RESUMO
The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log(10) reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC(90), 0.015 µg/ml) and coagulase-negative staphylococci (MIC(90), 0.12 µg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Assuntos
Antibacterianos/uso terapêutico , Proteínas de Bactérias/antagonistas & inibidores , Benzofuranos/uso terapêutico , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Pironas/uso terapêutico , Sepse/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Administração Oral , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Benzofuranos/farmacologia , Infecções Comunitárias Adquiridas/tratamento farmacológico , Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Esquema de Medicação , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Feminino , Humanos , Cinética , Camundongos , Testes de Sensibilidade Microbiana , Pironas/farmacologia , Sepse/microbiologia , Sepse/mortalidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Taxa de SobrevidaRESUMO
Spiropiperidine naphthyridinone inhibitors of Staphylococcus aureus and Escherichia coli FabI have been prepared. Compounds 14a and 14c were identified as having sub-nanomolar E. coli FabI activity and are among the most potent FabI inhibitors yet described. The structural model of 14a bound to E. coli FabI is shown.
Assuntos
Antibacterianos/farmacologia , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Escherichia coli/enzimologia , Naftiridinas/farmacologia , Piperidinas/farmacologia , Compostos de Espiro/farmacologia , Staphylococcus aureus/enzimologia , Antibacterianos/química , Domínio Catalítico , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Modelos Moleculares , Naftiridinas/química , Piperidinas/química , Ligação Proteica , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
In the search for new antibacterial agents, the enzyme FabI has been identified as an attractive target. Employing a structure guided approach, the previously reported ene-amide series of FabI inhibitors were expanded to include 2,3,4,5-tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines. These novel series incorporate additional H-bonding functions and can be more water soluble than their naphthyridinone progenitors; diazepine 16c is shown to be efficacious in a mouse infection model.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Azepinas/química , Azepinas/farmacologia , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Animais , Antibacterianos/uso terapêutico , Azepinas/uso terapêutico , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Infecções por Escherichia coli/tratamento farmacológico , Camundongos , Modelos Moleculares , Ligação Proteica , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologiaRESUMO
The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.
Assuntos
Antibacterianos/farmacologia , Mononucleotídeo de Flavina/farmacologia , Quinoxalinas/farmacologia , RNA Bacteriano/antagonistas & inibidores , Riboswitch , Antibacterianos/síntese química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Desenho de Fármacos , Mononucleotídeo de Flavina/síntese química , Mononucleotídeo de Flavina/química , Ligantes , Estrutura Molecular , Quinoxalinas/síntese química , Quinoxalinas/química , RNA Bacteriano/genética , Relação Estrutura-AtividadeRESUMO
AFN-1252 is a novel inhibitor of FabI, an essential enzyme in fatty acid biosynthesis in Staphylococcus spp. AFN-1252 exhibits typical MIC(90) values of ≤0·015 µg/ml against diverse clinical isolates of S. aureus, oral absorption, long elimination half-live and efficacy in animal models. We now report high binding (â¼95%) to serum proteins of mouse, rat, dog and humans, associated with an eight-fold increase in minimal inhibitory concentration (MIC) and which may be responsible for the long elimination half-lives on pharmacokinetic studies. Unlike daptomycin, AFN-1252 activity is not reduced in the presence of lung surfactant. AFN-1252 exhibits a short post-antibiotic effect of 1·1 hours against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) following a 4-hour exposure period. The AFN-1252 unique spectrum of activity is not compromised by interactions with major antibiotic classes, but demonstrates synergy with low concentrations of gentamicin against MSSA and MRSA. These studies support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Assuntos
Antibacterianos/sangue , Antibacterianos/farmacologia , Benzofuranos/sangue , Benzofuranos/farmacologia , Proteínas Sanguíneas/metabolismo , Pironas/sangue , Pironas/farmacologia , Animais , Daptomicina/sangue , Daptomicina/farmacologia , Cães , Gentamicinas/sangue , Gentamicinas/farmacologia , Meia-Vida , Humanos , Masculino , Meticilina/sangue , Meticilina/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Surfactantes Pulmonares/metabolismo , Ratos , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/efeitos dos fármacosRESUMO
An optimized synthetic route to prepare ring-locked retinoid 1a has been developed. We fully describe a purification protocol that provides isomerically pure 1a in support of on-going proof of concept studies for the development of therapeutic agents to treat human ADRP. Additionally, we have found that isomerically pure 1a can be stored in amber vials under argon at -20°C for use over time (up to six months) without degradation. Thus, enabling 1a to be an accessible and valuable biological tool.
RESUMO
MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.
RESUMO
Novel 2'-heteroaryl-2-(phenoxymethyl)imidazolines have been identified as potent agonists of the cloned human alpha(1)-adrenoceptors in vitro. The nature of the 2'-heteroaryl group can have significant effects on the potency, efficacy, and subtype selectivity in this series. alpha(1A) Subtype selective agonists have been identified.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1 , Agonistas alfa-Adrenérgicos/farmacologia , Imidazóis/síntese química , Imidazóis/farmacologia , Éteres Fenílicos/síntese química , Éteres Fenílicos/farmacologia , Humanos , Ligação de Hidrogênio , Receptores Adrenérgicos alfa 1 , Proteínas Recombinantes , Relação Estrutura-Atividade , Incontinência Urinária por Estresse/prevenção & controleRESUMO
The high-throughput manual solid-phase parallel synthesis of libraries comprising thousands of discrete samples using pellicular supports (i.e. SynPhase crowns and lanterns) and a suite of novel tools and techniques is described. Key aspects of this approach include the combination of a split-split-split synthesis strategy with spatial encoding to differentiate thousands of crowns, the rapid washing and filtration of up to 48 reaction vessels in parallel, the application of an inexpensive and environmentally friendly technique to remove trifluoroacetic acid from sixteen 96-well plates in parallel, and a high-throughput method for removing cleaved crowns from reusable pin racks. Tens of thousands of discrete samples have been produced in-house using this conceptually and operationally straightforward strategy.