RESUMEN
Cystine-dense peptides (CDPs) are a miniprotein class that can drug difficult targets with high affinity and low immunogenicity. Tools for their design, however, are not as developed as those for small-molecule and antibody drugs. CDPs have diverse taxonomic origins, but structural characterization is lacking. Here, we adapted Iterative Threading ASSEmbly Refinement (I-TASSER) and Rosetta protein modeling software for structural prediction of 4298 CDP scaffolds and performed in silico prescreening for CDP binders to targets of interest. Mammalian display screening of a library of docking-enriched, methionine and tyrosine scanned (DEMYS) CDPs against PD-L1 yielded binders from four distinct CDP scaffolds. One was affinity-matured, and cocrystallography yielded a high-affinity (KD = 202 pM) PD-L1-binding CDP that competes with PD-1 for PD-L1 binding. Its subsequent incorporation into a CD3-binding bispecific T cell engager produced a molecule with pM-range in vitro T cell killing potency and which substantially extends survival in two different xenograft tumor-bearing mouse models. Both in vitro and in vivo, the CDP-incorporating bispecific molecule outperformed a comparator antibody-based molecule. This CDP modeling and DEMYS technique can accelerate CDP therapeutic development.
Asunto(s)
Anticuerpos Biespecíficos , Linfocitos T , Animales , Humanos , Ratones , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antígeno B7-H1 , Complejo CD3 , Cistina , Modelos Animales de Enfermedad , Mamíferos , PéptidosRESUMEN
In the original version of this Article the colour key for the amino acid enrichment score was inadvertently omitted from the lower panel of Figure 5b during the production process. This has now been corrected in the PDF and HTML versions of the Article.
RESUMEN
Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. However, their exploitation has been hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression, screening and large-scale production of these peptides: 46 were successfully produced in multimilligram quantities, and >600 more were deemed expressible through stringent screening criteria. Many showed extreme resistance to temperature, proteolysis and/or reduction, and all displayed inhibitory activity against at least 1 of 20 ion channels tested, thus confirming their biological functionality. Crystal structures of 12 confirmed proper cystine topology and the utility of crystallography to study these molecules but also highlighted the need for rational classification. Previous categorization attempts have focused on limited subsets featuring distinct motifs. Here we present a global definition, classification and analysis of >700 structures of cystine-dense peptides, providing a unifying framework for these molecules.
Asunto(s)
Cistina/química , Péptidos/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Humanos , Canales Iónicos/antagonistas & inhibidores , Modelos Moleculares , Biosíntesis de Péptidos , Péptidos/clasificación , Péptidos/farmacologíaRESUMEN
Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology. Cystine-dense peptides have the potential to disrupt such interactions, and are used in drug-like roles by every clade of life, but their study has been hampered by a reputation for being difficult to produce, owing to their complex disulfide connectivity. Here we describe a platform for identifying target-binding cystine-dense peptides using mammalian surface display, capable of interrogating high quality and diverse scaffold libraries with verifiable folding and stability. We demonstrate the platform's capabilities by identifying a cystine-dense peptide capable of inhibiting the YAP:TEAD interaction at the heart of the oncogenic Hippo pathway, and possessing the potency and stability necessary for consideration as a drug development candidate. This platform provides the opportunity to screen cystine-dense peptides with drug-like qualities against targets that are implicated for the treatment of diseases, but are poorly suited for conventional approaches.
Asunto(s)
Cistina/análisis , Péptidos/química , Péptidos/farmacología , Mapas de Interacción de Proteínas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Descubrimiento de Drogas , Proteínas de Escherichia coli/química , Glicosilación , Humanos , Biblioteca de Péptidos , Péptidos/metabolismo , Unión Proteica , Pliegue de Proteína , Reproducibilidad de los Resultados , Proteínas de Saccharomyces cerevisiae/químicaAsunto(s)
Actinomycetales/metabolismo , Antraquinonas/farmacología , Antiinfecciosos/farmacología , Antraquinonas/aislamiento & purificación , Antiinfecciosos/aislamiento & purificación , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células Hep G2 , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Three new antibiotics, neopyrrolomycins B (1), C (2), and D (3), with potent activity against Gram-positive pathogens were discovered. They exhibited MIC values < 1 microg/mL versus a number of resistant strains. The compounds were obtained from the ethyl acetate extracts of a Streptomyces sp. after purification by column chromatography and RP-HPLC. Their structures were elucidated using X-ray crystallography (1) and NMR spectroscopy (2 and 3).
Asunto(s)
Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Streptomyces/química , Antibacterianos/química , Cristalografía por Rayos X , Farmacorresistencia Bacteriana/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Enterococcus faecium/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Pseudomonas aeruginosa/efectos de los fármacos , Pirroles/química , Pirroles/aislamiento & purificación , Pirroles/farmacología , Streptococcus pneumoniae/efectos de los fármacos , Vancomicina/farmacologíaRESUMEN
Two new xanthone antibiotics, citreamicin delta (1) and epsilon (2), with potent activity against Gram-positive pathogens including multidrug-resistant Staphylococcus aureus (MDRSA) were discovered. Compounds 1 and 2 exhibited MIC values < 1 microg/mL versus a number of resistant strains. The compounds were obtained from EtOAc extracts of Streptomyces vinaceus and were purified by countercurrent chromatography and reversed-phase HPLC. Their structures were elucidated using primarily NMR and mass spectroscopy.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Cromatografía Líquida de Alta Presión , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Oxazoles/química , Oxazoles/farmacologíaRESUMEN
Resistance to currently available antibiotics has become a widely recognized crisis in the medical community. To address this, many companies and researchers are refocusing their attention towards natural products, which have an excellent track record of producing effective antibacterial drugs. The AMRI natural product library was screened for activity against multi-drug resistant Staphylococcus aureus (MDRSA). The active samples were counter screened for cytotoxicity against the human hepatocellular carcinoma HepG2 cell line to determine an in vitro therapeutic index (in vitro TI). Those samples with a high in vitro TI were selected for fractionation and dereplication. This led to the discovery of a new anthracycline structure. This metabolite, named mutactimycin E (1), exhibited moderate activity against several gram positive organisms. Here we report the isolation, structure elucidation and biological activities of this new compound.