RESUMO
BACKGROUND: Bacteriophages (phages) have been used extensively as analytical tools to type bacterial cultures and recently for control of zoonotic foodborne pathogens in foods and in animal reservoirs. METHODS: We examined the host range, morphology, genome and proteome of the lytic E. coli O157 phage rV5, derived from phage V5, which is a member of an Escherichia coli O157:H7 phage typing set. RESULTS: Phage rV5 is a member of the Myoviridae family possessing an icosahedral head of 91 nm between opposite apices. The extended tail measures 121 x 17 nm and has a sheath of 44 x 20 nm and a 7 nm-wide core in the contracted state. It possesses a 137,947 bp genome (43.6 mol%GC) which encodes 233 ORFs and six tRNAs. Until recently this virus appeared to be phylogenetically isolated with almost 70% of its gene products ORFans. rV5 is closely related to coliphages Delta and vB-EcoM-FY3, and more distantly related to Salmonella phages PVP-SE1 and SSE-121, Cronobacter sakazakii phage vB_CsaM_GAP31, and coliphages phAPEC8 and phi92. A complete shotgun proteomic analysis was carried out on rV5, extending what had been gleaned from the genomic analyses. Host range studies revealed that rV5 is active against several other E. coli.
Assuntos
Bacteriófagos/genética , Escherichia coli O157/virologia , Genoma Viral , Especificidade de Hospedeiro , Myoviridae/fisiologia , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Genômica , Dados de Sequência Molecular , Myoviridae/classificação , Myoviridae/genética , Myoviridae/isolamento & purificação , Fases de Leitura Aberta , Filogenia , Proteômica , Proteínas Virais/genéticaRESUMO
Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.
Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Pró-Proteína Convertase 9/metabolismo , Proteólise/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Inibidores de Serina Proteinase/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
High-throughput screening (HTS) is a widespread method in early drug discovery for identifying promising chemical matter that modulates a target or phenotype of interest. Because HTS campaigns involve screening millions of compounds, it is often desirable to initiate screening with a subset of the full collection. Subsequently, virtual screening methods prioritize likely active compounds in the remaining collection in an iterative process. With this approach, orthogonal virtual screening methods are often applied, necessitating the prioritization of hits from different approaches. Here, we introduce a novel method of fusing these prioritizations and benchmark it prospectively on 17 screening campaigns using virtual screening methods in three descriptor spaces. We found that the fusion approach retrieves 15% to 65% more active chemical series than any single machine-learning method and that appropriately weighting contributions of similarity and machine-learning scoring techniques can increase enrichment by 1% to 19%. We also use fusion scoring to evaluate the tradeoff between screening more chemical matter initially in lieu of replicate samples to prevent false-positives and find that the former option leads to the retrieval of more active chemical series. These results represent guidelines that can increase the rate of identification of promising active compounds in future iterative screens.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Heurística , Interface Usuário-Computador , Aprendizado de MáquinaRESUMO
HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors.
Assuntos
Descoberta de Drogas/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Inibidores da Protease de HIV/farmacologia , Ensaios de Triagem em Larga Escala , Relação Dose-Resposta a Droga , Humanos , Cinética , Testes de Sensibilidade Microbiana , Especificidade por SubstratoRESUMO
As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)-MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥ 60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5-7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged <30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly, >99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas/métodos , Peptídeo Hidrolases/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/normas , Ensaios de Triagem em Larga Escala/normas , Humanos , Espectrometria de Massas/normas , Inibidores de Proteases/farmacologia , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
The development of chemically stable porous silicon (pSi) materials for DIOS (Desorption/Ionization on Silicon) mass spectrometry, covalent linkers cleaved in the DIOS laser pulse, and efficient methods for bond formation to immobilized species, allows for on-chip affinity purification and mass detection.
Assuntos
Silício/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Porosidade , Proteínas/análise , Propriedades de SuperfícieRESUMO
The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.
Assuntos
Anemia/tratamento farmacológico , Compostos Aza/síntese química , Hidantoínas/síntese química , Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Compostos de Espiro/síntese química , Animais , Compostos Aza/farmacocinética , Compostos Aza/farmacologia , Cães , Canal de Potássio ERG1 , Eritropoetina/biossíntese , Canais de Potássio Éter-A-Go-Go/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Hidantoínas/farmacocinética , Hidantoínas/farmacologia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Indóis/síntese química , Indóis/farmacocinética , Indóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Macaca mulatta , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Ratos , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Regulação para CimaRESUMO
To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the "straight" parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the "bent" structural isomer (parnafungin B).
Assuntos
Oxazolidinonas/química , Oxazolidinonas/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Cromatografia Líquida , Fungos/enzimologia , Humanos , Isomerismo , Ligantes , Espectrometria de Massas , Oxazolidinonas/análiseRESUMO
Affinity-based selection strategies have recently emerged as a complement to traditional high throughput screening for the rapid discovery of lead compounds for the large number of protein targets emerging from--omics technologies. Herein, we describe a method for the ranking of mixtures of ligands by affinity selection and apply it to rank order a set of inhibitors for the enzyme dipeptidyl peptidase IV.
Assuntos
Química Farmacêutica/métodos , Dipeptidil Peptidase 4/química , Inibidores Enzimáticos/síntese química , Ligantes , Diabetes Mellitus/tratamento farmacológico , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Modelos QuímicosRESUMO
A method for determining enantiomeric excess by mass spectrometry was employed to screen a family of chiral phosphite P,N-ligands for activity in the rhodium-catalyzed asymmetric hydrosilylation of ketones. The identification of an effective set of ligands was followed by preliminary studies of the reaction scope and mechanism. Asymmetric induction of 84-88% ee for larger-scale reactions was observed, which is close to the level of the best alternative catalysts previously discovered. The screening method was shown to be applicable to a variety of substrates without the need for special optimization.