RESUMEN
Staphylococcus aureus is the main aetiological agent responsible for the majority of human skin infections. Of particular concern is the methicillin-resistant variety, commonly known as MRSA. The extensive use of the first-line topical antibiotic of choice, mupirocin, has inevitably resulted in the emergence of resistant strains, signalling an urgent need for the development of new antibacterials with new mechanisms of action. In this work, we describe how we designed a novel cationic nonapeptide, containing only leucine and two lysine residues, with potent anti-MRSA activity and a rapid bactericidal mode of action. Coupled to a favourable safety profile towards human skin fibroblasts, we believe nonapeptide 11 has high potential for further development as a mupirocin replacement candidate to treat skin infections caused by MRSA.
Asunto(s)
Antibacterianos/farmacología , Diseño de Fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Nanoestructuras/química , Péptidos/química , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Antibacterianos/química , Supervivencia Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Mupirocina/farmacología , Péptidos/farmacología , Relación Estructura-ActividadRESUMEN
Patients lacking PYCR2, a mitochondrial enzyme that synthesizes proline, display postnatal degenerative microcephaly with hypomyelination. Here we report the crystal structure of the PYCR2 apo-enzyme and show that a novel germline p.Gly249Val mutation lies at the dimer interface and lowers its enzymatic activity. We find that knocking out Pycr2 in mice phenocopies the human disorder and depletes PYCR1 levels in neural lineages. In situ quantification of neurotransmitters in the brains of PYCR2 mutant mice and patients revealed a signature of encephalopathy driven by excessive cerebral glycine. Mechanistically, we demonstrate that loss of PYCR2 upregulates SHMT2, which is responsible for glycine synthesis. This hyperglycemia could be partially reversed by SHMT2 knockdown, which rescued the axonal beading and neurite lengths of cultured Pycr2 knockout neurons. Our findings identify the glycine metabolic pathway as a possible intervention point to alleviate the neurological symptoms of PYCR2-mutant patients.
Asunto(s)
Corteza Cerebral/metabolismo , Glicina Hidroximetiltransferasa/metabolismo , Glicina/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Pirrolina Carboxilato Reductasas/genética , Adolescente , Animales , Corteza Cerebral/patología , Preescolar , Femenino , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Lactante , Masculino , Ratones , Ratones Noqueados , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Linaje , Pirrolina Carboxilato Reductasas/deficienciaRESUMEN
Staphylococcus aureus is the pathogen responsible for the majority of human skin infections. In particular, the methicillin-resistant variety, MRSA, has become a global clinical concern. The extensive use of mupirocin, the first-line topical antibacterial drug of choice, has led to the emergence of mupirocin-resistant MRSA globally, resulting in the urgent need for a replacement. Antimicrobial peptides are deemed plausible candidates. Herein, we describe a structure-activity relationship approach in the design of an ultra-short peptide with potent anti-MRSA activity with a rapid, bactericidal mode of action. Coupled to a low cytotoxic activity, we believe our lead compound can be developed into a topical antibacterial agent to replace mupirocin as the first-line drug for treating MRSA skin infections.
Asunto(s)
Antibacterianos/farmacología , Diseño de Fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Péptidos/química , Secuencia de Aminoácidos , Antibacterianos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Mupirocina/farmacología , Péptidos/farmacología , Relación Estructura-ActividadRESUMEN
Even though many GyrB and ParE inhibitors have been reported in the literature, few possess activity against Gram-negative bacteria. This is primarily due to limited permeability across Gram-negative bacterial membrane as well as bacterial efflux mechanisms. Permeability of compounds across Gram-negative bacterial membranes depends on many factors including physicochemical properties of the inhibitors. Herein, we show the optimization of pyridylureas leading to compounds with potent activity against Gram-negative bacterial species such as P.aeruginosa, E.coli and A.baumannii.
Asunto(s)
Antibacterianos/farmacología , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , Descubrimiento de Drogas , Escherichia coli/efectos de los fármacos , Inhibidores de Topoisomerasa/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Topoisomerasa de ADN IV/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/enzimología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/enzimología , Relación Estructura-Actividad , Inhibidores de Topoisomerasa/síntesis química , Inhibidores de Topoisomerasa/químicaRESUMEN
Linear antimicrobial peptides, with their rapid bactericidal mode of action, are well-suited for development as topical antibacterial drugs. We recently designed a synthetic linear 4-residue peptide, BRBR-NH2, with potent bactericidal activity against Staphylococcus aureus (MIC 6.25⯵M), the main causative pathogen of human skin infections with an unknown mechanism of action. Herein, we describe a series of experiments conducted to gain further insights into its mechanism of action involving electron microscopy, artificial membrane dye leakage, solution- and solid-state NMR spectroscopy followed by molecular dynamics simulations. Experimental results point towards a SMART (Soft Membranes Adapt and Respond, also Transiently) mechanism of action, suggesting that the peptide can be developed as a topical antibacterial agent for treating drug-resistant Staphylococcus aureus infections.
Asunto(s)
Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Pared Celular/metabolismo , Secuencia de Aminoácidos , Antiinfecciosos/química , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Pared Celular/química , Liposomas/química , Liposomas/metabolismo , Espectroscopía de Resonancia Magnética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Microscopía Electrónica de Transmisión , Simulación de Dinámica MolecularRESUMEN
Vulvovaginal candidiasis (VVC) is a genital fungal infection afflicting approximately 75% of women globally and is primarily caused by the yeast Candida albicans. The extensive use of fluconazole, the first-line antifungal drug of choice, has led to the emergence of fluconazole-resistant C. albicans, creating a global clinical concern. This, coupled to the lack of new antifungal drugs entering the market over the past decade, has made it imperative for the introduction of new antifungal drug classes. Peptides with antifungal properties are deemed potential drug candidates due to their rapid membrane-disrupting mechanism of action. By specifically targeting and rapidly disrupting fungal membranes, they reduce the chances of resistance development and treatment duration. In a previous screening campaign involving an antimicrobial peptide library, we identified an octapeptide (IKIKIKIK-NH2) with potent activity against C. albicans. Herein, we report a structure-activity relationship study on this peptide with the aim of designing a more potent peptide for further development. The lead peptide was then tested against a panel of fluconazole-resistant C. albicans, subjected to a fungicidal/static determination assay, a human dermal fibroblast viability assay and a homozygous profiling assay to gain insights into its mechanism of action and potential for further development as a topical antifungal agent.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Fluconazol/farmacología , Péptidos/farmacología , Antifúngicos/síntesis química , Antifúngicos/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Relación Estructura-ActividadRESUMEN
Staphylococcus aureus is the primary pathogen responsible for the majority of human skin infections, and meticillin-resistant S. aureus (MRSA) currently presents a major clinical concern. The overuse of Mupirocin, the first-line topical antibacterial drug over 30 years, has led to the emergence of Mupirocin-resistant MRSA, creating a clinical concern. The antimicrobial peptide Omiganan was touted to be a promising antibacterial drug candidate due to its rapid membrane-disrupting bactericidal mode of action, entering clinical trials in 2005 as a topical gel to prevent catheter site infections. However, drug development ceased in 2009 due to a lack of efficacy. We postulate this to be due to proteolytic degradation caused by endogenous human skin proteases. Herein, we tested our hypothesis using Omiganan and its all-D enantiomer in a human skin protease stability assay, followed by anti-MRSA activity assay against of a panel of clinical MRSA isolates, a bactericidal/static determination and a time-kill assay to gauge all-D Omiganan's potential for further topical antibacterial drug development.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Mupirocina/farmacología , Administración Tópica , Antibacterianos/química , Antibacterianos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Péptido Hidrolasas/metabolismo , Estabilidad Proteica , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/microbiología , Enfermedades de la Piel/patología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , EstereoisomerismoRESUMEN
Vulvovaginal candidiasis/candidosis is a common fungal infection afflicting approximately 75% of women globally caused primarily by the yeast Candida albicans. Fluconazole is widely regarded as the antifungal drug of choice since its introduction in 1990 due to its high oral bioavailability, convenient dosing regimen and favourable safety profile. However, its widespread use has led to the emergence of fluconazole-resistant C. albicans, posing a universal clinical concern. Coupled to the dearth of new antifungal drugs entering the market, it is imperative to introduce new drug classes to counter this threat. Antimicrobial peptides (AMPs) are potential candidates due to their membrane-disrupting mechanism of action. By specifically targeting fungal membranes and being rapidly fungicidal, they can reduce the chances of resistance development and treatment duration. Towards this goal, we conducted a head-to-head comparison of 61 short linear AMPs from the literature to identify the peptide with the most potent activity against fluconazole-resistant C. albicans. The 11-residue peptide, P11-6, was identified and assayed against a panel of clinical C. albicans isolates followed by fungicidal/static determination and a time-kill assay to gauge its potential for further drug development. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Antifúngicos/síntesis química , Péptidos Catiónicos Antimicrobianos/síntesis química , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Agar , Secuencia de Aminoácidos , Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Candida albicans/crecimiento & desarrollo , Candida albicans/aislamiento & purificación , Candidiasis Vulvovaginal/microbiología , Femenino , Fluconazol/farmacología , Humanos , Miconazol/farmacología , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad CuantitativaRESUMEN
Pseudomonas aeruginosa infection is difficult to treat because of its drug resistance, but how it develops drug resistance remains largely unknown. In this study we investigated Ciprofloxacin resistance development in P. aeruginosa. Different Ciprofloxacin concentrations selected different low level resistant mutants, and high level resistant mutants emerged from low level resistant mutants if stressed further by Ciprofloxacin. A deep quantitative proteomic study of the Ciprofloxacin resistant mutants uncovered the cellular pathways that supported such resistances. The two low level resistant mutants had different molecular mechanisms. One was mainly due to switching to anaerobic respiration and overexpression of catalase and peroxidase, and the other was probably due to iron and polyamine uptake and DNA repair. High level of resistance involved the mexCD-oprJ efflux pump and the downregulation of PQS quorum sensing. Other pathways might also have contributed to high level resistance, like the arginine deiminase pathway, catalase, peroxidase, protein degradation and DNA repair. The intracellular Ciprofloxacin concentration assay indicated that only the mexCD-oprJ overexpressed mutants had low drug accumulation. This study provided a comprehensive overview of the proteomic landscape in the evolution of Ciprofloxacin resistance in P. aeruginosa, and might have implications in diagnosis and treatment of Ciprofloxacin resistant P. aeruginosa. Data are available via ProteomeXchange with identifier PXD004560. BIOLOGICAL SIGNIFICANCE: Pseudomonas aeruginosa infection is difficult to treat because of its drug resistance, but how it develops drug resistance remains largely unknown. In this study we investigated Ciprofloxacin resistance development in P. aeruginosa. We found that Ciprofloxacin resistance developed from low to high level. Two different low levels resistant molecular mechanisms were discovered from different mutants selected by different Ciprofloxacin concentrations, one was mainly due to switching to anaerobic respiration and overexpression of catalase and peroxidase, the other was probably due to iron, polyamine, and DNA repair. High level of Ciprofloxacin resistance all involved the efflux pump, mexCD-oprJ, and the downregulation of quorum sensing. The findings of this study provided insights into the evolution of Ciprofloxacin resistance in P. aeruginosa and should have implications in diagnosis and treatment of Ciprofloxacin resistant P. aeruginosa.
Asunto(s)
Ciprofloxacina/farmacología , Regulación Bacteriana de la Expresión Génica , Proteoma/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Anaerobiosis , Antibacterianos/farmacología , Catalasa/metabolismo , Reparación del ADN/genética , Farmacorresistencia Bacteriana , Hierro/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Peroxidasa/metabolismo , Poliaminas/metabolismo , Pseudomonas aeruginosa/genética , Percepción de QuorumRESUMEN
Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery.
Asunto(s)
Antivirales/farmacología , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/enzimología , Péptidos/farmacología , Inhibidores de Proteasas/farmacología , Proteínas Virales/antagonistas & inhibidores , Proteasas Virales 3C , Antivirales/química , Línea Celular , Cisteína Endopeptidasas , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Enterovirus/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Péptidos/química , Inhibidores de Proteasas/química , Replicación Viral/efectos de los fármacosRESUMEN
Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of ß1-α2 and residues in the loop of ß3-ß4 might improve the inhibitor potency.
Asunto(s)
Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Inhibidores de Topoisomerasa/química , Humanos , Indoles/química , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
The mosquito-borne West Nile virus (WNV) causes a wide range of symptoms ranging from fever to the often fatal viral encephalitis. To date, no vaccine or drug therapy is available. The trypsin-like WNV NS2B-NS3 protease is deemed a plausible drug target and was shown to be inhibited by bovine pancreatic trypsin inhibitor (BPTI), a 58-residue protein isolated from bovine lung. Herein, we report a protein truncation study that resulted in a novel 14-residue cyclic peptide with equipotent inhibitory activity to native BPTI. We believe our truncation strategy can be further applied in the development of peptide-based inhibitors targeting trypsin-like proteases.
Asunto(s)
Inhibidores de Proteasas/farmacología , Inhibidores de Tripsina/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Virus del Nilo Occidental/enzimología , Animales , Bovinos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Relación Estructura-Actividad , Tripsina/metabolismo , Inhibidores de Tripsina/síntesis química , Inhibidores de Tripsina/química , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/efectos de los fármacosRESUMEN
Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC50 value 3.4 ± 0.4 versus 7.3 ± 0.8 µM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities.
Asunto(s)
Enterovirus Humano A/enzimología , Peptidomiméticos/farmacología , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad , Proteínas Virales/antagonistas & inhibidores , Proteasas Virales 3C , Antivirales/química , Antivirales/farmacología , Técnicas de Química Sintética , Cristalografía por Rayos X , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Concentración 50 Inhibidora , Isoxazoles/química , Isoxazoles/farmacología , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Fenilalanina/análogos & derivados , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Pirrolidinonas/química , Pirrolidinonas/farmacología , Valina/análogos & derivados , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
The overuse and misuse of antibiotics has resulted in the emergence of drug-resistant pathogenic bacteria, including meticillin-resistant Staphylococcus aureus (MRSA), the primary pathogen responsible for human skin and soft-tissue infections. Antibacterial peptides are known to kill bacteria by rapidly disrupting their membranes and are deemed plausible alternatives to conventional antibiotics. One advantage of their membrane-targeting mode of action is that bacteria are unlikely to develop resistance as changing their cell membrane structure and morphology would likely involve extensive genetic mutations. However, major concerns in using peptides as antibacterial drugs include their instability towards plasma proteases, toxicity towards human cells due to their membrane-targeting mode of action and high manufacturing cost. These concerns can be mitigated by developing peptides as topical agents, by the judicial selection of amino acids and developing very short peptides respectively. In this preliminary report, we reveal a linear, non-hemolytic tetrapeptide with rapid bactericidal activity against MRSA developed from a structure-activity relationship study based on the antimicrobial hexapeptide WRWRWR-NH2. Our finding opens promising avenues for the development of ultra-short antibacterials to treat multidrug-resistant MRSA skin and soft tissue infections.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Descubrimiento de Drogas , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Oligopéptidos/química , Oligopéptidos/farmacología , Antibacterianos/síntesis química , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Humanos , Conformación Molecular , Oligopéptidos/síntesis química , Relación Estructura-ActividadRESUMEN
Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE.
Asunto(s)
Adenosina Trifosfato/metabolismo , Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/farmacología , Escherichia coli/enzimología , Adenosina Trifosfato/antagonistas & inhibidores , Secuencia de Aminoácidos , Antibacterianos/farmacología , Unión Competitiva , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/química , Diseño de Fármacos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
The lack of new antibacterial drugs entering the market and their misuse have resulted in the emergence of drug-resistant bacteria, posing a major health crisis worldwide. In particular, meticillin-resistant Staphylococcus aureus (MRSA), a pathogen responsible for numerous human infections, has become endemic in hospitals worldwide. Drug repurposing, the finding of new therapeutic indications for approved drugs, is deemed a plausible solution to accelerate drug discovery and development in this area. Towards this end, we screened 1163 drugs approved by the Food and Drug Administration (FDA) for bioactivities against MRSA in a 10 µM single-point assay. After excluding known antibiotics and antiseptics, six compounds were identified and their MICs were determined against a panel of clinical MRSA strains. A toxicity assay using human keratinocytes was also conducted to gauge their potential for repurposing as topical agents for treating MRSA skin infections.
RESUMEN
Bacterial resistance to antibiotics remains a serious threat to global health. The gyraseâ B enzyme is a well-validated target for developing antibacterial drugs. Despite being an attractive target for antibiotic development, there are currently no gyraseâ B inhibitory drugs on the market. A fragment screen using 1,800â compounds identified 14â fragments that bind to Escherichia coli (E.â coli) gyraseâ B. The detailed characterization of binding is described for all 14â fragments. With the aid of X-ray crystallography, modifications on a low-affinity fragment (KD =253â µM, IC50 =634â µM) has led to the development of a new class of potent phenyl aminopyrazole inhibitors against E.â coli gyraseâ B (IC50 =160â nM). The study presented here combines the use of a set of biophysical techniques including differential scanning fluorimetry, nuclear magnetic resonance, isothermal titration calorimetry, and X-ray crystallography to methodically identify, quantify, and optimize fragments into new chemical leads.
RESUMEN
The Murray Valley encephalitis virus (MVEV) and the West Nile virus (WNV) are mosquito-borne single-stranded RNA Flaviviruses responsible for many cases of viral encephalitis and deaths worldwide. The former is endemic in north Australia and Papua New Guinea while the latter has spread to different parts of the world and was responsible for a recent North American outbreak in 2012, resulting in 243 fatalities. There is currently no approved vaccines or drugs against MVEV and WNV viral infections. A plausible drug target is the viral non-structural NS2B/NS3 protease due to its role in viral replication. This trypsin-like serine protease recognizes and cleaves viral polyproteins at the C-terminal end of an arginine residue, opening an avenue for the development of peptide-based antivirals. This communication compares the P2 and P3 residue preferences of the MVEV and WNV NS2B/NS3 proteases using a series of C-terminal agmatine dipeptides. Our results revealed that both viral enzymes were highly specific toward lysines at the P2 and P3 positions, suggesting that a peptidomimetic viral protease inhibitor developed against one virus should also be active against the other.
Asunto(s)
Agmatina/química , Dipéptidos/química , Virus de la Encefalitis del Valle Murray/enzimología , Serina Endopeptidasas/química , Inhibidores de Serina Proteinasa/química , Proteínas no Estructurales Virales , Virus del Nilo Occidental/enzimología , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/químicaRESUMEN
Tankyrases constitute potential drug targets for cancer and myelin-degrading diseases. We have applied a structure- and biophysics-driven fragment-based ligand design strategy to discover a novel family of potent inhibitors for human tankyrases. Biophysical screening based on a thermal shift assay identified highly efficient fragments binding in the nicotinamide-binding site, a local hot spot for fragment binding. Evolution of the fragment hit 4-methyl-1,2-dihydroquinolin-2-one (2) along its 7-vector yields dramatic affinity improvements in the first cycle of expansion. A crystal structure of 7-(2-fluorophenyl)-4-methylquinolin-2(1H)-one (11) reveals that the nonplanar compound extends with its fluorine atom into a pocket, which coincides with a region of the active site where structural differences are seen between tankyrases and other poly(ADP-ribose) polymerase (PARP) family members. A further cycle of optimization yielded compounds with affinities and IC50 values in the low nanomolar range and with good solubility, PARP selectivity, and ligand efficiency.
Asunto(s)
Quinolinas/síntesis química , Tanquirasas/antagonistas & inhibidores , Cristalografía por Rayos X , Bases de Datos Factuales , Estabilidad de Medicamentos , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Quinolinas/química , Solubilidad , Relación Estructura-Actividad , Tanquirasas/química , TermodinámicaRESUMEN
Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, ß-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased ß-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics.