ABSTRACT
BACKGROUND: During antifungal prophylaxis, micafungin is generally infused IV once daily over 1 h. In practice, less-frequent dosing could improve the quality of life in patients requiring long-term treatment or prophylaxis. The feasibility of this approach was assessed using humanized doses of daily or infrequent micafungin regimens. OBJECTIVES: To evaluate the effectiveness of intermittent high-dose micafungin, simulating human exposure, for prophylaxis of invasive candidiasis in a rat model of chronic Candida albicans gastrointestinal colonization and systemic dissemination. METHODS: Two weeks post-infection with an oral challenge of C. albicans, Sprague-Dawley rats were immunocompromised with a cytotoxic drug and a steroid. Rats received IV infusions of: daily vehicle control; daily subcutaneous micafungin (20 mg/kg SC); high-dose micafungin (20 mg/kg bolus SC + 80 mg/kg infusion/72 h, to simulate intermittent human dosing of 300 mg/72 h); or daily fluconazole by mouth (10 mg/kg PO). The effects of antifungal prophylaxis on faecal fungal burden and systemic C. albicans dissemination were evaluated. RESULTS: A rat model of chronic C. albicans gastrointestinal colonization and systemic dissemination was established, characterized by a sustained microbiological burden over 29 days and fungal recovery from normally sterile tissues. Using this model, intermittent high-dose micafungin (delivered via iPrecio pumps) to simulate humanized doses of 300 mg/72 h was significantly more effective than vehicle control, as effective as once-daily micafungin and similar to daily fluconazole at reducing faecal burden and preventing systemic dissemination. CONCLUSIONS: These data indicate that intermittent high-dose micafungin can be as effective as daily therapy, supporting clinical assessment in high-risk patients requiring long-term antifungal prophylaxis.
Subject(s)
Candida albicans , Echinocandins , Animals , Antifungal Agents/therapeutic use , Humans , Lipopeptides , Micafungin , Quality of Life , Rats , Rats, Sprague-DawleyABSTRACT
Clostridium difficile causes infections of the colon in susceptible patients. Specifically, gut dysbiosis induced by treatment with broad-spectrum antibiotics facilitates germination of ingested C. difficile spores, expansion of vegetative cells, and production of symptom-causing toxins TcdA and TcdB. The current standard of care for C. difficile infections (CDI) consists of administration of antibiotics such as vancomycin that target the bacterium but also perpetuate gut dysbiosis, often leading to disease recurrence. The monoclonal antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) are currently in development for the prevention of recurrent CDI. In this study, the effects of vancomycin or actoxumab/bezlotoxumab treatment on progression and resolution of CDI were assessed in mice and hamsters. Rodent models of CDI are characterized by an early severe phase of symptomatic disease, associated with high rates of morbidity and mortality; high intestinal C. difficile burden; and a disrupted intestinal microbiota. This is followed in surviving animals by gradual recovery of the gut microbiota, associated with clearance of C. difficile and resolution of disease symptoms over time. Treatment with vancomycin prevents disease initially by inhibiting outgrowth of C. difficile but also delays microbiota recovery, leading to disease relapse following discontinuation of therapy. In contrast, actoxumab/bezlotoxumab treatment does not impact the C. difficile burden but rather prevents the appearance of toxin-dependent symptoms during the early severe phase of disease, effectively preventing disease until the microbiota (the body's natural defense against C. difficile) has fully recovered. These data provide insight into the mechanism of recurrence following vancomycin administration and into the mechanism of recurrence prevention observed clinically with actoxumab/bezlotoxumab.
Subject(s)
Anti-Bacterial Agents/adverse effects , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antitoxins/pharmacology , Clostridium Infections/drug therapy , Vancomycin/adverse effects , Animals , Anti-Bacterial Agents/administration & dosage , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/biosynthesis , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/biosynthesis , Broadly Neutralizing Antibodies , Clostridioides difficile/drug effects , Clostridioides difficile/growth & development , Clostridioides difficile/pathogenicity , Clostridium Infections/immunology , Clostridium Infections/microbiology , Clostridium Infections/mortality , Convalescence , Cricetulus , Disease Models, Animal , Disease Progression , Enterotoxins/antagonists & inhibitors , Enterotoxins/biosynthesis , Gastrointestinal Microbiome/drug effects , Gastrointestinal Microbiome/immunology , Humans , Mice , Mice, Inbred C57BL , Survival Analysis , Vancomycin/administration & dosageABSTRACT
UNLABELLED: The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a ß-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of ß-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the ß-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general. IMPORTANCE: HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., TyrâPhe or AspâGlu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.
Subject(s)
Hepacivirus/enzymology , Hepacivirus/physiology , RNA-Dependent RNA Polymerase/metabolism , Transcription Elongation, Genetic , Transcription Initiation, Genetic , Virus Replication , Crystallography, X-Ray , DNA Mutational Analysis , Hepacivirus/chemistry , Hepacivirus/genetics , Humans , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Conformation , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/geneticsABSTRACT
OBJECTIVES: SMT19969 is a novel narrow-spectrum antimicrobial under development for the treatment of Clostridium difficile infection (CDI). The objectives were to assess the relative efficacies of SMT19969, vancomycin and fidaxomicin in the hamster model of CDI. METHODS: Hamsters were infected with either C. difficile BI1 (ribotype 027) or C. difficile 630 (ribotype 012) prior to treatment with vehicle, SMT19969, fidaxomicin or vancomycin for 5 days. Animals were further monitored through to day 28 and survival recorded. Plasma and gastrointestinal concentrations of SMT19969 following single and repeat administration in infected hamsters were determined. RESULTS: Following infection with C. difficile BI1, treatment with SMT19969, vancomycin and fidaxomicin resulted in 100% survival during the 5 day dosing period, with 90%-100% of animals receiving SMT19969 and fidaxomicin surviving during the post-dosing follow-up period. Whilst protective during treatment, onset of mortality was observed on day 11 in animals treated with vancomycin, with a 10% survival recorded by day 28. Similar results were observed for SMT19969 and vancomycin following infection with C. difficile 630, with day 28 survival rates of 80%-100% and 0%, respectively. Fidaxomicin protected animals infected with C. difficile 630 from mortality during dosing, although day 28 survival rates varied from 0% to 40% depending on dose. Plasma levels of SMT19969 were typically below the limit of quantification, but levels in the gastrointestinal tract remained far in excess of the MIC. CONCLUSIONS: These data show that SMT19969 is highly effective at treating both acute infection and preventing recurrent disease and support continued investigation of SMT19969 as a potential therapy for CDI.
Subject(s)
Aminoglycosides/administration & dosage , Anti-Bacterial Agents/administration & dosage , Benzimidazoles/administration & dosage , Clostridioides difficile/drug effects , Clostridium Infections/drug therapy , Pyridines/administration & dosage , Vancomycin/administration & dosage , Animals , Anti-Bacterial Agents/pharmacokinetics , Benzimidazoles/pharmacokinetics , Clostridium Infections/microbiology , Disease Models, Animal , Fidaxomicin , Gastrointestinal Tract/chemistry , Mesocricetus , Microbial Sensitivity Tests , Plasma/chemistry , Pyridines/pharmacokinetics , Recurrence , Survival Analysis , Treatment OutcomeABSTRACT
Substitution at the 7-position of the chromen-4-one pharmacophore of 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one NU7441, a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, with allyl, n-propyl or methyl enabled the resolution by chiral HPLC of atropisomers. Biological evaluation against DNA-PK of each pair of atropisomers showed a marked difference in potency, with biological activity residing exclusively in the laevorotatory enantiomer.
Subject(s)
Chromones/chemistry , Chromones/pharmacology , DNA-Activated Protein Kinase/antagonists & inhibitors , Morpholines/chemistry , Morpholines/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , Binding Sites , Chromones/chemical synthesis , DNA-Activated Protein Kinase/chemistry , DNA-Activated Protein Kinase/metabolism , Humans , Models, Molecular , Morpholines/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Stereoisomerism , Structure-Activity Relationship , SwineABSTRACT
Rampant rise of multidrug resistant strains among Gram-negative bacteria has necessitated investigation of alternative antimicrobial agents with novel modes of action including antimicrobial proteins such as bacteriocins. The main hurdle in the clinical development of bacteriocin biologics is their narrow specificity and limited strain activity spectrum. Genome mining of bacteria for broadly active bacteriocins have identified a number of promising candidates but attempts to improve these natural multidomain proteins further, for example by combining domains of different origin, have so far met with limited success. We have found that domain swapping of Pseudomonas bacteriocins of porin type, when carried out between phylogenetically related molecules with similar mechanism of activity, allows the generation of highly active molecules with broader spectrum of activity, for example by abolishing strain resistance due to the presence of immunity proteins. The most broadly active chimera engineered in this study, S5-PmnH, exhibits excellent control of Pseudomonas aeruginosa infection in validated murine keratitis and lung infection models.
Subject(s)
Anti-Infective Agents , Bacteriocins , Keratitis , Pseudomonas Infections , Animals , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/metabolism , Bacteriocins/genetics , Bacteriocins/metabolism , Bacteriocins/pharmacology , Chimera/metabolism , Keratitis/drug therapy , Lung/metabolism , Mice , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolismABSTRACT
This study describes a novel series of UDP-N-acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC50) <5 nM) and cellular activity against P. aeruginosa (best minimal inhibitory concentration (MIC) of 4 µg/mL). Lack of activity against E. coli was maintained (IC50 > 20 µM and MIC > 128 µg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa.
Subject(s)
Acyltransferases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Drug Discovery , Enzyme Inhibitors/pharmacology , Pseudomonas aeruginosa/drug effects , Anti-Bacterial Agents/chemistry , Crystallography, X-Ray , Drug Resistance, Bacterial/drug effects , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity RelationshipABSTRACT
A novel series of P2-P4 macrocyclic HCV NS3/4A protease inhibitors with α-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around α-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action.
Subject(s)
Boron Compounds/chemistry , Boronic Acids/chemistry , Macrocyclic Compounds/chemistry , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Binding Sites , Boron Compounds/chemical synthesis , Boron Compounds/pharmacology , Catalytic Domain , Crystallography, X-Ray , Hepacivirus/drug effects , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Protein Structure, Tertiary , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolismABSTRACT
We have designed and synthesized a novel series of alpha-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described.
Subject(s)
Boronic Acids/chemical synthesis , Hepacivirus/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Boronic Acids/pharmacology , Boronic Acids/therapeutic use , Catalytic Domain , Drug Design , Hepacivirus/enzymology , Molecular Structure , Serine/chemistry , Structure-Activity RelationshipABSTRACT
PURPOSE: Clostridium difficile infection (CDI) is an increasing cause of nosocomial diarrhoea worldwide, which has been partly attributed to the emergence of hypervirulent strains including C. difficile BI/NAP1/ribotype 027 and BK/NAP7/ribotype 078. Cadazolid is a new antibiotic currently in late-stage clinical studies for the treatment of CDI. The present study evaluated the in vitro bactericidal effect of cadazolid and comparator antibiotics against four C. difficile strains. The data demonstrate the potent and bactericidal activity of cadazolid against different ribotypes of C. difficile. METHODOLOGY: MICs for test antibiotics were determined in brain- heart infusion-supplemented broth (BHIS) containing 5 g l-1 yeast extract and 0.025â% (w/v) l-cysteine. Time-kill kinetics to investigate the rate of killing of each antibiotic at sub- and supra-MIC concentrations were performed at concentrations of 0.5, 1, 2, 4, 8 or 16× the MIC of cadazolid, vancomycin and fidaxomicin at intervals over a 48 h period.Results/key findings. Cadazolid-mediated killing of C. difficile was faster and occurred at lower concentrations than observed for vancomycin, while potency and killing was largely comparable to those observed for fidaxomicin. Notably, cadazolid also displayed a potent bactericidal effect against fluoroquinolone-resistant hypervirulent ribotype 027 and 078 strains. C. difficile spore formation was largely inhibited by all three antibiotics at concentrations >1× MIC; however, none were able to eliminate spores effectively, which were present at the start of the experiment. CONCLUSION: The data presented here demonstrate the potent in vitro bactericidal activity of cadazolid against different ribotypes of C. difficile, although on a limited set of strains.
Subject(s)
Anti-Bacterial Agents/pharmacology , Clostridioides difficile/drug effects , Oxazolidinones/pharmacology , Aminoglycosides/pharmacology , Clostridioides difficile/chemistry , Clostridioides difficile/classification , Clostridioides difficile/growth & development , Clostridium Infections/microbiology , Fidaxomicin , Humans , Kinetics , Microbial Sensitivity Tests , Ribotyping , Vancomycin/pharmacologyABSTRACT
Optimization of a pyrrolidine-based template using structure-based design and physicochemical considerations has provided a development candidate 20b (3082) with submicromolar potency in the HCV replicon and good pharmacokinetic properties.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/drug effects , Pyrrolidines/chemical synthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Biological Availability , Chlorocebus aethiops , Hepacivirus/enzymology , Models, Molecular , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , RNA-Dependent RNA Polymerase/chemistry , Rats , Stereoisomerism , Structure-Activity Relationship , Vero CellsABSTRACT
UNLABELLED: The emerging epidemic of drug resistance places the development of efficacious and safe antibiotics in the spotlight of current research. Here, we report the design of next-generation aminoglycosides. Discovery efforts were driven by rational synthesis focusing on 4' alkylations of the aminoglycoside paromomycin, with the goal to alleviate the most severe and disabling side effect of aminoglycosides-irreversible hearing loss. Compounds were evaluated for target activity in in vitro ribosomal translation assays, antibacterial potency against selected pathogens, cytotoxicity against mammalian cells, and in vivo ototoxicity. The results of this study produced potent compounds with excellent selectivity at the ribosomal target, promising antibacterial activity, and little, if any, ototoxicity upon chronic administration. The favorable biocompatibility profile combined with the promising antibacterial activity emphasizes the potential of next-generation aminoglycosides in the treatment of infectious diseases without the risk of ototoxicity. IMPORTANCE: The ever-widening epidemic of multidrug-resistant infectious diseases and the paucity of novel antibacterial agents emerging from modern screening platforms mandate the reinvestigation of established drugs with an emphasis on improved biocompatibility and overcoming resistance mechanisms. Here, we describe the preparation and evaluation of derivatives of the established aminoglycoside antibiotic paromomycin that effectively remove its biggest deficiency, ototoxicity, and overcome certain bacterial resistance mechanisms.
Subject(s)
Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Aminoglycosides/chemical synthesis , Animals , Anti-Bacterial Agents/chemical synthesis , Bacterial Infections/drug therapy , Escherichia coli/drug effects , Guinea Pigs , Hexosamines/chemical synthesis , Hexosamines/pharmacology , Male , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , NIH 3T3 Cells , Phylogeny , RNA, Ribosomal, 16S/genetics , Ribosomes/drug effects , Staphylococcus aureus/drug effectsABSTRACT
Clinical use of 2-deoxystreptamine aminoglycoside antibiotics, which target the bacterial ribosome, is compromised by adverse effects related to limited drug selectivity. Here we present a series of 4',6'-O-acetal and 4'-O-ether modifications on glucopyranosyl ring I of aminoglycosides. Chemical modifications were guided by measuring interactions between the compounds synthesized and ribosomes harbouring single point mutations in the drug-binding site, resulting in aminoglycosides that interact poorly with the drug-binding pocket of eukaryotic mitochondrial or cytosolic ribosomes. Yet, these compounds largely retain their inhibitory activity for bacterial ribosomes and show antibacterial activity. Our data indicate that 4'-O-substituted aminoglycosides possess increased selectivity towards bacterial ribosomes and little activity for any of the human drug-binding pockets.
Subject(s)
Aminoglycosides/chemistry , Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Aminoglycosides/therapeutic use , Animals , Anti-Bacterial Agents/therapeutic use , Base Sequence , Cell-Free System , Crystallography, X-Ray , Disease Models, Animal , Drug Interactions , Escherichia coli/drug effects , Escherichia coli/isolation & purification , Humans , Inhibitory Concentration 50 , Male , Mice , Microbial Sensitivity Tests , Molecular Sequence Data , Mycobacterium smegmatis/drug effects , Nucleic Acid Conformation , Protein Biosynthesis/drug effects , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , Ribosomes/metabolism , Sepsis/drug therapy , Staphylococcus aureus/drug effectsABSTRACT
A boronic acid moiety was found to be a critical pharmacophore for enhanced in vitro potency against wild-type hepatitis C replicons and known clinical polymorphic and resistant HCV mutant replicons. The synthesis, optimization, and structure-activity relationships associated with inhibition of HCV replication in a subgenomic replication system for a series of non-nucleoside boron-containing HCV RNA-dependent RNA polymerase (NS5B) inhibitors are described. A summary of the discovery of 3 (GSK5852), a molecule which entered clinical trials in subjects infected with HCV in 2011, is included.
Subject(s)
Antiviral Agents/pharmacology , Boronic Acids/chemistry , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Antiviral Agents/chemistry , Drug Discovery , Drug Resistance, Viral/genetics , Hepacivirus/enzymology , Hepacivirus/genetics , Magnetic Resonance Spectroscopy , Models, Molecular , Structure-Activity Relationship , Viral Nonstructural Proteins/antagonists & inhibitorsABSTRACT
ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound 6 (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC50 of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC50 of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Morpholines/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemical synthesis , Animals , Antineoplastic Agents/therapeutic use , Ataxia Telangiectasia Mutated Proteins , Crystallography, X-Ray , Drug Discovery , Female , HeLa Cells , Humans , Mice , Models, Molecular , Morpholines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Analogues of (dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441), a potent inhibitor of DNA-dependent protein kinase (DNA-PK; IC50 = 42 ± 2 nM), have been synthesized in which water-solubilizing groups [NHCO(CH2)nNR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines, e.g., morpholine] were placed at the 1-position. Several of the newly synthesized compounds exhibited high potency against DNA-PK and potentiated the cytotoxicity of ionizing radiation (IR) in vitro 10-fold or more (e.g., 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thio-phen-1-yl)acetamide, 39; DNA-PK IC50 = 5.0 ± 1 nM, IR dose modification ratio = 13). Furthermore, 39 was shown to potentiate not only IR in vitro but also DNA-inducing cytotoxic anticancer agents, both in vitro and in vivo. Counter-screening against other members of the phosphatidylinositol 3-kinase (PI-3K) related kinase (PIKK) family unexpectedly revealed that some of the compounds were potent mixed DNA-PK and PI-3K inhibitors.
Subject(s)
DNA-Activated Protein Kinase/antagonists & inhibitors , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , HeLa Cells , Humans , Morpholines/chemistryABSTRACT
DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 µmol/L (MCF7 cells) and 0.17 µmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 µmol/L (MCF7 cells) and more than 10 µmol/L (SW620 cells). Five-day exposure to 1 µmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Chromones/pharmacology , DNA-Activated Protein Kinase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Thiophenes/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Chromones/administration & dosage , Drug Resistance, Neoplasm , Enzyme Activation , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Female , Humans , MCF-7 Cells , Mice , Neoplasms/drug therapy , Neoplasms/metabolism , Thiophenes/administration & dosage , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND/AIMS: The development of new therapies for hepatitis C virus (HCV) infection has been hampered by the lack of a small animal model. GB virus B (GBV-B), which infects new world monkeys, has been proposed as a surrogate system for HCV replication. Despite their short genetic distance, however, difficulties exist when extrapolating results from GBV-B to the HCV system. One way of addressing this is the creation of chimeric GBV-B containing HCV elements. METHODS: Construction and analysis of GBV-B chimeras in which the p13 ion channel was replaced by its HCV counterpart, p7. RESULTS: Replacing all, or part of, the GBV-B p13 protein with HCV p7 resulted in viable chimeras which replicated at wild-type levels in marmosets following intra-hepatic RNA injection. Serum from one animal injected with chimeric RNA was infectious in three naïve recipients, indicating that chimeras formed fully infectious virions. Amantadine, which blocks the ion channel activity of both HCV and GBV-B proteins in vitro, also inhibited GBV-B replication in primary hepatocytes. CONCLUSIONS: These viruses highlight the potential for chimeric GBV-B in the development of HCV-specific therapies and will provide a means of developing HCV p7 as a therapeutic target.
Subject(s)
GB virus B/genetics , Hepacivirus/genetics , Hepatitis C, Chronic/virology , Recombinant Fusion Proteins/genetics , Viral Proteins/genetics , Amantadine/pharmacology , Animals , Antiviral Agents/pharmacology , Callithrix , Cell Line , Disease Models, Animal , Drug Design , Genome, Viral , Hepatitis C, Chronic/drug therapy , Hepatocytes/cytology , Hepatocytes/virology , Humans , Kidney/cytology , RNA, Viral/blood , RNA, Viral/genetics , TransfectionABSTRACT
The SAR development is described for a series of N-acyl pyrrolidine inhibitors of the Hepatitis C virus RNA-dependent RNA polymerase, NS5B, from tractable Delta21 enzyme inhibitors to an example with antiviral activity in a cellular assay (HCV replicon).
Subject(s)
Antiviral Agents/pharmacology , Chemistry, Pharmaceutical/methods , Hepacivirus/chemistry , Hepacivirus/genetics , Pyrrolidines/antagonists & inhibitors , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Replicon/genetics , Viral Nonstructural Proteins/pharmacology , Antiviral Agents/chemistry , Drug Design , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , RNA, Viral/chemistry , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effectsABSTRACT
A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-trans-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the trans-lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.