RÉSUMÉ
Burkholderia pseudomallei is the causative agent of melioidosis, which is increasingly being reported worldwide. Mortality rates as high as 40% have been reported based on clinical patient outcomes in the endemic areas of Australia and Thailand. Novel therapies are needed to reduce treatment duration and adverse effects and improve treatment outcomes. Epetraborole, a novel antibiotic, targets leucyl-tRNA synthetase (LeuRS), an essential enzyme that catalyzes the attachment of leucine to transfer RNA. Epetraborole was evaluated for in vitro activity and efficacy in a murine model to assess clinical relevance against Burkholderia pseudomallei infections for possible treatment of melioidosis. Epetraborole was tested against 13 clinically derived and three reference B. pseudomallei strains that have a broad spectrum of susceptibilities to the standard-of-care (SoC) drugs for melioidosis, which showed that epetraborole exhibited minimal inhibitory concentrations of 0.25-4 µg/mL. Ex vivo studies using THP-1 macrophages confirmed the potency of epetraborole and demonstrated synergy between epetraborole and ceftazidime. In the acute pulmonary murine infection model of melioidosis, epetraborole demonstrated equivalent efficacy when delivered orally or subcutaneously, which compared well with the standard-of-care drug ceftazidime. In addition, adding epetraborole to ceftazidime significantly improved antimicrobial activity in this animal model. This work warrants further exploration of epetraborole as a candidate for treating melioidosis and substantiates LeuRS as a clinically relevant drug target in B. pseudomallei.
Sujet(s)
Amino acyl-tRNA synthetases , Burkholderia pseudomallei , Mélioïdose , Animaux , Souris , Humains , Ceftazidime/pharmacologie , Ceftazidime/usage thérapeutique , Mélioïdose/traitement médicamenteux , Antibactériens/pharmacologie , Antibactériens/usage thérapeutique , Amino acyl-tRNA synthetases/pharmacologie , Amino acyl-tRNA synthetases/usage thérapeutiqueRÉSUMÉ
AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.
Sujet(s)
Antituberculeux/pharmacologie , Composés aza/pharmacologie , Composés du bore/pharmacologie , Hydrocarbures fluorés/pharmacologie , Inhibines/antagonistes et inhibiteurs , Mycobacterium tuberculosis/effets des médicaments et des substances chimiques , Tuberculose pulmonaire/traitement médicamenteux , Animaux , Charge bactérienne/effets des médicaments et des substances chimiques , Modèles animaux de maladie humaine , Développement de médicament , Femelle , Isoniazide/pharmacologie , Poumon/anatomopathologie , Souris , Souris de lignée C3H , Tests de sensibilité microbienne , Tuberculose pulmonaire/microbiologieRÉSUMÉ
African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.
Sujet(s)
Facteur de spécificité de clivage et polyadénylation/antagonistes et inhibiteurs , Protéines de protozoaire/antagonistes et inhibiteurs , Trypanocides/pharmacologie , Trypanosoma brucei brucei/physiologie , Maladie du sommeil/prévention et contrôle , Animaux , Benzamides/pharmacologie , Benzamides/usage thérapeutique , Composés du bore/pharmacologie , Composés du bore/usage thérapeutique , Systèmes CRISPR-Cas , Noyau de la cellule/génétique , Noyau de la cellule/métabolisme , Facteur de spécificité de clivage et polyadénylation/génétique , Facteur de spécificité de clivage et polyadénylation/métabolisme , Résistance aux substances/effets des médicaments et des substances chimiques , Résistance aux substances/génétique , Techniques de knock-down de gènes , Banque de gènes , Tests de criblage à haut débit/méthodes , Humains , Simulation de docking moléculaire , Protéines de protozoaire/génétique , Protéines de protozoaire/métabolisme , Maturation post-transcriptionnelle des ARN/effets des médicaments et des substances chimiques , ARN messager/métabolisme , ARN des protozoaires/métabolisme , Trypanocides/usage thérapeutique , Trypanosoma brucei brucei/effets des médicaments et des substances chimiques , Maladie du sommeil/transmission , Maladie du sommeil/médecine vétérinaire , Valine/analogues et dérivés , Valine/pharmacologie , Valine/usage thérapeutiqueRÉSUMÉ
We identified a series of novel 7-phenyl benzoxaborole compounds with activity against Mycobacterium tuberculosis. Compounds had a range of activity with inhibitory concentrations (IC90) as low as 5.1 µM and no cytotoxicity against eukaryotic cells (IC50 > 50 µM). Compounds were active against intracellular mycobacteria cultured in THP-1 macrophages. We isolated and characterized resistant mutants with mutations in NADH dehydrogenase (Ndh) or the regulatory protein Mce3R. Mutations suggest that Ndh may be the target of this series.
Sujet(s)
Composés du bore/pharmacologie , Composés hétérocycliques bicycliques/pharmacologie , Mycobacterium tuberculosis/effets des médicaments et des substances chimiques , Protéines bactériennes/antagonistes et inhibiteurs , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Composés du bore/composition chimique , Composés du bore/toxicité , Composés hétérocycliques bicycliques/composition chimique , Composés hétérocycliques bicycliques/toxicité , Relation dose-effet des médicaments , Résistance bactérienne aux médicaments , Humains , Tests de sensibilité microbienne , Mutation , Mycobacterium tuberculosis/enzymologie , Mycobacterium tuberculosis/génétique , NADH dehydrogenase/antagonistes et inhibiteurs , NADH dehydrogenase/génétique , NADH dehydrogenase/métabolisme , Cellules THP-1RÉSUMÉ
There is an urgent need to develop new and safer antitubercular agents that possess a novel mode of action. We synthesized and evaluated a novel series of 3-aminomethyl 4-halogen benzoxaboroles as Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (LeuRS) inhibitors. A number of Mtb LeuRS inhibitors were identified that demonstrated good antitubercular activity with high selectivity over human mitochondrial and cytoplasmic LeuRS. Further evaluation of these Mtb LeuRS inhibitors by in vivo pharmacokinetics (PK) and murine tuberculosis (TB) efficacy models led to the discovery of GSK3036656 (abbreviated as GSK656). This molecule shows potent inhibition of Mtb LeuRS (IC50 = 0.20 µM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 µM). Additionally, it is highly selective for the Mtb LeuRS enzyme with IC50 of >300 µM and 132 µM for human mitochondrial LeuRS and human cytoplasmic LeuRS, respectively. In addition, it exhibits remarkable PK profiles and efficacy against Mtb in mouse TB infection models with superior tolerability over initial leads. This compound has been progressed to clinical development for the treatment of tuberculosis.
Sujet(s)
Antituberculeux/synthèse chimique , Antituberculeux/pharmacologie , Composés du bore/pharmacologie , Antienzymes/synthèse chimique , Antienzymes/pharmacologie , Composés hétérobicycliques/pharmacologie , Leucine-tRNA ligase/antagonistes et inhibiteurs , Mycobacterium tuberculosis/effets des médicaments et des substances chimiques , Animaux , Antituberculeux/pharmacocinétique , Composés du bore/synthèse chimique , Composés du bore/pharmacocinétique , Découverte de médicament , Antienzymes/pharmacocinétique , Femelle , Composés hétérobicycliques/synthèse chimique , Humains , Souris , Souris de lignée C57BL , Mycobacterium tuberculosis/enzymologie , Relation structure-activité , Spécificité du substratRÉSUMÉ
We identified a novel 6-benzyl ether benzoxaborole with potent activity against Mycobacterium tuberculosis The compound had an MIC of 2 µM in liquid medium. The compound was also able to prevent growth on solid medium at 0.8 µM and was active against intracellular bacteria (50% inhibitory concentration [IC50] = 3.6 µM) without cytotoxicity against eukaryotic cells (IC50 > 100 µM). We isolated resistant mutants (MIC ≥ 100 µM), which had mutations in Rv1683, Rv3068c, and Rv0047c.
Sujet(s)
Antituberculeux/pharmacologie , Mycobacterium tuberculosis/effets des médicaments et des substances chimiques , Tuberculose multirésistante/traitement médicamenteux , Tuberculose pulmonaire/traitement médicamenteux , Antituberculeux/effets indésirables , Lignée cellulaire tumorale , Résistance bactérienne aux médicaments/génétique , Cellules HepG2 , Humains , Tests de sensibilité microbienne , Mycobacterium tuberculosis/génétique , Mycobacterium tuberculosis/croissance et développement , Tuberculose multirésistante/microbiologie , Tuberculose pulmonaire/microbiologieRÉSUMÉ
Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC50 32 nM), Ugandan field isolates (mean ex vivo IC50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED90 0.34 and 0.57 mg kg-1, respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target.
Sujet(s)
Antipaludiques/pharmacologie , Composés du bore/pharmacologie , Facteur de spécificité de clivage et polyadénylation/composition chimique , Plasmodium falciparum/effets des médicaments et des substances chimiques , Protéines de protozoaire/composition chimique , ARN messager/génétique , Séquence d'acides aminés , Animaux , Antipaludiques/synthèse chimique , Composés du bore/synthèse chimique , Systèmes CRISPR-Cas , Domaine catalytique , Facteur de spécificité de clivage et polyadénylation/antagonistes et inhibiteurs , Facteur de spécificité de clivage et polyadénylation/génétique , Facteur de spécificité de clivage et polyadénylation/métabolisme , Résistance aux substances/génétique , Érythrocytes/effets des médicaments et des substances chimiques , Érythrocytes/parasitologie , Édition de gène/méthodes , Humains , Paludisme/traitement médicamenteux , Paludisme/parasitologie , Paludisme à Plasmodium falciparum/traitement médicamenteux , Paludisme à Plasmodium falciparum/parasitologie , Souris , Simulation de docking moléculaire , Mutation , Plasmodium berghei/effets des médicaments et des substances chimiques , Plasmodium berghei/génétique , Plasmodium berghei/croissance et développement , Plasmodium berghei/métabolisme , Plasmodium falciparum/génétique , Plasmodium falciparum/croissance et développement , Plasmodium falciparum/métabolisme , Liaison aux protéines , Motifs et domaines d'intéraction protéique , Structure secondaire des protéines , Protéines de protozoaire/antagonistes et inhibiteurs , Protéines de protozoaire/génétique , Protéines de protozoaire/métabolisme , ARN messager/métabolisme , Alignement de séquences , Similitude de séquences d'acides aminés , Trophozoïtes/effets des médicaments et des substances chimiques , Trophozoïtes/génétique , Trophozoïtes/croissance et développement , Trophozoïtes/métabolismeRÉSUMÉ
The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.
Sujet(s)
Antituberculeux/pharmacologie , Leucine-tRNA ligase/antagonistes et inhibiteurs , Mycobacterium tuberculosis/effets des médicaments et des substances chimiques , Inhibiteurs de la synthèse protéique/pharmacologie , Administration par voie orale , Animaux , Antituberculeux/administration et posologie , Antituberculeux/composition chimique , Antituberculeux/pharmacocinétique , Modèles animaux de maladie humaine , Évaluation préclinique de médicament/méthodes , Femelle , Humains , Leucine-tRNA ligase/composition chimique , Leucine-tRNA ligase/génétique , Souris de lignée BALB C , Souris de lignée C57BL , Lignées consanguines de souris , Tests de sensibilité microbienne , Mycobacterium smegmatis/effets des médicaments et des substances chimiques , Mycobacterium smegmatis/génétique , Mycobacterium tuberculosis/génétique , Inhibiteurs de la synthèse protéique/administration et posologie , Inhibiteurs de la synthèse protéique/composition chimique , Inhibiteurs de la synthèse protéique/pharmacocinétique , Relation structure-activité , Tuberculose/traitement médicamenteux , Cellules VeroRÉSUMÉ
The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNA(Leu) in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.
Sujet(s)
Antiprotozoaires/pharmacologie , Composés du bore/pharmacologie , Cryptosporidium parvum/effets des médicaments et des substances chimiques , Leucine-tRNA ligase/antagonistes et inhibiteurs , Leucine-tRNA ligase/composition chimique , Toxoplasma/effets des médicaments et des substances chimiques , Animaux , Antiprotozoaires/composition chimique , Antiprotozoaires/métabolisme , Composés du bore/composition chimique , Cristallographie aux rayons X , Chiens , Évaluation préclinique de médicament/méthodes , Antienzymes/composition chimique , Antienzymes/pharmacologie , Fibroblastes/effets des médicaments et des substances chimiques , Fibroblastes/parasitologie , Humains , Leucine-tRNA ligase/métabolisme , Cellules rénales canines Madin-Darby/parasitologie , Simulation de docking moléculaire , Conformation des protéinesRÉSUMÉ
There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.
Sujet(s)
Antipaludiques/pharmacologie , Leucine-tRNA ligase/métabolisme , Paludisme à Plasmodium falciparum/traitement médicamenteux , Plasmodium falciparum/effets des médicaments et des substances chimiques , Composés du bore/pharmacologie , Résistance aux substances/effets des médicaments et des substances chimiques , Concentration inhibitrice 50 , Paludisme à Plasmodium falciparum/parasitologie , Plasmodium falciparum/métabolismeRÉSUMÉ
Tavaborole topical solution, 5% (tavaborole) is a novel, boron-based, antifungal pharmaceutical agent indicated for treatment of toenail onychomycosis due to the dermatophytes Trichophyton rubrum or Trichophyton mentagrophytes. In preclinical studies, tavaborole effectively penetrated through full-thickness, non-diseased cadaver fingernails, including those with up to four layers of nail polish. Limited systemic absorption was observed following topical application of tavaborole. In phase III clinical trials involving patients with distal subungual onychomycosis affecting 20-60% of a target great toenail, significantly more patients treated with tavaborole versus vehicle achieved completely clear nail with negative mycology following daily application for 48 weeks. Treatment-emergent adverse events reported by at least 1% of patients treated with tavaborole and at a greater frequency versus vehicle included ingrown toenail, exfoliation, erythema and dermatitis. Treatment discontinuations were uncommon. Results from preclinical studies and phase III clinical trials establish tavaborole as a safe and efficacious treatment for toenail onychomycosis.
Sujet(s)
Composés du bore/administration et posologie , Composés hétérocycliques bicycliques/administration et posologie , Dermatoses du pied/traitement médicamenteux , Onychomycose/traitement médicamenteux , Administration par voie topique , Antifongiques/administration et posologie , Composés du bore/effets indésirables , Composés du bore/pharmacocinétique , Composés du bore/pharmacologie , Composés hétérocycliques bicycliques/effets indésirables , Composés hétérocycliques bicycliques/pharmacocinétique , Composés hétérocycliques bicycliques/pharmacologie , Interactions médicamenteuses , Humains , SolutionsRÉSUMÉ
AN3365 (MIC(50/90), 0.5/1 µg/ml) was active against Enterobacteriaceae, including a subset of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains (MIC(50/90), 1/2 µg/ml). AN3365 inhibited 98.0 and 92.2% of wild-type (MIC(50/90), 2/8 µg/ml) and carbapenem-resistant (MIC(50/90), 4/8 µg/ml) Pseudomonas aeruginosa strains, respectively, at ≤ 8 µg/ml. AN3365 also demonstrated activity against wild-type Acinetobacter baumannii (MIC(50/90), 2/8 µg/ml) and Stenotrophomonas maltophilia (MIC(50/90), 2/4 µg/ml), while it was less active against multidrug-resistant A. baumannii (MIC50/90, 8/16 µg/ml) and Burkholderia cepacia (MIC(50/90), 8/32 µg/ml).
Sujet(s)
Antibactériens/pharmacologie , Bore/composition chimique , Enterobacteriaceae/effets des médicaments et des substances chimiques , Bactéries à Gram négatif/effets des médicaments et des substances chimiques , Tests de sensibilité microbienne/normes , Inhibiteurs de la synthèse protéique/pharmacologie , Antibactériens/composition chimique , Multirésistance bactérienne aux médicaments , Enterobacteriaceae/classification , Enterobacteriaceae/isolement et purification , Bactéries à Gram négatif/classification , Bactéries à Gram négatif/isolement et purification , Infections bactériennes à Gram négatif/microbiologie , Humains , Inhibiteurs de la synthèse protéique/composition chimiqueRÉSUMÉ
Gram-negative bacteria cause approximately 70% of the infections in intensive care units. A growing number of bacterial isolates responsible for these infections are resistant to currently available antibiotics and to many in development. Most agents under development are modifications of existing drug classes, which only partially overcome existing resistance mechanisms. Therefore, new classes of Gram-negative antibacterials with truly novel modes of action are needed to circumvent these existing resistance mechanisms. We have previously identified a new a way to inhibit an aminoacyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS), in fungi via the oxaborole tRNA trapping (OBORT) mechanism. Herein, we show how we have modified the OBORT mechanism using a structure-guided approach to develop a new boron-based antibiotic class, the aminomethylbenzoxaboroles, which inhibit bacterial leucyl-tRNA synthetase and have activity against Gram-negative bacteria by largely evading the main efflux mechanisms in Escherichia coli and Pseudomonas aeruginosa. The lead analogue, AN3365, is active against Gram-negative bacteria, including Enterobacteriaceae bearing NDM-1 and KPC carbapenemases, as well as P. aeruginosa. This novel boron-based antibacterial, AN3365, has good mouse pharmacokinetics and was efficacious against E. coli and P. aeruginosa in murine thigh infection models, which suggest that this novel class of antibacterials has the potential to address this unmet medical need.
Sujet(s)
Amino acyl-tRNA synthetases/antagonistes et inhibiteurs , Antibactériens/pharmacologie , Composés du bore/pharmacologie , Escherichia coli/effets des médicaments et des substances chimiques , Infections bactériennes à Gram négatif/traitement médicamenteux , Pseudomonas aeruginosa/effets des médicaments et des substances chimiques , Amino acyl-tRNA synthetases/métabolisme , Animaux , Antibactériens/synthèse chimique , Antibactériens/pharmacocinétique , Protéines bactériennes/antagonistes et inhibiteurs , Protéines bactériennes/métabolisme , Composés du bore/synthèse chimique , Composés du bore/pharmacocinétique , Cristallographie aux rayons X , Découverte de médicament , Multirésistance bactérienne aux médicaments/effets des médicaments et des substances chimiques , Escherichia coli/enzymologie , Femelle , Infections bactériennes à Gram négatif/microbiologie , Humains , Leucine/métabolisme , Souris , Tests de sensibilité microbienne , Simulation de docking moléculaire , Pseudomonas aeruginosa/enzymologie , Relation structure-activité , Cuisse/microbiologie , Inhibiteurs des bêta-lactamases , bêta-Lactamases/métabolismeRÉSUMÉ
We have designed and synthesized a novel class of compounds based on fluoroquinolone antibacterial prototype. The design concept involved the replacement of the 3-carboxylic acid in ciprofloxacin with an oxaborole-fused ring as an acid-mimicking group. The synthetic method employed in this work provides a good example of incorporating boron atom in complex molecules with multiple functional groups. The antibacterial activity of the newly synthesized compounds has been evaluated.
Sujet(s)
Antibactériens/synthèse chimique , Antibactériens/pharmacologie , Composés du bore/synthèse chimique , Composés du bore/pharmacologie , Fluoroquinolones/synthèse chimique , Fluoroquinolones/pharmacologie , Composés hétérocycliques bicycliques/synthèse chimique , Composés hétérocycliques bicycliques/pharmacologie , Ciprofloxacine/composition chimique , Ciprofloxacine/pharmacologie , Relation structure-activitéRÉSUMÉ
We have used boron-based molecules to create novel, competitive, reversible inhibitors of phosphodiesterase 4 (PDE4). The co-crystal structure reveals a binding configuration which is unique compared to classical catechol PDE4 inhibitors, with boron binding to the activated water in the bimetal center. These phenoxybenzoxaboroles can be optimized to generate submicromolar potency enzyme inhibitors, which inhibit TNF-α, IL-2, IFN-γ, IL-5 and IL-10 activities in vitro and show safety and efficacy for topical treatment of human psoriasis. They provide a valuable new route for creating novel potent anti-PDE4 inhibitors.
Sujet(s)
Composés du bore/composition chimique , Composés du bore/pharmacologie , Cyclic Nucleotide Phosphodiesterases, Type 4/composition chimique , Cyclic Nucleotide Phosphodiesterases, Type 4/métabolisme , Inhibiteurs de la phosphodiestérase/composition chimique , Inhibiteurs de la phosphodiestérase/pharmacologie , Anti-inflammatoires non stéroïdiens/composition chimique , Anti-inflammatoires non stéroïdiens/pharmacologie , Fixation compétitive , Composés hétérocycliques bicycliques/composition chimique , Composés hétérocycliques bicycliques/pharmacologie , Domaine catalytique , Cristallographie aux rayons X , Cyclic Nucleotide Phosphodiesterases, Type 4/génétique , Cytokines/biosynthèse , Humains , Techniques in vitro , Isoenzymes/antagonistes et inhibiteurs , Isoenzymes/composition chimique , Isoenzymes/génétique , Isoenzymes/métabolisme , Cinétique , Agranulocytes/effets des médicaments et des substances chimiques , Agranulocytes/immunologie , Métaux/composition chimique , Modèles moléculaires , Protéines recombinantes/antagonistes et inhibiteurs , Protéines recombinantes/composition chimique , Protéines recombinantes/génétique , Protéines recombinantes/métabolismeRÉSUMÉ
The broad-spectrum benzoxaborole antifungal AN2690 blocks protein synthesis by inhibiting leucyl-tRNA synthetase (LeuRS) via a novel oxaborole tRNA trapping mechanism in the editing site. Herein, one set of resistance mutations is at Asp487 outside the LeuRS hydrolytic editing pocket, in a region of unknown function. It is located within a eukaryote/archaea specific insert I4, which forms part of a cap over a benzoxaborole-AMP that is bound in the LeuRS CP1 domain editing active site. Mutational and biochemical analysis at Asp487 identified a salt bridge between Asp487 and Arg316 in the hinge region of the I4 cap of yeast LeuRS that is critical for tRNA deacylation. We hypothesize that this electrostatic interaction stabilizes the cap during binding of the editing substrate for hydrolysis.
Sujet(s)
Composés du bore/métabolisme , Composés hétérocycliques bicycliques/métabolisme , Résistance des champignons aux médicaments/génétique , Leucine-tRNA ligase/composition chimique , Leucine-tRNA ligase/génétique , Mutation , Coiffes des ARN/composition chimique , Édition des ARN , Séquence d'acides aminés , Antifongiques/composition chimique , Antifongiques/pharmacologie , Composés du bore/composition chimique , Composés hétérocycliques bicycliques/composition chimique , Analyse de mutations d'ADN , Leucine-tRNA ligase/antagonistes et inhibiteurs , Leucine-tRNA ligase/métabolisme , Données de séquences moléculaires , Structure moléculaire , Mutagenèse dirigée , Structure tertiaire des protéines , Saccharomyces cerevisiae/enzymologie , Saccharomyces cerevisiae/génétique , Alignement de séquences , Électricité statiqueRÉSUMÉ
A new class of benzoxaborole ß-lactamase inhibitors were designed and synthesized. 6-Aryloxy benzoxaborole 22 inhibited AmpC P99 and CMY-2 with K(i) values in the low nanomolar range. Compound 22 restored antibacterial activity of ceftazidime against Enterobacter cloacae P99 expressing AmpC, a class C ß-lactamase enzyme. The SAR around the arylbenzoxaboroles, which included the influence of linker and substitutions was also established.
Sujet(s)
Antibactériens/synthèse chimique , Benzoxazoles/composition chimique , Composés du bore/composition chimique , Antienzymes/synthèse chimique , Pyrazines/synthèse chimique , Inhibiteurs des bêta-lactamases , Antibactériens/composition chimique , Antibactériens/pharmacologie , Composés du bore/synthèse chimique , Composés du bore/pharmacologie , Enterobacter cloacae/effets des médicaments et des substances chimiques , Antienzymes/composition chimique , Antienzymes/pharmacologie , Tests de sensibilité microbienne , Pyrazines/composition chimique , Pyrazines/pharmacologie , Relation structure-activité , bêta-Lactamases/métabolismeRÉSUMÉ
Leucyl-tRNA synthetase (LeuRS) specifically links leucine to the 3' end of tRNA(leu) isoacceptors. The overall accuracy of the two-step aminoacylation reaction is enhanced by an editing domain that hydrolyzes mischarged tRNAs, notably ile-tRNA(leu). We present crystal structures of the editing domain from two eukaryotic cytosolic LeuRS: human and fungal pathogen Candida albicans. In comparison with previous structures of the editing domain from bacterial and archeal kingdoms, these structures show that the LeuRS editing domain has a conserved structural core containing the active site for hydrolysis, with distinct bacterial, archeal, or eukaryotic specific peripheral insertions. It was recently shown that the benzoxaborole antifungal compound AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-1,2-benzoxaborole) inhibits LeuRS by forming a covalent adduct with the 3' adenosine of tRNA(leu) at the editing site, thus locking the enzyme in an inactive conformation. To provide a structural basis for enhancing the specificity of these benzoxaborole antifungals, we determined the structure at 2.2 A resolution of the C. albicans editing domain in complex with a related compound, AN3018 (6-(ethylamino)-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol), using AMP as a surrogate for the 3' adenosine of tRNA(leu). The interactions between the AN3018-AMP adduct and C. albicans LeuRS are similar to those previously observed for bacterial LeuRS with the AN2690 adduct, with an additional hydrogen bond to the extra ethylamine group. However, compared to bacteria, eukaryotic cytosolic LeuRS editing domains contain an extra helix that closes over the active site, largely burying the adduct and providing additional direct and water-mediated contacts. Small differences between the human domain and the fungal domain could be exploited to enhance fungal specificity.
Sujet(s)
Antifongiques/composition chimique , Antifongiques/pharmacologie , Composés du bore/composition chimique , Composés du bore/pharmacologie , Composés hétérocycliques bicycliques/composition chimique , Composés hétérocycliques bicycliques/pharmacologie , Candida albicans/enzymologie , Leucine-tRNA ligase/antagonistes et inhibiteurs , Leucine-tRNA ligase/composition chimique , Séquence d'acides aminés , Cristallographie aux rayons X , Drogues fabriquées clandestinement , Humains , Modèles moléculaires , Données de séquences moléculaires , Phylogenèse , Structure tertiaire des protéines , Alignement de séquencesRÉSUMÉ
Approximately one-third of the world's population carries Staphylococcus aureus. The recent emergence of extreme drug resistant strains that are resistant to the "antibiotic of last resort", vancomycin, has caused a further increase in the pressing need to discover new drugs against this organism. The S. aureus enoyl reductase, saFabI, is a validated target for drug discovery. To drive the development of potent and selective saFabI inhibitors, we have studied the mechanism of the enzyme and analyzed the interaction of saFabI with triclosan and two related diphenyl ether inhibitors. Results from kinetic assays reveal that saFabI is NADPH-dependent, and prefers acyl carrier protein substrates carrying fatty acids with long acyl chains. On the basis of product inhibition studies, we propose that the reaction proceeds via an ordered sequential ternary complex, with the ACP substrate binding first, followed by NADPH. The interaction of NADPH with the enzyme has been further explored by site-directed mutagenesis, and residues R40 and K41 have been shown to be involved in determining the specificity of the enzyme for NADPH compared to NADH. Finally, in preliminary inhibition studies, we have shown that triclosan, 5-ethyl-2-phenoxyphenol (EPP), and 5-chloro-2-phenoxyphenol (CPP) are all nanomolar slow-onset inhibitors of saFabI. These compounds inhibit the growth of S. aureus with MIC values of 0.03-0.06 microg/mL. Upon selection for resistance, three novel safabI mutations, A95V, I193S, and F204S, were identified. Strains containing these mutations had MIC values approximately 100-fold larger than that of the wild-type strain, whereas the purified mutant enzymes had K i values 5-3000-fold larger than that of wild-type saFabI. The increase in both MIC and K i values caused by the mutations supports the proposal that saFabI is the intracellular target for the diphenyl ether-based inhibitors.
Sujet(s)
Enoyl-(acyl-carrier protein) reductase (NADH)/composition chimique , Enoyl-(acyl-carrier protein) reductase (NADH)/métabolisme , Antienzymes/pharmacologie , Staphylococcus aureus/enzymologie , Séquence d'acides aminés , Catalyse , Séquence conservée , Enoyl-(acyl-carrier protein) reductase (NADH)/génétique , Enoyl-(acyl-carrier protein) reductase (NADH)/isolement et purification , Activation enzymatique/effets des médicaments et des substances chimiques , Cinétique , Modèles moléculaires , Données de séquences moléculaires , Structure moléculaire , Mutation/génétique , Éthers phényliques/pharmacologie , Alignement de séquences , Staphylococcus aureus/génétique , Spécificité du substratRÉSUMÉ
Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.