RESUMO
We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.
Assuntos
Ensaios de Triagem em Larga Escala , Espectrometria de Massas por Ionização por Electrospray , AcústicaRESUMO
The respiratory syncytial virus (RSV) L protein is a viral RNA-dependent RNA polymerase that contains multiple enzyme activities required for RSV replication. The RSV L inhibitors described in literature are limited by their cytotoxicity or the lack of RSV B subtype coverage. Here, we characterize a new RSV L inhibitor with strong antiviral activity against both RSV A and B subtypes and no detectable cytotoxicity. This compound, AZ-27, was equally active against RSV live viruses and subgenomic replicons and demonstrated advantages over other classes of RSV inhibitors in time-of-addition and cell line dependency studies. Resistance studies identified a dominant mutation in the putative capping enzyme domain of L protein, which conferred strong resistance to the AZ-27 series but not other classes of RSV inhibitors, supporting RSV L protein as the direct target for AZ-27. This novel and broad-spectrum RSV L polymerase inhibitor may pave the way toward an efficacious RSV therapeutic and provide a new tool for interrogation of the L protein function.
Assuntos
Antivirais/farmacologia , Benzazepinas/farmacologia , Ciclopropanos/farmacologia , Niacinamida/análogos & derivados , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Sequência de Aminoácidos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Farmacorresistência Viral , Humanos , Dados de Sequência Molecular , Niacinamida/farmacologia , Reação em Cadeia da Polimerase , Replicon/genéticaRESUMO
Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Inibidores de Proteases , SARS-CoV-2 , Humanos , Animais , Camundongos , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/farmacocinética , Antivirais/uso terapêutico , Antivirais/química , Administração Oral , Inibidores de Proteases/farmacologia , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/uso terapêutico , Inibidores de Proteases/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Ratos , COVID-19/virologiaRESUMO
Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.
Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Vírus Sinciciais Respiratórios/enzimologia , Proteínas Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Azepinas/síntese química , Azepinas/química , Azepinas/farmacologia , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Humanos , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.
Assuntos
Fígado , Membranas Mitocondriais , Humanos , Fígado/metabolismo , Membranas Mitocondriais/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/química , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Sequência de AminoácidosRESUMO
The incidence of hospital-acquired infections with multidrug-resistant (MDR) Gram-negative pathogens is increasing at an alarming rate. Equally alarming is the overall lack of efficacious therapeutic options for clinicians, which is due primarily to the acquisition and development of various antibiotic resistance mechanisms that render these drugs ineffective. Among these mechanisms is the reduced permeability of the outer membrane, which prevents many marketed antibiotics from traversing this barrier. To circumvent this, recent drug discovery efforts have focused on conjugating a siderophore moiety to a pharmacologically active compound that has been designed to hijack the bacterial siderophore transport system and trick cells into importing the active drug by recognizing it as a nutritionally beneficial compound. MC-1, a novel siderophore-conjugated ß-lactam that promotes its own uptake into bacteria, has exquisite activity against many Gram-negative pathogens. While the inclusion of the siderophore was originally designed to facilitate outer membrane penetration into Gram-negative cells, here we show that this structural moiety also renders other clinically relevant antibiotic resistance mechanisms unable to affect MC-1 efficacy. Resistance frequency determinations and subsequent characterization of first-step resistant mutants identified PiuA, a TonB-dependent outer membrane siderophore receptor, as the primary means of MC-1 entry into Pseudomonas aeruginosa. While the MICs of these mutants were increased 32-fold relative to the parental strain in vitro, we show that this resistance phenotype is not relevant in vivo, as alternative siderophore-mediated uptake mechanisms compensated for the loss of PiuA under iron-limiting conditions.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/fisiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , beta-Lactamas/farmacologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Western Blotting , Clonagem Molecular , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Biblioteca Gênica , Camundongos , Porinas/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Sepse/tratamento farmacológico , Sepse/microbiologia , Sideróforos , beta-Lactamases/biossíntese , beta-Lactamases/genéticaRESUMO
Vero cells are widely used for antiviral tests and virology research for SARS-CoV-2 as well as viruses from various other families. However, Vero cells generally express high levels of multi-drug resistance 1 (MDR1) or Pgp protein, the efflux transporter of foreign substances including many antiviral compounds, affecting the antiviral activity as well as interpretation of data. To address this, a Pgp gene knockout VeroE6 cell line (VeroE6-Pgp-KO) was generated using CRISPR-CAS9 technology. These cells no longer expressed the Pgp protein as indicated by flow cytometry analysis following staining with a Pgp-specific monoclonal antibody. They also showed significantly reduced efflux transporter activity in the calcein acetoxymethyl ester (calcein AM) assay. The VeroE6-Pgp-KO cells and the parental VeroE6 cells were each infected with SARS-CoV-2 to test antiviral activities of remdesivir and nirmatrelvir, two known Pgp substrates, in the presence or absence of a Pgp inhibitor. The compounds showed antiviral activities in VeroE6-Pgp-KO cells similar to that observed in the presence of the Pgp inhibitor. Thus, the newly established VeroE6-Pgp-KO cell line adds a new in vitro virus infection system for SARS-CoV-2 and possibly other viruses to test antiviral therapies without a need to control the Pgp activity. Removal of the Pgp inhibitor for antiviral assays will lead to less data variation and prevent failed assays.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Chlorocebus aethiops , Animais , SARS-CoV-2/genética , Antivirais/farmacologia , Técnicas de Inativação de Genes , Células Vero , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Linhagem CelularRESUMO
The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.
Assuntos
Tratamento Farmacológico da COVID-19 , Lactamas/farmacologia , Lactamas/uso terapêutico , Leucina/farmacologia , Leucina/uso terapêutico , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Prolina/farmacologia , Prolina/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Inibidores de Protease Viral/farmacologia , Inibidores de Protease Viral/uso terapêutico , Administração Oral , Animais , COVID-19/virologia , Ensaios Clínicos Fase I como Assunto , Coronavirus/efeitos dos fármacos , Modelos Animais de Doenças , Quimioterapia Combinada , Humanos , Lactamas/administração & dosagem , Lactamas/farmacocinética , Leucina/administração & dosagem , Leucina/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Nitrilas/administração & dosagem , Nitrilas/farmacocinética , Prolina/administração & dosagem , Prolina/farmacocinética , Ensaios Clínicos Controlados Aleatórios como Assunto , Ritonavir/administração & dosagem , Ritonavir/uso terapêutico , SARS-CoV-2/fisiologia , Inibidores de Protease Viral/administração & dosagem , Inibidores de Protease Viral/farmacocinética , Replicação Viral/efeitos dos fármacosRESUMO
COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.
Assuntos
Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/administração & dosagem , Indóis/administração & dosagem , Leucina/administração & dosagem , Pirrolidinonas/administração & dosagem , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/efeitos adversos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacocinética , Alanina/administração & dosagem , Alanina/efeitos adversos , Alanina/análogos & derivados , Alanina/farmacocinética , Animais , COVID-19/virologia , Chlorocebus aethiops , Coronavirus Humano 229E/efeitos dos fármacos , Coronavirus Humano 229E/enzimologia , Inibidores de Protease de Coronavírus/efeitos adversos , Inibidores de Protease de Coronavírus/farmacocinética , Modelos Animais de Doenças , Desenho de Fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Células HeLa , Humanos , Indóis/efeitos adversos , Indóis/farmacocinética , Infusões Intravenosas , Leucina/efeitos adversos , Leucina/farmacocinética , Camundongos , Pirrolidinonas/efeitos adversos , Pirrolidinonas/farmacocinética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Células VeroRESUMO
COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro , and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.
RESUMO
Multidrug-resistant Gram-negative pathogens are an emerging threat to human health, and addressing this challenge will require development of new antibacterial agents. This can be achieved through an improved molecular understanding of drug-target interactions combined with enhanced delivery of these agents to the site of action. Herein we describe the first application of siderophore receptor-mediated drug uptake of lactivicin analogues as a strategy that enables the development of novel antibacterial agents against clinically relevant Gram-negative bacteria. We report the first crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from Pseudomonas aeruginosa and characterize the reactivity of lactivicin and ß-lactam core structures. Results from drug sensitivity studies with ß-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed.
Assuntos
Bactérias Gram-Negativas/metabolismo , Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Sideróforos/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Testes de Sensibilidade Microbiana , Peptídeos/efeitos dos fármacos , Peptídeos Cíclicos , beta-Lactamases/metabolismoRESUMO
The three SAT serotype viruses, endemic in Africa, are well known for their difficulty to adapt to cell culture. The viral mechanism involved in foot-and-mouth disease virus (FMDV) tissue tropism and cell-entry is not well understood. A recombinant, small plaque-forming virus (vSAT1tc), derived from a tissue culture-adapted SAT1 virus (SAR/9/81tc), revealed four amino acid substitutions (VP3 Asp192âTyr; VP3 Ser217âIle; VP1 Ala69âGly and VP1 Asn110âLys) in the capsid, compared to the SAR/9/81wt isolate collected from infected impala epithelium. One substitution added a positively charged lysine residue to the short ßF-ßG loop of VP1. Furthermore, vSAT1tc displayed a high affinity for CHO-K1 cells possibly via interaction with negatively charged sulphated polysaccharides while SAT1 impala strain relied strongly on α(V)ß6 integrin receptors for cell entry. The cell culture adaptation and small plaque phenotype of vSAT1tc was accompanied by differences in particle aggregation and significant differences in acid stability. Based on limited cross neutralization data, the antigenic features seem to be unchanged. Thus, acquisition of positively charged residues in the virion may be beneficial for adaptation of SAT type field strains to cell culture.