RESUMO
RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone. CONCLUSIONS: In the fibrotic lung, dual inhibition of integrins αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.
Assuntos
Antifibróticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Integrina alfa6beta1/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Receptores de Vitronectina/antagonistas & inibidores , Animais , Bleomicina , Linhagem Celular , Técnicas de Cocultura , Cadeia alfa 1 do Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Integrina alfa6beta1/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Fosforilação , Receptores de Vitronectina/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismoRESUMO
The optimization of a series of benzimidazole-benzoxaborole hybrid molecules linked via a ketone that exhibit good activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness, is described. The lead identified in this series, 21 (AN15470), was found to have acceptable pharmacokinetic properties to enable an evaluation following oral dosing in an animal model of onchocerciasis. Compound 21was effective in killing worms implanted in Mongolian gerbils when dosed orally as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 7 days.
Assuntos
Benzimidazóis/uso terapêutico , Compostos de Boro/uso terapêutico , Cetonas/química , Oncocercose Ocular/tratamento farmacológico , Administração Oral , Animais , Benzimidazóis/farmacocinética , Compostos de Boro/farmacocinética , Modelos Animais de Doenças , Feminino , Filaricidas/farmacocinética , Filaricidas/uso terapêutico , Gerbillinae , MasculinoRESUMO
A series of benzimidazole-benzoxaborole hybrid molecules linked via an amide linker are described that exhibit good in vitro activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness. The lead identified in this series, 8a (AN8799), was found to have acceptable pharmacokinetic properties to enable evaluation in animal models of human filariasis. Compound 8a was effective in killing Brugia malayi, B. pahangi, and Litomosoides sigmodontis worms present in Mongolian gerbils when dosed subcutaneously as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 28 days. The measurement of plasma levels of 8a at the end of the dosing period and at the time of sacrifice revealed an interesting dependence of activity on the extended exposure for both 8a and the positive control, flubendazole.
Assuntos
Benzimidazóis/uso terapêutico , Compostos de Boro/uso terapêutico , Brugia/efeitos dos fármacos , Oncocercose/tratamento farmacológico , Amidas , Animais , Benzimidazóis/farmacocinética , Compostos de Boro/farmacocinética , Feminino , Filaricidas/farmacocinética , Filaricidas/uso terapêutico , Gerbillinae , Masculino , Onchocerca volvulus/efeitos dos fármacosRESUMO
A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti- Wolbachia antibiotics and, as such, may be useful in the treatment of filarial infections caused by Onchocerca volvulus, resulting in Onchocerciasis or river blindness, or Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in lymphatic filariasis. These two important neglected tropical diseases disproportionately impact patients in the developing world. The lead preclinical candidate compound containing 7-fluoro-6-oxybenzoxaborole (15, AN11251) was shown to have good in vitro anti- Wolbachia activity and physicochemical and pharmacokinetic properties providing high exposure in plasma. The lead was effective in reducing the Wolbachia load in filarial worms following oral administration to mice.
Assuntos
Boro/farmacologia , Diterpenos/farmacologia , Filariose Linfática/tratamento farmacológico , Filaricidas/uso terapêutico , Oncocercose/tratamento farmacológico , Compostos Policíclicos/farmacologia , Wolbachia/efeitos dos fármacos , Wuchereria bancrofti/efeitos dos fármacos , Animais , Boro/química , Diterpenos/química , Filaricidas/farmacocinética , Filaricidas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Compostos Policíclicos/química , PleuromutilinasRESUMO
Onychomycosis is a fungal infection of the nail primarily caused by the dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes. The topical-based treatment of onychomycosis remains a challenge because of the difficulty associated with penetrating the dense, protective structure of the keratinized nail plate. Tavaborole is a novel small-molecule antifungal agent recently approved in the United States for the topical treatment of toenail onychomycosis. The low molecular weight, slight water solubility, and boron chemistry of tavaborole maximize nail penetration after topical application, allowing for effective targeting of the infection in the nail bed. The efficacy of tavaborole is associated with its novel mechanism of action, whereby it inhibits the fungal leucyl-tRNA synthetase (LeuRS) enzyme. Because LeuRS is an essential component in fungal protein synthesis, inhibition of LeuRS ultimately leads to fungal cell death. Tavaborole is the first boron-based antifungal medication approved for the treatment of mild-to-moderate onychomycosis and presents patients with a new topical option. Previously, ciclopirox and efinaconazole were the only approved topical treatments for onychomycosis. This article details the properties that are at the core of the clinical benefits associated with tavaborole.
Assuntos
Compostos de Boro/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Onicomicose/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Unhas/efeitos dos fármacos , Unhas/microbiologia , Onicomicose/metabolismo , Resultado do TratamentoRESUMO
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.
Assuntos
Antituberculosos/síntese química , Antituberculosos/farmacologia , Compostos de Boro/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Leucina-tRNA Ligase/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Animais , Antituberculosos/farmacocinética , Compostos de Boro/síntese química , Compostos de Boro/farmacocinética , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Feminino , Compostos Heterocíclicos com 2 Anéis/síntese química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/enzimologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer's disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). METHODOLOGY/PRINCIPAL FINDINGS: A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite's genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target. CONCLUSIONS/SIGNIFICANCE: The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A's contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets.
Assuntos
Anti-Helmínticos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Animais , Animais Geneticamente Modificados/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Domínio Catalítico , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Locomoção/efeitos dos fármacos , Schistosoma mansoni/anatomia & histologia , Schistosoma mansoni/fisiologiaRESUMO
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.
Assuntos
Antituberculosos/farmacologia , Leucina-tRNA Ligase/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Administração Oral , Animais , Antituberculosos/administração & dosagem , Antituberculosos/química , Antituberculosos/farmacocinética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Leucina-tRNA Ligase/química , Leucina-tRNA Ligase/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Inibidores da Síntese de Proteínas/administração & dosagem , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacocinética , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico , Células VeroRESUMO
Psoriasis and atopic dermatitis are skin diseases affecting millions of patients. Here, we characterize benzoxaborole phosphodiesterase (PDE)-4 inhibitors, a new topical class that has demonstrated therapeutic benefit for psoriasis and atopic dermatitis in phase 2 or phase 3 studies. Crisaborole [AN2728, 4-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)oxy)benzonitrile], compd2 [2-ethoxy-6-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)oxy)nicotinonitrile], compd3 [6-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)oxy)-2-(2-isopropoxyethoxy)nicotinonitrile], and compd4 [5-chloro-6-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)oxy)-2-((4-oxopentyl)oxy)nicotinonitrile] are potent PDE4 inhibitors with similar affinity for PDE4 isoforms and equivalent inhibition on the catalytic domain and the full-length enzyme. These benzoxaboroles are less active on other PDE isozymes. Compd4 binds to the catalytic domain of PDE4B2 with the oxaborole group chelating the catalytic bimetal and overlapping with the phosphate in cAMP during substrate hydrolysis, and the interaction extends into the adenine pocket. In cell culture, benzoxaborole PDE4 inhibitors suppress the release of tumor necrosis factor-α, interleukin (IL)-23, IL-17, interferon-γ, IL-4, IL-5, IL-13, and IL-22, and these cytokines contribute to the pathologic changes in skin structure and barrier functions as well as immune dysregulation in atopic dermatitis and psoriasis. Treatment with compd3 or N(6),2'-O-dibutyryladenosine 3',5'-cyclic monophosphate increases cAMP response element binding protein phosphorylation in human monocytes and decreases extracellular signal-regulated kinase phosphorylation in human T cells; these changes lead to reduced cytokine production and are among the mechanisms by which compd3 blocks cytokine release. Topical compd3 penetrates the skin and suppresses phorbol myristate acetate-induced IL-13, IL-22, IL-17F, and IL-23 transcription and calcipotriol-induced thymic stromal lymphopoietin expression in mouse skin. Skin thinning is a major dose-limiting side effect of glucocorticoids. By contrast, repeated application of compd3 did not thin mouse skin. These findings show the potential benefits and safety of benzoxaborole PDE4 inhibitors for the treatment of psoriasis and atopic dermatitis.
Assuntos
Compostos de Boro/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Dermatite Atópica/tratamento farmacológico , Inibidores da Fosfodiesterase 4/farmacologia , Psoríase/tratamento farmacológico , Pele/efeitos dos fármacos , Pele/patologia , Administração Tópica , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Domínio Catalítico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Citocinas/metabolismo , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/uso terapêutico , Fosforilação/efeitos dos fármacos , Psoríase/metabolismo , Psoríase/patologia , Pele/metabolismo , Linfopoietina do Estroma do TimoRESUMO
Benzoxaboroles are a novel class of drug-like compounds that have been rich sources of novel inhibitors for various enzymes and of new drugs. While examining benzoxaborole activity in phenotypic screens, our attention was attracted by the (aminomethylphenoxy)benzoxaborole family, which potently inhibited Toll-like receptor-stimulated cytokine secretion from leukocytes. After considering their structure-activity relationships and the central role of kinases in leukocyte biology, we performed a kinome-wide screen to investigate the members of the (aminomethylphenoxy)benzoxaborole family. This technique identified Rho-activated kinase (ROCK) as a target. We showed competitive behavior, with respect to ATP, and then determined the ROCK2-drug cocrystal structure. The drug occupies the ATP site in which the oxaborole moiety provides hydrogen bond donors and acceptors to the hinge, and the aminomethyl group interacts with the magnesium/ATP-interacting aspartic acid common to protein kinases. The series exhibits excellent selectivity against most of the kinome, with greater than 15-fold selectivity against the next best member of the AGC protein kinase subfamily. Medicinal chemistry efforts with structure-based design resulted in a compound with a Ki of 170 nM. Cellular studies revealed strong enzyme inhibition rank correlation with suppression of intracellular phosphorylation of a ROCK substrate. The biochemical potencies of these compounds also translated to functional activity, causing smooth muscle relaxation in rat aorta and guinea pig trachea. The series exhibited oral availability and one member reduced rat blood pressure, consistent with ROCK's role in smooth muscle contraction. Thus, the benzoxaborole moiety represents a novel hinge-binding kinase scaffold that may have potential for therapeutic use.
Assuntos
Compostos de Boro/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Citocinas/sangue , Humanos , Células Jurkat , Modelos Moleculares , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/química , Proteína Fosfatase 1/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Traqueia/efeitos dos fármacos , Quinases Associadas a rho/genéticaRESUMO
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.
Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Antibacterianos/farmacologia , Compostos de Boro/farmacologia , Escherichia coli/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Aminoacil-tRNA Sintetases/metabolismo , Animais , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Compostos de Boro/síntese química , Compostos de Boro/farmacocinética , Cristalografia por Raios X , Descoberta de Drogas , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/enzimologia , Feminino , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Leucina/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa/enzimologia , Relação Estrutura-Atividade , Coxa da Perna/microbiologia , Inibidores de beta-Lactamases , beta-Lactamases/metabolismoRESUMO
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.
Assuntos
Compostos de Boro/química , Compostos de Boro/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Ligação Competitiva , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Citocinas/biossíntese , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Metais/química , Modelos Moleculares , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Antibacterianos/síntese química , Benzoxazóis/química , Compostos de Boro/química , Inibidores Enzimáticos/síntese química , Pirazinas/síntese química , Inibidores de beta-Lactamases , Antibacterianos/química , Antibacterianos/farmacologia , Compostos de Boro/síntese química , Compostos de Boro/farmacologia , Enterobacter cloacae/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Testes de Sensibilidade Microbiana , Pirazinas/química , Pirazinas/farmacologia , Relação Estrutura-Atividade , beta-Lactamases/metabolismoRESUMO
African trypanosomiasis, caused by the proto zoal pathogen Trypanosoma brucei (T. brucei), is one of the most neglected tropical diseases that are in great need of new drugs. We report the design and synthesis of T. brucei leucyl-tRNA synthetase (TbLeuRS) inhibitors and their structure--activity relationship. Benzoxaborole was used as the core structure and C(6) was modified to achieve improved affinity based on docking results that showed further binding space at this position. Indeed, compounds with C(7) substitutions showed diminished activity due to clash with the eukaryote specific I4ae helix while substitutions at C(6) gave enhanced affinity. TbLeuRS inhibitors with IC(50) as low as 1.6 µM were discovered, and the structure-activity relationship was discussed. The most potent enzyme inhibitors also showed excellent T. brucei parasite growth inhibition activity. This is the first time that TbLeuRS inhibitors are reported, and this study suggests that leucyl-tRNA synthetase (LeuRS) could be a potential target for antiparasitic drug development.
Assuntos
Ácidos Borônicos/síntese química , Leucina-tRNA Ligase/antagonistas & inibidores , Tripanossomicidas/síntese química , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Linhagem Celular , Desenho de Fármacos , Camundongos , Modelos Moleculares , Estereoisomerismo , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Inibidores Enzimáticos/farmacologia , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Catálise , Sequência Conservada , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/isolamento & purificação , Ativação Enzimática/efeitos dos fármacos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação/genética , Éteres Fenílicos/farmacologia , Alinhamento de Sequência , Staphylococcus aureus/genética , Especificidade por SubstratoRESUMO
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.