aminopurine/pharmacology , Adenocarcinoma/blood supply , Adenosine/pharmacology , Allantois/blood supply , Allantois/drug effects , Animals , Antineoplastic Agents/pharmacology , Aorta/cytology , Brain/blood supply , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cattle , Cells, Cultured , Chick Embryo , Chorion/blood supply , Chorion/drug effects , Cornea/blood supply , Cornea/drug effects , Depression, Chemical , Endometrial Neoplasms/blood supply , Endothelium, Vascular/drug effects , Female , Fibroblast Growth Factor 2/antagonists & inhibitors , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/pharmacology , Humans , Mercaptopurine/analogs & derivatives , Mercaptopurine/pharmacology , Mice , Mitogen-Activated Protein Kinase 1 , Neoplasm Transplantation , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Rabbits , Recombinant Fusion Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Transfection
12.
Invest New Drugs ; 15(2): 139-45, 1997.
Article in English | MEDLINE | ID: mdl-9220293

ABSTRACT

The results of several clinical trials support the hypothesis that biochemical modulation may enhance the antitumor activity of 5-Fluorouracil (5-FU). We have performed a phase I trial using a combination of three different biochemical modulators at the optimal dose established in previous clinical trials. The modulators include: phosphonacetyl-l-aspartate (PALA), which may increase 5-FU incorporation into RNA; leucovorin, which potentiates thymidylate synthase inhibition; and 6-methylmercaptopurine riboside (MMPR), which promotes the intracellular retention of fluorinated nucleotides. The treatment regimen consisted of PALA 250 mg/m2 day 1, followed 24 h later by MMPR 150 mg/m2 as an iv bolus, and the initiation of a 24-hour infusion of 5-FU along with leucovorin 50 mg/m2. This regimen was repeated weekly. Doses of 5-FU were escalated in cohorts of four or more patients from 2,000 to 2,600 mg/m2. Among 20 patients entered, the majority had colorectal cancer, and most had received prior 5-FU treatment. Toxicity was predominantly gastrointestinal, and diarrhea was dose-limiting at a 5-FU dose of 2600 mg/m2. There were three partial remissions observed, two of whom had colorectal cancer. Emerging data that casts doubt on the modulation value of PALA at this dose and schedule suggests that revision of this regimen be considered before Phase II trial.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Aspartic Acid/analogs & derivatives , Fluorouracil/therapeutic use , Leucovorin/pharmacology , Methylthioinosine/pharmacology , Phosphonoacetic Acid/analogs & derivatives , Adult , Aged , Antimetabolites, Antineoplastic/administration & dosage , Aspartic Acid/administration & dosage , Aspartic Acid/pharmacology , Colorectal Neoplasms/drug therapy , Drug Interactions , Female , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Fluorouracil/pharmacokinetics , Humans , Leucovorin/administration & dosage , Male , Methylthioinosine/administration & dosage , Middle Aged , Phosphonoacetic Acid/administration & dosage , Phosphonoacetic Acid/pharmacology
14.
Mol Biol Cell ; 4(1): 71-8, 1993 Jan.
Article in English | MEDLINE | ID: mdl-7680248

ABSTRACT

Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.


Subject(s)
Protein Kinase C , Protein Kinases/metabolism , Receptors, Nerve Growth Factor/metabolism , 2-Aminopurine/pharmacology , Animals , Cell Line , Methylthioinosine/pharmacology , Myelin Basic Protein/metabolism , Phosphorylation , Rats , Thioguanine/pharmacology
15.
J Biol Chem ; 267(16): 11038-45, 1992 Jun 05.
Article in English | MEDLINE | ID: mdl-1597445

ABSTRACT

The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylases in which N10-formyltetrahydrofolate is the one-carbon donor. We have found that the antifolates methotrexate (MTX) and piritrexim (PTX) completely block the de novo purine pathway in mouse L1210 leukemia cells growing in culture but with only minor accumulations of GAR and AICAR to less than 5% of the polyphosphate derivatives of N-formylglycinamide ribotide (FGAR) which accumulate when the pathway is blocked completely by azaserine. This azaserine-induced accumulation of FGAR polyphosphates is completely abolished by MTX, indicating that inhibition of the pathway is at or before GAR transformylase (reaction 3; Lyons, S. D., and Christopherson, R. I. (1991) Biochem. Int. 24, 187-197). Three h after the addition of MTX (0.1 microM), cellular 5-phosphoribosyl-1-pyrophosphate has accumulated 3.4-fold while 6-methyl-mercaptopurine riboside (25 microM) induces a 6.3-fold accumulation. These data suggest that amido phosphoribosyltransferase catalyzing reaction 1 of the pathway is the primary site of inhibition. In support of this conclusion, we have found that dihydrofolate-Glu5, which accumulates in MTX-treated cells, is a noncompetitive inhibitor of amido phosphoribosyltransferase with a dissociation constant of 3.41 +/- 0.08 microM for interaction with the enzyme-glutamine complex in vitro. Folate-Glu5, MTX-Glu5, PTX, dihydrotriazine benzenesulfonyl fluoride, and AICAR also inhibit amido phosphoribosyltransferase.


Subject(s)
Amidophosphoribosyltransferase/antagonists & inhibitors , Folic Acid Antagonists/pharmacology , Leukemia, Experimental/enzymology , Methotrexate/pharmacology , Pyrimidines/pharmacology , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/metabolism , Animals , Azaserine/pharmacology , Leukemia, Experimental/metabolism , Methylthioinosine/pharmacology , Mice , Purines/metabolism , Ribonucleotides/metabolism , Tumor Cells, Cultured
16.
J Biol Chem ; 267(7): 4317-21, 1992 Mar 05.
Article in English | MEDLINE | ID: mdl-1311306

ABSTRACT

Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.


Subject(s)
B-Lymphocytes/metabolism , Hypoxanthine Phosphoribosyltransferase/deficiency , Purine Nucleotides/biosynthesis , Ribose-Phosphate Pyrophosphokinase/metabolism , Aminopterin/pharmacology , B-Lymphocytes/enzymology , Humans , Hypoxanthine Phosphoribosyltransferase/metabolism , Kinetics , Methylthioinosine/pharmacology
17.
J Neurochem ; 58(2): 700-8, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1309569

ABSTRACT

Protein kinase N (PKN) is a soluble, apparently novel serine protein kinase that is activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma cells as well as in several nonneuronal cell lines. Purine analogs, such as 6-thioguanine and 2-aminopurine, have been found to inhibit PKN in vitro. When applied to intact cells, these compounds suppress certain biological responses to NGF, but not others, a findings suggesting the presence of multiple pathways in the NGF mechanism. We report here that 6-methylmercaptopurine riboside (6-MMPR) inhibits NGF-stimulated PKN activity in vitro with an apparent Ki of approximately 5 nM. This is approximately 1,000-fold lower than the Ki of the most potent purine inhibitor of PKN. Compounds similar to 6-MMPR, but lacking the methyl or riboside groups, were much less potent as PKN inhibitors. A survey of six additional purified protein kinases shows no inhibitory effect of 6-MMPR, thus indicating a good degree of specificity of this compound for PKN. In contrast to NGF-stimulated PKN, a PKN-like activity stimulated in PC12 cells in response to activation of cyclic AMP-dependent protein kinase was nearly insensitive to 6-MMPR. Application of 6-MMPR to intact PC12 cells resulted in blockade of several responses to NGF (neurite regeneration and ornithine decarboxylase induction) but not of several others (rapid enhancement of tyrosine hydroxylase phosphorylation and PKN activation). These findings suggest that 6-MMPR is a potent and selective agent for characterizing PKN in vitro and for assessing its potential role in the multiple pathways of the NGF mechanism of action.


Subject(s)
Methylthioinosine/pharmacology , Nerve Growth Factors/pharmacology , Protein Kinase C , Protein Kinase Inhibitors , Animals , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Induction , Ornithine Decarboxylase/metabolism , PC12 Cells/drug effects , PC12 Cells/enzymology , Phosphotransferases/antagonists & inhibitors , Protein Kinases/metabolism
20.
Mol Biochem Parasitol ; 28(3): 181-7, 1988 Apr.
Article in English | MEDLINE | ID: mdl-2838751

ABSTRACT

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.


Subject(s)
Adenosine Kinase/metabolism , Leishmania donovani/enzymology , Phosphotransferases/metabolism , Adenosine/metabolism , Adenosine Diphosphate/pharmacology , Adenosine Kinase/antagonists & inhibitors , Adenosine Monophosphate/pharmacology , Adenosine Triphosphate/metabolism , Animals , Kinetics , Methylthioinosine/metabolism , Methylthioinosine/pharmacology , Substrate Specificity , Tubercidin/metabolism , Tubercidin/pharmacology
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