RESUMO
Taniborbactam is a novel cyclic boronate ß-lactamase inhibitor in clinical development in combination with cefepime. We assessed the in vitro activity of cefepime-taniborbactam and comparators against a 2018-2020 collection of Enterobacterales (n = 13,731) and Pseudomonas aeruginosa (n = 4,619) isolates cultured from infected patients attending hospitals in 56 countries. MICs were determined by CLSI broth microdilution. Taniborbactam was tested at a fixed concentration of 4 µg/mL. Isolates with cefepime-taniborbactam MICs of ≥16 µg/mL underwent whole-genome sequencing. ß-lactamase genes were identified in meropenem-resistant isolates by PCR/Sanger sequencing. Against Enterobacterales, taniborbactam reduced the cefepime MIC90 value by >64-fold (from >16 to 0.25 µg/mL). At ≤16 µg/mL, cefepime-taniborbactam inhibited 99.7% of all Enterobacterales isolates; >97% of isolates with multidrug-resistant (MDR) and ceftolozane-tazobactam-resistant phenotypes; ≥90% of isolates with meropenem-resistant, difficult-to-treat-resistant (DTR), meropenem-vaborbactam-resistant, and ceftazidime-avibactam-resistant phenotypes; 100% of VIM-positive, AmpC-positive, and KPC-positive isolates; 98.7% of extended-spectrum ß-lactamase (ESBL)-positive; 98.8% of OXA-48-like-positive; and 84.6% of NDM-positive isolates. Against P. aeruginosa, taniborbactam reduced the cefepime MIC90 value by 4-fold (from 32 to 8 µg/mL). At ≤16 µg/mL, cefepime-taniborbactam inhibited 97.4% of all P. aeruginosa isolates; ≥85% of isolates with meropenem-resistant, MDR, and meropenem-vaborbactam-resistant phenotypes; >75% of isolates with DTR, ceftazidime-avibactam-resistant, and ceftolozane-tazobactam-resistant phenotypes; and 87.4% of VIM-positive isolates. Multiple potential mechanisms, including carriage of IMP, certain alterations in PBP3, permeability (porin) defects, and possibly, upregulation of efflux were present in most isolates with cefepime-taniborbactam MICs of ≥16 µg/mL. We conclude that cefepime-taniborbactam exhibited potent in vitro activity against Enterobacterales and P. aeruginosa and inhibited most carbapenem-resistant isolates, including those carrying serine carbapenemases or NDM/VIM metallo-ß-lactamases (MBLs).
Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Cefepima/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Meropeném/farmacologia , Tazobactam/farmacologia , beta-Lactamases/genética , Pseudomonas aeruginosa , Bactérias Gram-Negativas , Compostos Azabicíclicos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Ceftibuten-ledaborbactam etzadroxil is a cephalosporin-boronate ß-lactamase inhibitor prodrug combination under development as an oral treatment for complicated urinary tract infections caused by multidrug-resistant (MDR) Enterobacterales producing serine ß-lactamases (Ambler class A, C, and D). In vivo, ledaborbactam etzadroxil (formerly VNRX-7145) is cleaved to the active inhibitor ledaborbactam (formerly VNRX-5236). To more completely define the breadth of ceftibuten-ledaborbactam's activity against important antimicrobial-resistant pathogens, we assessed its in vitro activity against phenotypic and genotypic subsets from a 2018-2020 global culture collection of 3,889 clinical isolates of Enterobacterales, including MDR organisms, extended-spectrum-ß-lactamase (ESBL)-positive organisms, and organisms that are nonsusceptible and resistant to other antimicrobials. MICs were determined by CLSI broth microdilution and interpreted using both CLSI and EUCAST breakpoints. Ledaborbactam was tested at a fixed concentration of 4 µg/mL. ß-Lactamase genes were characterized by PCR followed by Sanger sequencing or whole-genome sequencing for selected ß-lactam-resistant isolate subsets. At ≤1 µg/mL, ceftibuten-ledaborbactam (MIC90, 0.25 µg/mL) inhibited 89.7% of MDR isolates, 98.3% of isolates with a presumptive ESBL-positive phenotype, and 92.6% of trimethoprim-sulfamethoxazole-nonsusceptible, 91.7% of levofloxacin-nonsusceptible, 88.1% of amoxicillin-clavulanate-nonsusceptible, 85.7% of ceftibuten-resistant (MIC >1 µg/mL), and 54.1% of carbapenem-nonsusceptible isolates. Against specific ESBL genotype-positive isolates (AmpC negative, serine carbapenemase negative, and metallo-ß-lactamase negative), ceftibuten-ledaborbactam inhibited 96.3% of CTX-M-9 group (MIC90, 0.25 µg/mL), 91.5% of CTX-M-1 group (MIC90, 0.5 µg/mL), and 88.2% of SHV-positive (MIC90, 2 µg/mL) isolates at ≤1 µg/mL. Against specific serine carbapenemase genotype-positive isolates, ceftibuten-ledaborbactam inhibited 85.9% of KPC-positive (MIC90, 2 µg/mL) and 82.9% of OXA-48-group-positive (MIC90, 2 µg/mL) isolates at ≤1 µg/mL. Continued development of ceftibuten-ledaborbactam appears warranted.
Assuntos
Antibacterianos , beta-Lactamases , Ceftibuteno/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , beta-Lactamases/genética , Testes de Sensibilidade Microbiana , Serina , Compostos Azabicíclicos/farmacologiaRESUMO
There is an urgent need for oral agents to combat resistant Gram-negative pathogens. Here, we describe the characterization of VNRX-5236, a broad-spectrum boronic acid ß-lactamase inhibitor (BLI), and its orally bioavailable etzadroxil prodrug, VNRX-7145. VNRX-7145 is being developed in combination with ceftibuten, an oral cephalosporin, to combat strains of Enterobacterales expressing extended-spectrum ß-lactamases (ESBLs) and serine carbapenemases. VNRX-5236 is a reversible covalent inhibitor of serine ß-lactamases, with inactivation efficiencies on the order of 104 M-1 · sec-1, and prolonged active site residence times (t1/2, 5 to 46 min). The spectrum of inhibition includes Ambler class A ESBLs, class C cephalosporinases, and class A and D carbapenemases (KPC and OXA-48, respectively). Rescue of ceftibuten by VNRX-5236 (fixed at 4 µg/ml) in isogenic strains of Escherichia coli expressing class A, C, or D ß-lactamases demonstrated an expanded spectrum of activity relative to oral comparators, including investigational penems, sulopenem, and tebipenem. VNRX-5236 rescued ceftibuten activity in clinical isolates of Enterobacterales expressing ESBLs (MIC90, 0.25 µg/ml), KPCs (MIC90, 1 µg/ml), class C cephalosporinases (MIC90, 1 µg/ml), and OXA-48-type carbapenemases (MIC90, 1 µg/ml). Frequency of resistance studies demonstrated a low propensity for recovery of resistant variants at 4× the MIC of the ceftibuten/VNRX-5236 combination. In vivo, whereas ceftibuten alone was ineffective (50% effective dose [ED50], >128 mg/kg), ceftibuten/VNRX-7145 administered orally protected mice from lethal septicemia caused by Klebsiella pneumoniae producing KPC carbapenemase (ED50, 12.9 mg/kg). The data demonstrate potent, broad-spectrum rescue of ceftibuten activity by VNRX-5236 in clinical isolates of cephalosporin-resistant and carbapenem-resistant Enterobacterales.
Assuntos
Cefalosporinas , Inibidores de beta-Lactamases , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias , Carbapenêmicos/farmacologia , Ceftibuteno , Cefalosporinas/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Serina , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
As shifts in the epidemiology of ß-lactamase-mediated resistance continue, carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are the most urgent threats. Although approved ß-lactam (BL)-ß-lactamase inhibitor (BLI) combinations address widespread serine ß-lactamases (SBLs), such as CTX-M-15, none provide broad coverage against either clinically important serine-ß-lactamases (KPC, OXA-48) or clinically important metallo-ß-lactamases (MBLs; e.g., NDM-1). VNRX-5133 (taniborbactam) is a new cyclic boronate BLI that is in clinical development combined with cefepime for the treatment of infections caused by ß-lactamase-producing CRE and CRPA. Taniborbactam is the first BLI with direct inhibitory activity against Ambler class A, B, C, and D enzymes. From biochemical and structural analyses, taniborbactam exploits substrate mimicry while employing distinct mechanisms to inhibit both SBLs and MBLs. It is a reversible covalent inhibitor of SBLs with slow dissociation and a prolonged active-site residence time (half-life, 30 to 105 min), while in MBLs, it behaves as a competitive inhibitor, with inhibitor constant (Ki ) values ranging from 0.019 to 0.081 µM. Inhibition is achieved by mimicking the transition state structure and exploiting interactions with highly conserved active-site residues. In microbiological testing, taniborbactam restored cefepime activity in 33/34 engineered Escherichia coli strains overproducing individual enzymes covering Ambler classes A, B, C, and D, providing up to a 1,024-fold shift in the MIC. Addition of taniborbactam restored the antibacterial activity of cefepime against all 102 Enterobacterales clinical isolates tested and 38/41 P. aeruginosa clinical isolates tested with MIC90s of 1 and 4 µg/ml, respectively, representing ≥256- and ≥32-fold improvements, respectively, in antibacterial activity over that of cefepime alone. The data demonstrate the potent, broad-spectrum rescue of cefepime activity by taniborbactam against clinical isolates of CRE and CRPA.
Assuntos
Antibacterianos/farmacologia , Ácidos Borínicos/farmacologia , Ácidos Carboxílicos/farmacologia , Inibidores de beta-Lactamases/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cefepima/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
V-073, a small-molecule capsid inhibitor originally developed for nonpolio enterovirus indications is considerably more potent against polioviruses. All poliovirus isolates tested to date (n = 45), including wild, vaccine, vaccine-derived, and laboratory strains, are susceptible to the antiviral capsid inhibitor V-073. We grew poliovirus in the presence of V-073 to allow for the identification of variants with reduced susceptibility to the drug. Sequence analysis of 160 independent resistant variants (80 isolates of poliovirus type 1, 40 isolates each of types 2 and 3) established that V-073 resistance involved a single amino acid change in either of two virus capsid proteins, VP1 (67 of 160 [42%]) or VP3 (93 of 160 [58%]). In resistant variants with a VP1 change, the majority (53 of 67 [79%]) exhibited a substitution of isoleucine at position 194 (equivalent position 192 in type 3) with either methionine or phenylalanine. Of those with a VP3 change, alanine at position 24 was replaced with valine in all variants (n = 93). The resistance phenotype was relatively stable upon passage of viruses in cell culture in the absence of drug. Single-step growth studies showed no substantial differences between drug-resistant variants and the virus stocks from which they were derived, while the resistant viruses were generally more thermally labile than the corresponding drug-susceptible parental viruses. These studies provide a foundation from which to build a greater understanding of resistance to antiviral compound V-073.
Assuntos
Antivirais/farmacologia , Proteínas do Capsídeo/genética , Farmacorresistência Viral/efeitos dos fármacos , Éteres Difenil Halogenados/farmacologia , Mutação , Poliovirus/genética , Substituição de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Capsídeo/química , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Farmacorresistência Viral/genética , Humanos , Macaca mulatta , Éteres Fenílicos , Poliovirus/efeitos dos fármacos , Poliovirus/metabolismo , Ensaio de Placa ViralRESUMO
A major antimicrobial resistance mechanism in Gram-negative bacteria is the production of ß-lactamase enzymes. The increasing emergence of ß-lactamase-producing multi-drug-resistant "superbugs" has resulted in increases in costly hospital Emergency Department (ED) visits and hospitalizations due to the requirement for parenteral antibiotic therapy for infections caused by these difficult-to-treat bacteria. To address the lack of outpatient treatment, we initiated an iterative program combining medicinal chemistry, biochemical testing, microbiological profiling, and evaluation of oral pharmacokinetics. Lead optimization focusing on multiple smaller, more lipophilic active compounds, followed by an exploration of oral bioavailability of a variety of their respective prodrugs, provided 36 (VNRX-7145/VNRX-5236 etzadroxil), the prodrug of the boronic acid-containing ß-lactamase inhibitor 5 (VNRX-5236). In vitro and in vivo studies demonstrated that 5 restored the activity of the oral cephalosporin antibiotic ceftibuten against Enterobacterales expressing Ambler class A extended-spectrum ß-lactamases, class A carbapenemases, class C cephalosporinases, and class D oxacillinases.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Enterobacteriaceae/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Enterobacteriaceae/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/químicaRESUMO
A major resistance mechanism in Gram-negative bacteria is the production of ß-lactamase enzymes. Originally recognized for their ability to hydrolyze penicillins, emergent ß-lactamases can now confer resistance to other ß-lactam drugs, including both cephalosporins and carbapenems. The emergence and global spread of ß-lactamase-producing multi-drug-resistant "superbugs" has caused increased alarm within the medical community due to the high mortality rate associated with these difficult-to-treat bacterial infections. To address this unmet medical need, we initiated an iterative program combining medicinal chemistry, structural biology, biochemical testing, and microbiological profiling to identify broad-spectrum inhibitors of both serine- and metallo-ß-lactamase enzymes. Lead optimization, beginning with narrower-spectrum, weakly active compounds, provided 20 (VNRX-5133, taniborbactam), a boronic-acid-containing pan-spectrum ß-lactamase inhibitor. In vitro and in vivo studies demonstrated that 20 restored the activity of ß-lactam antibiotics against carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Enterobacteriaceae. Taniborbactam is the first pan-spectrum ß-lactamase inhibitor to enter clinical development.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Ácidos Borínicos/química , Ácidos Borínicos/farmacologia , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Ácidos Borínicos/síntese química , Ácidos Borínicos/uso terapêutico , Carbapenêmicos/farmacologia , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/uso terapêutico , Humanos , Camundongos , Modelos Moleculares , Resistência beta-Lactâmica , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/uso terapêuticoRESUMO
V-073, an enterovirus capsid inhibitor, was evaluated for its spectrum of antipoliovirus activity. V-073 inhibited all 45 polioviruses tested in a virus-induced cytopathic effect protection assay, with 50% effective concentration (EC50) values ranging from 0.003 to 0.126 microM. Ninety percent of the polioviruses tested were inhibited at EC(50)s of < or = 0.076 microM (MIC90 = 32 ng/ml). V-073 is a promising antiviral candidate for the posteradication management of poliovirus incidents.
Assuntos
Antivirais/farmacologia , Poliovirus/efeitos dos fármacos , Antivirais/química , Estrutura MolecularRESUMO
A series of novel, potent orthopoxvirus egress inhibitors was identified during high-throughput screening of the ViroPharma small molecule collection. Using structure--activity relationship information inferred from early hits, several compounds were synthesized, and compound 14 was identified as a potent, orally bioavailable first-in-class inhibitor of orthopoxvirus egress from infected cells. Compound 14 has shown comparable efficaciousness in three murine orthopoxvirus models and has entered Phase I clinical trials.
Assuntos
Antivirais/síntese química , Benzamidas/síntese química , Indóis/síntese química , Orthopoxvirus/efeitos dos fármacos , Administração Oral , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Benzamidas/farmacocinética , Benzamidas/farmacologia , Disponibilidade Biológica , Linhagem Celular , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Indóis/farmacocinética , Indóis/farmacologia , Isoindóis , Macaca fascicularis , Camundongos , Estrutura Molecular , Orthopoxvirus/fisiologia , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67-78% identical to Junín virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junín, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.
Assuntos
Antivirais/química , Arenavirus do Novo Mundo/efeitos dos fármacos , Chumbo/química , Proteínas Virais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/virologia , Chlorocebus aethiops , Efeito Citopatogênico Viral , Febres Hemorrágicas Virais/tratamento farmacológico , Febres Hemorrágicas Virais/virologia , Humanos , Chumbo/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Sulfonamidas/química , Sulfonamidas/farmacologia , Ureia/análogos & derivados , Ureia/química , Ureia/farmacologia , Células Vero , Proteínas Virais/metabolismoRESUMO
Recent phylogenetic analyses of the deduced amino acid sequence of the major viral capsid protein (VP1) of all human rhinovirus (HRV) serotypes revealed two distinct species within the genus: species A (75 serotypes) and species B (25 serotypes). Pleconaril is a novel capsid inhibitor of HRVs. All 75 species A serotypes and 18 of the 25 species B serotypes are susceptible to inhibition by pleconaril in cell culture. The seven resistant serotypes are HRV-4, -5, -42, -84, -93, -97 and -99. We were interested in understanding the genetic basis for phenotypic resistance to pleconaril among these naturally occurring viruses. We compared the 25 amino acids of VP1 that comprise the drug-binding pocket of susceptible and resistant species B viruses. A consistent difference was observed at two positions: the vast majority of susceptible viruses had tyrosine and valine at VP1 residues 152 and 191, respectively (Y(152) and V(191)); all resistant viruses had phenylalanine and leucine at these positions (F(152) and L(191)). HRV-14, a pleconaril susceptible virus, has a drug-binding pocket amino acid composition that differs from the naturally resistant HRV-5 and HRV-42 only at these two positions. To gain further insight into the role of these specific residues in natural resistance to pleconaril, we substituted the amino acids at these two positions individually and in combination in an infectious clone of HRV-14 and tested the rescued virus for susceptibility to pleconaril and virion stability. The results indicate that substitution of V(191) to Leu in HRV-14 has a profound negative impact on drug susceptibility but that full resistance to pleconaril is only seen when combined with Phe at position 152 in a HRV-14 double variant (F(152), L(191)). These data identify L(191) in species B HRV as a potentially key residue in conferring significantly reduced susceptibility to pleconaril. These results may be useful in distinguishing naturally occurring viral resistance to pleconaril from treatment-emergent resistance.
Assuntos
Antivirais/farmacologia , Proteínas do Capsídeo/genética , Farmacorresistência Viral/genética , Oxidiazóis/farmacologia , Rhinovirus/efeitos dos fármacos , Antivirais/metabolismo , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/química , Humanos , Oxazóis , Rhinovirus/genéticaRESUMO
VP14637, the lead compound in a series of substituted bis-tetrazole-benzhydrylphenols developed by ViroPharma Incorporated, was evaluated for antiviral efficacy against respiratory syncytial virus (RSV) in vitro in cell culture and in vivo in cotton rats. A selective index of >3000 (> or =2000 times greater than that observed for ribavirin) was determined in the in vitro studies for this compound against both RSV A and B subtypes. In cotton rats, animals given as little as 126 microg drug/kg by small droplet aerosol in divided doses starting 1 day after experimental virus infection with either a RSV A or B subtype consistently had significantly lower mean pulmonary RSV titers and reduced histopathological findings than mock-treated animals or cotton rats given placebo (vehicle-treated animals). No cotton rat treated with aerosols of VP14637 during these studies manifested any evident untoward responses. Thus, VP14637 exhibited good selective antiviral efficacy both in vitro and in vivo.
Assuntos
Antivirais/administração & dosagem , Antivirais/farmacologia , Compostos Benzidrílicos/administração & dosagem , Compostos Benzidrílicos/farmacologia , Fenóis/administração & dosagem , Fenóis/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Tetrazóis/administração & dosagem , Tetrazóis/farmacologia , Aerossóis , Animais , Antivirais/química , Compostos Benzidrílicos/química , Linhagem Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Pulmão/patologia , Masculino , Fenóis/química , Infecções por Vírus Respiratório Sincicial/patologia , Sigmodontinae , Tetrazóis/químicaRESUMO
Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA-dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695-residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical "core," although overall sequence identity is low. Eighty-four expression clones of the BVDV polymerase were designed by fine-sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high-throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96-well format, and two of these proteins were successfully crystallized.
Assuntos
Vírus da Diarreia Viral Bovina/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Cristalização , Cristalografia , Vírus da Diarreia Viral Bovina/genética , Escherichia coli/enzimologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Tripsina/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
The novel capsid-binding antiviral pleconaril inhibits in vitro replication of most rhinoviruses and enteroviruses. Oral pleconaril treatment was studied in 2 parallel randomized, double-blind, placebo-controlled trials. Among 1363 picornavirus-infected participants (65%) in the studies combined, the median time to alleviation of illness was 1 day shorter for pleconaril recipients than for placebo recipients (P<.001). Cold symptom scores and frequency of picornavirus cultured from nasal mucus specimens were lower among pleconaril recipients by day 2 of treatment. No treatment effects were seen in those without picornavirus infection. Pleconaril was associated with a higher incidence of nausea (6% vs. 4%) and diarrhea (9% vs. 7%) and with small increases in mean serum cholesterol levels and platelet counts, compared with baseline measurements. A subsequent 6-week prophylaxis study found that pleconaril induces cytochrome P-450 3A enzymes, which metabolize a variety of drugs, including ethinyl estradiol. Early pleconaril treatment was well tolerated and significantly reduced the duration and severity of colds due to picornaviruses in adults.
Assuntos
Antivirais/uso terapêutico , Resfriado Comum/tratamento farmacológico , Oxidiazóis/uso terapêutico , Infecções por Picornaviridae/tratamento farmacológico , Administração Oral , Adulto , Antivirais/efeitos adversos , Diarreia/induzido quimicamente , Método Duplo-Cego , Feminino , Humanos , Masculino , Náusea/induzido quimicamente , Oxidiazóis/efeitos adversos , Oxazóis , Picornaviridae/efeitos dos fármacos , Estudos Prospectivos , Resultado do TratamentoRESUMO
The chemokine receptor CCR5 provides a portal of entry for human immunodeficiency virus type 1 (HIV-1) into susceptible CD4(+) cells. Both monoclonal antibody (MAb) and small-molecule CCR5 inhibitors have entered human clinical testing, but little is known regarding their potential interactions. We evaluated the interactions between CCR5 MAbs, small-molecule CCR5 antagonists, and inhibitors of HIV-1 gp120, gp41, and reverse transcriptase in vitro. Inhibition data were analyzed for cooperative effects using the combination index (CI) method and stringent statistical criteria. Potent, statistically significant antiviral synergy was observed between the CCR5 MAb PRO 140 and the small-molecule CCR5 antagonists maraviroc (UK-427,857), vicriviroc (SCH-D), and TAK-779. High-level synergy was observed consistently across various assay systems, HIV-1 envelopes, CCR5 target cells, and inhibition levels. CI values ranged from 0.18 to 0.64 and translated into in vitro dose reductions of up to 14-fold. Competition binding studies revealed nonreciprocal patterns of CCR5 binding by MAb and small-molecule CCR5 inhibitors, suggesting that synergy occurs at the level of receptor binding. In addition, both PRO 140 and maraviroc synergized with the chemokine RANTES, a natural ligand for CCR5; however, additive effects were observed for both small-molecule CCR5 antagonists and PRO 140 in combination with other classes of HIV-1 inhibitors. The findings provide a rationale for clinical exploration of MAb and small-molecule CCR5 inhibitors in novel dual-CCR5 regimens for HIV-1 therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Antagonistas dos Receptores CCR5 , Cicloexanos/farmacologia , Anticorpos Anti-HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Triazóis/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Ligação Competitiva , Linhagem Celular , Cicloexanos/metabolismo , Sinergismo Farmacológico , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/imunologia , Inibidores da Fusão de HIV/metabolismo , Células HeLa , Humanos , Maraviroc , Fusão de Membrana/efeitos dos fármacos , Piperazinas/metabolismo , Pirimidinas/metabolismo , Receptores CCR5/imunologia , Inibidores da Transcriptase Reversa/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Triazóis/metabolismoRESUMO
Pleconaril, a specific inhibitor of human picornaviruses, showed therapeutic efficacy against community-acquired colds caused by rhinoviruses in two placebo-controlled trials. Virological assessments were conducted during these trails, including virus culture and drug susceptibility testing. Nasal mucus samples collected from the enrolled patients were tested for the presence of picornavirus by reverse transcriptase PCR and culture. In total, 827 baseline nasal mucus samples were positive by virus culture (420 in the placebo group and 407 in the pleconaril group). Pleconaril treatment was associated with a more rapid loss of culturable virus. By study day 3, the number of samples positive by culture fell to 282 for the placebo-treated subjects and 202 for the pleconaril-treated subjects (P < 0.0001); and by day 6, the number of samples in the two groups positive by culture fell to 196 and 165, respectively (P = 0.07). The clinical benefit correlated strongly with the pleconaril susceptibility of the baseline virus isolate. Pleconaril-treated subjects infected with the more highly susceptible viruses (50% effective concentration < or = 0.38 microg/ml) experienced a median 1.9- to 3.9-day reduction in symptom duration compared with that for the placebo-treated subjects. By contrast, subjects whose baseline virus isolate susceptibility was >0.38 microg/ml did not benefit from pleconaril treatment. These results indicate that the magnitude of symptomatic improvement in pleconaril-treated subjects with community-acquired colds is related to the drug susceptibility of the infecting virus, clearly linking the antiviral effects of the drug to clinical efficacy. Post-baseline virus isolates with reduced susceptibility or full resistance to pleconaril were recovered from 10.7% and 2.7% of drug-treated subjects, respectively. These patients shed low levels of virus and had no unusual clinical outcomes. Nevertheless, studies on the biologic properties and transmissibility of these variant viruses are warranted.
Assuntos
Antivirais/uso terapêutico , Resfriado Comum/tratamento farmacológico , Oxidiazóis/uso terapêutico , Resfriado Comum/virologia , Método Duplo-Cego , Farmacorresistência Bacteriana , Humanos , Oxazóis , Rhinovirus/efeitos dos fármacosRESUMO
ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.
Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Indóis/farmacologia , Orthopoxvirus/efeitos dos fármacos , Infecções por Poxviridae/prevenção & controle , Administração Oral , Sequência de Aminoácidos , Animais , Antivirais/efeitos adversos , Antivirais/química , Benzamidas/efeitos adversos , Benzamidas/química , Efeito Citopatogênico Viral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Vírus da Ectromelia/isolamento & purificação , Ectromelia Infecciosa/prevenção & controle , Feminino , Indóis/efeitos adversos , Indóis/química , Isoindóis , Fígado/virologia , Pulmão/virologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Orthopoxvirus/isolamento & purificação , Orthopoxvirus/fisiologia , Infecções por Poxviridae/virologia , Alinhamento de Sequência , Baço/virologia , Vacínia/prevenção & controle , Proteínas do Envelope Viral/efeitos dos fármacos , Proteínas do Envelope Viral/genética , Ensaio de Placa Viral , Montagem de Vírus/efeitos dos fármacosRESUMO
Biochemical characterization of hepatitis C virus (HCV) replication using purified, membrane-associated replication complexes is hampered by the presence of endogenous nuclease activity that copurifies with the replication complex. In this study, pulse-chase analyses were used to demonstrate that newly synthesized replicon RNA was protected from nuclease activity by a factor(s) that was sensitive to 0.5% NP-40 or protease treatment. Nuclease susceptibility was not related to disruption of lipid membranes, since NP-40 did not significantly affect the buoyant density of HCV replication complexes or protease susceptibility of HCV NS3 and NS5A proteins. These results suggest that a protease-sensitive factor(s) protects newly synthesized RNA from nuclease degradation.
Assuntos
Hepacivirus/genética , RNA Viral/genética , RNA Viral/metabolismo , Linhagem Celular , Endopeptidases/metabolismo , Hepacivirus/fisiologia , Humanos , Octoxinol , Polietilenoglicóis/farmacologia , Replicon , Ribonucleases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Rhinoviruses are the most common infectious agents of humans. They are the principal etiologic agents of afebrile viral upper-respiratory-tract infections (the common cold). Human rhinoviruses (HRVs) comprise a genus within the family Picornaviridae. There are >100 serotypically distinct members of this genus. In order to better understand their phylogenetic relationship, the nucleotide sequence for the major surface protein of the virus capsid, VP1, was determined for all known HRV serotypes and one untyped isolate (HRV-Hanks). Phylogenetic analysis of deduced amino acid sequence data support previous studies subdividing the genus into two species containing all but one HRV serotype (HRV-87). Seventy-five HRV serotypes and HRV-Hanks belong to species HRV-A, and twenty-five HRV serotypes belong to species HRV-B. Located within VP1 is a hydrophobic pocket into which small-molecule antiviral compounds such as pleconaril bind and inhibit functions associated with the virus capsid. Analyses of the amino acids that constitute this pocket indicate that the sequence correlates strongly with virus susceptibility to pleconaril inhibition. Further, amino acid changes observed in reduced susceptibility variant viruses recovered from patients enrolled in clinical trials with pleconaril were distinct from those that confer natural phenotypic resistance to the drug. These observations suggest that it is possible to differentiate rhinoviruses naturally resistant to capsid function inhibitors from those that emerge from susceptible virus populations as a result of antiviral drug selection pressure based on sequence analysis of the drug-binding pocket.