RESUMO
Although more than 98% of the human genome is non-coding1, nearly all of the drugs on the market target one of about 700 disease-related proteins. The historical reluctance to invest in non-coding RNA stems partly from requirements for drug targets to adopt a single stable conformation2. Most RNAs can adopt several conformations of similar stabilities. RNA structures also remain challenging to determine3. Nonetheless, an increasing number of diseases are now being attributed to non-coding RNA4 and the ability to target them would vastly expand the chemical space for drug development. Here we devise a screening strategy and identify small molecules that bind the non-coding RNA prototype Xist5. The X1 compound has drug-like properties and binds specifically the RepA motif6 of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that RepA can adopt multiple conformations but favours one structure in solution. X1 binding reduces the conformational space of RepA, displaces cognate interacting protein factors (PRC2 and SPEN), suppresses histone H3K27 trimethylation, and blocks initiation of X-chromosome inactivation. X1 inhibits cell differentiation and growth in a female-specific manner. Thus, RNA can be systematically targeted by drug-like compounds that disrupt RNA structure and epigenetic function.
Assuntos
Cromossomos Humanos X , RNA Longo não Codificante , Inativação do Cromossomo X , Diferenciação Celular , Cromossomos Humanos X/genética , Feminino , Histonas/metabolismo , Humanos , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genéticaRESUMO
Most drugs are developed through iterative rounds of chemical synthesis and biochemical testing to optimize the affinity of a particular compound for a protein target of therapeutic interest. This process is challenging because candidate molecules must be selected from a chemical space of more than 1060 drug-like possibilities 1 , and a single reaction used to synthesize each molecule has more than 107 plausible permutations of catalysts, ligands, additives and other parameters 2 . The merger of a method for high-throughput chemical synthesis with a biochemical assay would facilitate the exploration of this enormous search space and streamline the hunt for new drugs and chemical probes. Miniaturized high-throughput chemical synthesis3-7 has enabled rapid evaluation of reaction space, but so far the merger of such syntheses with bioassays has been achieved with only low-density reaction arrays, which analyse only a handful of analogues prepared under a single reaction condition8-13. High-density chemical synthesis approaches that have been coupled to bioassays, including on-bead 14 , on-surface 15 , on-DNA 16 and mass-encoding technologies 17 , greatly reduce material requirements, but they require the covalent linkage of substrates to a potentially reactive support, must be performed under high dilution and must operate in a mixture format. These reaction attributes limit the application of transition-metal catalysts, which are easily poisoned by the many functional groups present in a complex mixture, and of transformations for which the kinetics require a high concentration of reactant. Here we couple high-throughput nanomole-scale synthesis with a label-free affinity-selection mass spectrometry bioassay. Each reaction is performed at a 0.1-molar concentration in a discrete well to enable transition-metal catalysis while consuming less than 0.05 milligrams of substrate per reaction. The affinity-selection mass spectrometry bioassay is then used to rank the affinity of the reaction products to target proteins, removing the need for time-intensive reaction purification. This method enables the primary synthesis and testing steps that are critical to the invention of protein inhibitors to be performed rapidly and with minimal consumption of starting materials.
Assuntos
Nanotecnologia/métodos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Proteínas/química , Bioensaio , Catálise , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/química , Avaliação Pré-Clínica de Medicamentos , Cinética , Ligantes , Espectrometria de Massas , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas/antagonistas & inibidores , Especificidade por SubstratoRESUMO
Macrocyclic α-helical peptides have emerged as a compelling new therapeutic modality to tackle targets confined to the intracellular compartment. Within the scope of hydrocarbon-stapling there has been significant progress to date, including the first stapled α-helical peptide to enter into clinical trials. The principal design concept of stapled α-helical peptides is to mimic a cognate (protein) ligand relative to binding its target via an α-helical interface. However, it was the proclivity of such stapled α-helical peptides to exhibit cell permeability and proteolytic stability that underscored their promise as unique macrocyclic peptide drugs for intracellular targets. This perspective highlights key learnings as well as challenges in basic research with respect to structure-based design, innovative chemistry, cell permeability and proteolytic stability that are essential to fulfill the promise of stapled α-helical peptide drug development.
Assuntos
Descoberta de Drogas/métodos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Animais , Humanos , Compostos Macrocíclicos/farmacocinética , Modelos Moleculares , Peptídeos/farmacocinética , Conformação Proteica em alfa-HéliceRESUMO
Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.
Assuntos
Doenças Autoimunes , Janus Quinase 1 , Janus Quinase 3 , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos/métodos , Humanos , Isoenzimas , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Monitorização Imunológica/métodos , Inibidores de Proteínas Quinases/farmacologia , RatosRESUMO
Affinity selection-mass spectrometry (AS-MS) is a high-throughput screening (HTS) technique for drug discovery that enables rapid screening of large collections of compounds to identify ligands for a specific biomolecular target. AS-MS is a binding assay that is insensitive to the functional effects a ligand might have, which is important because it lets us identify novel ligands irrespective of their binding site. This approach is gaining popularity, notably due to its role in the emergence of useful agents for targeted protein degradation. This Perspective highlights the use of AS-MS techniques to explore broad chemical space and identify small-molecule ligands for biological targets that have proven challenging to address with other screening paradigms. We present chemical structures of reported AS-MS hits to illustrate the potential of this screening approach to deliver high-quality hits for further optimization. AS-MS has, thus, evolved from being an infrequent alternative to traditional HTS or DNA-encoded library strategies to now firmly establishing itself as a HTS approach for drug discovery.
RESUMO
Although the potential value of RNA as a target for new small molecule therapeutics is becoming increasingly credible, the physicochemical properties required for small molecules to selectively bind to RNA remain relatively unexplored. To investigate the druggability of RNAs with small molecules, we have employed affinity mass spectrometry, using the Automated Ligand Identification System (ALIS), to screen 42 RNAs from a variety of RNA classes, each against an array of chemically diverse drug-like small molecules (~50,000 compounds) and functionally annotated tool compounds (~5100 compounds). The set of RNA-small molecule interactions that was generated was compared with that for protein-small molecule interactions, and naïve Bayesian models were constructed to determine the types of specific chemical properties that bias small molecules toward binding to RNA. This set of RNA-selective chemical features was then used to build an RNA-focused set of ~3800 small molecules that demonstrated increased propensity toward binding the RNA target set. In addition, the data provide an overview of the specific physicochemical properties that help to enable binding to potential RNA targets. This work has increased the understanding of the chemical properties that are involved in small molecule binding to RNA, and the methodology used here is generally applicable to RNA-focused drug discovery efforts.
Assuntos
Descoberta de Drogas , Terapia de Alvo Molecular , RNA/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Ligantes , Espectrometria de Massas , Preparações Farmacêuticas , RNA/genética , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Membrana/metabolismo , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , HumanosRESUMO
The Myc oncogene is overexpressed in many cancers, yet targeting it for cancer therapy has remained elusive. One strategy for inhibition of Myc expression is through stabilization of the G-quadruplex (G4), a G-rich DNA secondary structure found within the Myc promoter; stabilization of G4s has been shown to halt transcription of downstream gene products. Here we used the Automated Ligand Identification System (ALIS), an affinity selection-mass spectrometry method, to identify compounds that bind to the Myc G4 out of a pool of compounds that had previously been shown to inhibit Myc expression in a reporter screen. Using an ALIS-based screen, we identified hits that bound to the Myc G4, a small subset of which bound preferentially relative to G4s from the promoters of five other genes. To determine functionality and specificity of the Myc G4-binding compounds in cell-based assays, we compared inhibition of Myc expression in cells with and without Myc G4 regulation. Several compounds inhibited Myc expression only in the Myc G4-containing line, and one compound was verified to function through Myc G4 binding. Our study demonstrates that ALIS can be used to identify selective nucleic acid-binding compounds from phenotypic screen hits, increasing the pool of drug targets beyond proteins.
Assuntos
Quadruplex G , Espectrometria de Massas/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular , Proliferação de Células , Avaliação Pré-Clínica de Medicamentos , Éxons/genética , Humanos , Ligantes , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA. Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA. Here, we report the first application of the Automated Ligand Detection System (ALIS), an indirect AS-MS technique, for the selective detection of small molecule-ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds (structurally distinct from both FMN and ribocil) that target the FMN riboswitch. Crystal structures reveal that different compounds induce various conformations of the FMN riboswitch, leading to different activity profiles. Our findings validate the ALIS platform for HTS screening for RNA-binding small molecules and further demonstrate that ncRNA can be broadly targeted by chemically diverse yet selective small molecules as therapeutics.
Assuntos
Descoberta de Drogas , Espectrometria de Massas/métodos , RNA/metabolismo , Bibliotecas de Moléculas Pequenas , Cristalografia por Raios X , Mononucleotídeo de Flavina/metabolismo , Ligantes , Estrutura Molecular , Pirimidinas/metabolismo , Pirimidinas/farmacologia , RiboswitchRESUMO
Inhibitors of hepatitis C virus (HCV) protease have shown marked antiviral activity in short-term clinical studies in HCV-infected individuals. The interaction of the investigational HCV protease inhibitors VX-950 and SCH 503034 with ritonavir, a potent inhibitor of cytochrome P450 3A, was studied in vitro and in vivo. In rat and human liver microsomes, the metabolism of VX-950 and SCH 503034 was strongly inhibited by the presence of 4 microM ritonavir. Upon co-dosing either VX-950 or SCH 503034 with ritonavir in rats, plasma exposure of the HCV protease inhibitors was increased by > 15-fold, and plasma concentrations 8 h after dosing were increased by > 50-fold. A human pharmacokinetic model of VX-950 co-administered with low-dose ritonavir suggested that improved efficacy and/or dosing convenience may be feasible by pharmacokinetic enhancement with ritonavir.
Assuntos
Antivirais/farmacologia , Antivirais/farmacocinética , Oligopeptídeos/farmacocinética , Prolina/análogos & derivados , Inibidores de Proteases/farmacocinética , Ritonavir/farmacologia , Animais , Hepacivirus/efeitos dos fármacos , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Animais , Oligopeptídeos/sangue , Plasma/química , Prolina/sangue , Prolina/farmacocinética , Inibidores de Proteases/sangue , Ratos , Ratos Sprague-Dawley , Ritonavir/administração & dosagemRESUMO
Due to recent advances in high throughput organic synthesis, discovery teams now need to profile increased numbers of analogs in vitro for their absorption, distribution, metabolism, and excretion (ADME) properties. Consequently, pharmaceutical companies are developing lower cost and higher throughput methods for ADME testing. As demands for metabolic stability testing have increased in our laboratory, the time required to analyze samples using high-pressure liquid chromatography-mass spectrometry (HPLC-MS) has grown rapidly and ultimately limited our data output. In this study we show that solid phase extraction-mass spectrometry (SPE-MS) is a viable alternative to HPLC-MS for monitoring small molecule stability in liver microsomes. Using the SPE-MS approach, samples can be analyzed in 24 s compared to 2.5 min on the HPLC-MS without compromising data quality, thereby alleviating the analytical bottleneck.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Microssomos Hepáticos/metabolismo , Extração em Fase Sólida/métodos , Animais , Cães , Humanos , Taxa de Depuração Metabólica , CamundongosRESUMO
Though phenotypic and target-based high-throughput screening approaches have been employed to discover new antibiotics, the identification of promising therapeutic candidates remains challenging. Each approach provides different information, and understanding their results can provide hypotheses for a mechanism of action (MoA) and reveal actionable chemical matter. Here, we describe a framework for identifying efficacy targets of bioactive compounds. High throughput biophysical profiling against a broad range of targets coupled with machine learning was employed to identify chemical features with predicted efficacy targets for a given phenotypic screen. We validate the approach on data from a set of 55â¯000 compounds in 24 historical internal antibacterial phenotypic screens and 636 bacterial targets screened in high-throughput biophysical binding assays. Models were built to reveal the relationships between phenotype, target, and chemotype, which recapitulated mechanisms for known antibacterials. We also prospectively identified novel inhibitors of dihydrofolate reductase with nanomolar antibacterial efficacy against Mycobacterium tuberculosis. Molecular modeling provided structural insight into target-ligand interactions underlying selective killing activity toward mycobacteria over human cells.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
A potent and selective Factor IXa (FIXa) inhibitor was subjected to a series of liver microsomal incubations, which generated a number of metabolites. Using automated ligand identification system-affinity selection (ALIS-AS) methodology, metabolites in the incubation mixture were prioritized by their binding affinities to the FIXa protein. Microgram quantities of the metabolites of interest were then isolated through microisolation analytical capabilities, and structurally characterized using MicroCryoProbe heteronuclear 2D NMR techniques. The isolated metabolites recovered from the NMR experiments were then submitted directly to an in vitro FIXa enzymatic assay. The order of the metabolites' binding affinity to the Factor IXa protein from the ALIS assay was completely consistent with the enzymatic assay results. This work showcases an innovative and efficient approach to uncover structure-activity relationships (SARs) and guide drug design via microisolation-structural characterization and ALIS capabilities.
Assuntos
Automação , Desenho de Fármacos , Fator IXa/antagonistas & inibidores , Fibrinolíticos/farmacologia , Ressonância Magnética Nuclear Biomolecular , Animais , Relação Dose-Resposta a Droga , Fator IXa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/metabolismo , Humanos , Ligantes , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala/métodos , NF-kappa B/antagonistas & inibidores , Relação Estrutura-Atividade , Ligantes , Espectrometria de Massas/métodos , NF-kappa B/química , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Fator 5 Associado a Receptor de TNF/antagonistas & inibidores , Fator 5 Associado a Receptor de TNF/química , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/químicaRESUMO
The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact. After filtering luciferase inhibitors and several known classes of antibiotics, approximately 50 hits remained. These compounds were examined for their ability to inhibit Escherichia coli TT, uncoupled S. pneumoniae translation or transcription, rabbit reticulocyte translation, and in vitro toxicity in human and bacterial cells. One of these compounds had the desired profile of broad-spectrum biochemical activity in bacteria and selectivity versus mammalian biochemical and whole-cell assays.
Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Biossíntese de Proteínas , Streptococcus pneumoniae/efeitos dos fármacos , Transcrição Gênica , Antibacterianos/efeitos adversos , Sequência de Bases , Linhagem Celular Tumoral , DNA Bacteriano , Genes Reporter , Humanos , Luciferases/genética , Dados de Sequência Molecular , Streptococcus pneumoniae/genéticaRESUMO
Our HTS effort yielded a preferential mGluR1 pyrimidinone antagonist 1 with lead-like characteristics. Rapid hit to lead (HTL) study identified compounds with improved functional activity and selectivity such as 1b with little improvements in ADME properties. Addition of an aminosulfonyl group on the N-1 aromatic ring led to 2f, a compound with similar in vitro biochemical profiles as those of 1b but drastically improved in vitro ADME properties. These improvements were paralleled by rat PK study characterized by low clearance and quantitative bioavailability. Compound 2f represented a true lead-like molecule that is amenable for further lead optimization (LO) evaluation.
Assuntos
Pirazóis/química , Piridinas/química , Pirimidinonas/farmacologia , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Avaliação Pré-Clínica de Medicamentos , Pirimidinonas/química , Pirimidinonas/farmacocinética , RatosRESUMO
A series of substituted thienopyridine ureas was prepared and evaluated for enzymatic and cellular inhibition of KDR kinase activity. Several of these analogs, such as 2, are potent inhibitors of KDR (<10 nM) in both enzymatic and cellular assays. Further characterization of inhibitor 2 indicated that this analog possessed excellent in vivo potency (ED50 2.1 mg/kg) as measured in an estradiol-induced mouse uterine edema model.
Assuntos
Piridinas/síntese química , Ureia/síntese química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Edema/induzido quimicamente , Estradiol , Feminino , Camundongos , Modelos Moleculares , Piridinas/farmacologia , Relação Estrutura-Atividade , Ureia/química , Ureia/farmacologia , Doenças Uterinas/patologiaRESUMO
The novel ribosome inhibitors (NRIs) are a broad-spectrum naphthyridine class that selectively inhibits bacterial protein synthesis (P. J. Dandliker et al., Antimicrob. Agents Chemother. 47:3831-3839, 2003). Footprinting experiments, using a range of NRIs and chemical modification agents on Escherichia coli ribosomes, revealed no evidence for direct protection of rRNA. In the presence of tRNA, however, we found that NRIs enhanced the known ribosomal footprinting pattern of tRNA in a dose-dependent manner. The most prominent increase in protection, at A1492/3 and A1413 in helix-44 of 16S RNA, strictly required the presence of tRNA and poly(U), and the effect was correlated with the potency of the inhibitor. Radioligand binding studies with inhibitor [(3)H]A-424902 showed that the compound binds to tRNA, either in its charged or uncharged form. The dissociation constant for [(3)H]A-424902 binding to Phe-tRNA(Phe) was determined to be 1.8 microM, near its translation inhibition potency of 1.6 muM in a cell-free S. pneumoniae extract assay. The compound did not change the binding of radiolabeled tRNA to the 30S ribosomal subunit. Taken together, these results imply that the NRIs exert their effects on protein synthesis by structurally perturbing the tRNA/30S complex at the decoding site.
Assuntos
Naftiridinas/farmacologia , RNA Ribossômico 16S/efeitos dos fármacos , RNA de Transferência/biossíntese , Ribossomos/efeitos dos fármacos , Autorradiografia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Primers do DNA , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Biossíntese de Proteínas , Pegadas de Proteínas , RNA de Transferência/genética , Ensaio Radioligante , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genéticaRESUMO
A series of 5-methoxy- and 5-hydroxy-6-fluoro-1,8-naphthyridone-3-carboxylic acid derivatives were prepared and evaluated for cell-free bacterial protein synthesis inhibition and whole cell antibacterial activity. When compared to the analogous 5-hydrogen compounds, the presence of the 5-OH group negatively affects biochemical potency. However, a tolerance of the 5-methoxy group is indicated. Only moderate whole cell antibacterial activity is seen, but this could be due to poor cellular penetration. Because only a few 7-position variants were made for this study, further investigation into this novel series combining a broader range of 7-amino derivatives with these 5-position modifications is warranted.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologiaRESUMO
High-throughput metabolic screening has been requested routinely to keep pace with high-throughput organic synthesis. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a fast gradient has become the method of choice for the task due to its sensitivity and selectivity. We have developed an automated system that consists of a robotic system for in vitro incubation and a commercially available software package for automatic MS/MS method development. A short, generic LC gradient and MS conditions that are applicable to most compounds have been developed to minimize the method development time and data analysis. This system has been used to support a number of in vitro screening assays in early drug discovery phase including microsomal stability and protein binding.