RESUMEN
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/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
The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a (1)H-(15)N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.
Asunto(s)
Virus del Dengue/enzimología , Complejos Multienzimáticos/química , Proteínas no Estructurales Virales/química , Cristalografía por Rayos X , Virus del Dengue/genética , Humanos , Espectroscopía de Resonancia Magnética , Complejos Multienzimáticos/genética , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , ARN Helicasas/química , ARN Helicasas/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
The SARS-CoV-2 pandemic is currently causing an unprecedented global health emergency since its emergence in December 2019. In December 2021, the FDA granted emergency use authorization to nirmatrelvir, a SARS-CoV-2 main protease inhibitor, for treating infected patients. This peptidomimetic is designed with a nitrile warhead, which forms a covalent bond to the viral protease. Herein, we investigate nirmatrelvir analogs with different warheads and their inhibitory activities. In addition, antiviral activities against human alphacoronavirus 229E was also investigated along with a cell-based assay. We discovered that the hydroxymethylketone and ketobenzothiazole warheads were equipotent to the nitrile warhead, suggesting that these analogs can also be used for treating coronavirus infections.
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
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
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
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
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
The human TEAD family of transcription factors (TEAD1-4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD's co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small-molecule inhibitors. Our X-ray crystallography studies reveal that flufenamic acid, a non-steroidal anti-inflammatory drug (NSAID), binds to the central pocket of TEAD2 YBD. Our biochemical and functional analyses further demonstrate that binding of NSAIDs to TEAD inhibits TEAD-YAP-dependent transcription, cell migration, and proliferation, indicating that the central pocket is important for TEAD function. Therefore, our studies discover a novel way of targeting TEAD transcription factors and set the stage for therapeutic development of specific TEAD-YAP inhibitors against human cancers.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Proteínas de Unión al ADN/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Antiinflamatorios no Esteroideos/química , Antineoplásicos/química , Sitios de Unión , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Unión Proteica , Factores de Transcripción de Dominio TEA , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
This communication describes the synthesis and inhibitory activities of thirty-seven novel C-terminal agmatine dipeptides used as screening compounds to study the structure-activity relationship between active-site peptidomimetics and the West Nile virus (WNV) NS2B/NS3 serine protease. Our efforts lead to the discovery of a novel agmatine dipeptide inhibitor (compound 33, IC50 2.6 ± 0.3 µM) with improved inhibitory activity in comparison to the most potent inhibitor described in our recent report [IC50 4.7 ± 1.2 µM; Lim et al., Eur. J. Med. Chem. 46 (2011) 3130-3134]. In addition, our study cleared the contention surrounding the previous X-ray co-crystallization study and an enzyme inhibition report on the binding conformation adopted by active-site peptide aldehydes. Our data should provide valuable insights into the design of future peptidomimetic antivirals against WNV infections.
Asunto(s)
Agmatina/farmacología , Dipéptidos/farmacología , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/efectos de los fármacos , Virus del Nilo Occidental/enzimología , Agmatina/síntesis química , Agmatina/química , Cristalografía por Rayos X , Dipéptidos/síntesis química , Dipéptidos/química , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , ARN Helicasas/efectos de los fármacos , Serina Endopeptidasas/efectos de los fármacos , Programas Informáticos , Relación Estructura-Actividad , Virus del Nilo Occidental/efectos de los fármacosRESUMEN
Murray Valley encephalitis is an infectious disease spread by a mosquito-borne virus endemic in Papua New Guinea and northern Australia. In the past decade, it has spread to various regions of Australia and there is currently no therapeutic treatment against this disease. An attractive drug target is the viral serine protease NS2B/NS3, a critical enzyme involved in viral replication. Herein, we report the inhibitory activities of 37 C-terminal agmatine peptidomimetic inhibitors which led to the design of a novel structurally-constrained competitive inhibitor 38 possessing a Ki of 2.5±0.5 µM. We believe our data provides crucial insights into the viral protease active site specificity which could be used to facilitate drug design against Murray Valley encephalitis viral infections.
Asunto(s)
Virus de la Encefalitis del Valle Murray/enzimología , Peptidomiméticos/farmacología , Serina Proteasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Inhibidores de Serina Proteinasa/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
West Nile virus (WNV) NS2B-NS3 protease is an important drug target since it is an essential protein for the replication of the virus. In order to determine the minimum pharmacophore for protease inhibition, a series of dipeptide aldehydes were synthesized. The 50% inhibitory concentration (IC(50)) measurements revealed that a simple acetyl-KR-aldehyde was only threefold less active than 4-phenyl-phenylacetyl-KKR-aldehyde (1) (Stoermer et al., 2008) that was used as the reference compound. The ligand efficiency of 0.40 kcal/mol/HA (HA=heavy atom) for acetyl-KR-aldehyde is much improved compared to the reference compound 1 (0.23 kcal/mol/HA). The binding of the inhibitors was examined using (1)H-(15)N-HSQC experiments and differential chemical shifts were used to map the ligand binding sites. The biophysical studies show that the conformational mobility of WNV protease has a major impact on the design of novel inhibitors, since the protein conformation changes profoundly depending on the structure of the bound ligand.