RESUMEN
Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor ß-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN), complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2, with preclinical evidence to support their efficacy in T cell malignancies.
Asunto(s)
Neoplasias , Linfocitos T , Humanos , Inmunoterapia , Receptores de Antígenos de Linfocitos TRESUMEN
Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , COVID-19/prevención & control , COVID-19/terapia , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Técnicas de Visualización de Superficie Celular/métodos , Minería de Datos/métodos , Epítopos/inmunología , Humanos , Inmunización Pasiva/métodos , Sueroterapia para COVID-19RESUMEN
The early phase of protein drug development has traditionally focused on target binding properties leading to a desired mode of therapeutic action. As more protein therapeutics pass through the development pipeline; however, it is clear that non-optimal biophysical properties can emerge, particularly as proteins are formulated at high concentrations, causing aggregation or polyreactivity. Such late-stage "developability" problems can lead to delay or failure in traversing the development process. Aggregation propensity is also correlated with increased immunogenicity, resulting in expensive, late-stage clinical failures. Using nucleases-directed integration, we have constructed large mammalian display libraries where each cell contains a single antibody gene/cell inserted at a single locus, thereby achieving transcriptional normalization. We show a strong correlation between poor biophysical properties and display level achieved in mammalian cells, which is not replicated by yeast display. Using two well-documented examples of antibodies with poor biophysical characteristics (MEDI-1912 and bococizumab), a library of variants was created based on surface hydrophobic and positive charge patches. Mammalian display was used to select for antibodies that retained target binding and permitted increased display level. The resultant variants exhibited reduced polyreactivity and reduced aggregation propensity. Furthermore, we show in the case of bococizumab that biophysically improved variants are less immunogenic than the parental molecule. Thus, mammalian display helps to address multiple developability issues during the earliest stages of lead discovery, thereby significantly de-risking the future development of protein drugs.
Asunto(s)
Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Afinidad de Anticuerpos/genética , Técnicas de Visualización de Superficie Celular , Células HEK293 , HumanosRESUMEN
The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization. The utility of the system is illustrated by the affinity maturation of a PD-1-blocking antibody through the systematic mutation and functional survey of 4-mer variants within a 16 amino acid paratope region. Mutating VH CDR3 only, we identified a dominant "solution" involving substitution of a central tyrosine to histidine. This appears to be a local affinity maximum, and this variant was surpassed by a lysine substitution when light chain variants were introduced. We achieve this comprehensive and quantitative interrogation of sequence space by combining high-throughput oligonucleotide synthesis with mammalian display and flow cytometry operating at the multi-million scale.
Asunto(s)
Anticuerpos Monoclonales Humanizados/genética , Afinidad de Anticuerpos , Sitios de Unión de Anticuerpos/genética , Animales , Sitios de Unión de Anticuerpos/inmunología , Células CHO , Sistemas CRISPR-Cas , Regiones Determinantes de Complementariedad/genética , Cricetulus , Endodesoxirribonucleasas , Citometría de Flujo , Edición Génica , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Mutagénesis Sitio-Dirigida , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
Small-molecule inhibitors of the mycobacterial transcriptional repressor EthR have previously been shown to act as boosters of the second-line antituberculosis drug ethionamide. Fragment-based drug discovery approaches have been used in the past to make highly potent EthR inhibitors with ethionamide boosting activity both in vitro and ex vivo. Herein, we report the development of fragment-sized EthR ligands with nanomolar minimum effective concentration values for boosting the ethionamide activity in Mycobacterium tuberculosis whole-cell assays.
Asunto(s)
Etionamida/farmacología , Mycobacterium tuberculosis/enzimología , Proteínas Represoras/antagonistas & inhibidores , Antituberculosos , Proteínas Bacterianas , Descubrimiento de Drogas , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Etionamida/uso terapéutico , Ligandos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacosRESUMEN
Thymidylate synthase X (ThyX) represents an attractive target for tuberculosis drug discovery. Herein, we selected 16 compounds through a virtual screening approach. We solved the first X-ray crystal structure of Thermatoga maritima (Tm) ThyX in complex with a nonsubstrate analog inhibitor. Given the active site similarities between Mycobacterium tuberculosis ThyX (Mtb-ThyX) and Tm-ThyX, our crystal structure paves the way for a structure-based design of novel antimycobacterial compounds. The 1H-imidazo[4,5-d]pyridazine was identified as scaffold for the development of Mtb-ThyX inhibitors.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Mycobacterium tuberculosis/enzimología , Piridazinas/química , Piridazinas/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Flavinas/metabolismo , Humanos , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Tuberculosis/tratamiento farmacológicoRESUMEN
The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 µM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Sistema Enzimático del Citocromo P-450/química , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Ligandos , Mycobacterium tuberculosis/enzimología , Unión Proteica , Estructura Terciaria de Proteína , Tuberculosis/microbiologíaRESUMEN
With the ever-increasing instances of resistance to frontline TB drugs there is the need to develop novel strategies to fight the worldwide TB epidemic. Boosting the effect of the existing second-line antibiotic ethionamide by inhibiting the mycobacterial transcriptional repressor protein EthR is an attractive therapeutic strategy. Herein we report the use of a fragment based drug discovery approach for the structure-guided systematic merging of two fragment molecules, each binding twice to the hydrophobic cavity of EthR from M. tuberculosis. These together fill the entire binding pocket of EthR. We elaborated these fragment hits and developed small molecule inhibitors which have a 100-fold improvement of potency in vitro over the initial fragments.
Asunto(s)
Etionamida/química , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Represoras/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Cristalografía por Rayos X , Etionamida/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
A combination of chemical genetic and biochemical assays was applied to investigate the mechanism of action of the anticancer drug 5-fluorouracil (5-FU), against Mycobacterium tuberculosis (Mtb). 5-FU resistance was associated with mutations in upp or pyrR. Upp-catalyzed conversion of 5-FU to FUMP was shown to constitute the first step in the mechanism of action, and resistance conferred by nonsynonymous SNPs in pyrR shown to be due to derepression of the pyr operon and rescue from the toxic effects of FUMP and downstream antimetabolites through de novo production of UMP. 5-FU-derived metabolites identified in Mtb were consistent with the observed incorporation of 5-FU into RNA and DNA and the reduced amount of mycolyl arabinogalactan peptidoglycan in 5-FU-treated cells. Conditional depletion of the essential thymidylate synthase ThyX resulted in modest hypersensitivity to 5-FU, implicating inhibition of ThyX by fluorodeoxyuridylate as a further component of the mechanism of antimycobacterial action of this drug.
Asunto(s)
Antimetabolitos/metabolismo , Fluorouracilo/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antimetabolitos/química , Antimetabolitos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Radioisótopos de Carbono/química , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Fluorouracilo/química , Fluorouracilo/farmacología , Marcaje Isotópico , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Operón , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
A structure-guided fragment-based approach was used to target the lipophilic allosteric binding site of Mycobacterium tuberculosis EthR. This elongated channel has many hydrophobic residues lining the binding site, with few opportunities for hydrogen bonding. We demonstrate that a fragment-based approach involving the inclusion of flexible fragments in the library leads to an efficient exploration of chemical space, that fragment binding can lead to an extension of the cavity, and that fragments are able to identify hydrogen-bonding opportunities in this hydrophobic environment that are not exploited in Nature. In the present paper, we report the identification of a 1 µM affinity ligand obtained by structure-guided fragment linking.
Asunto(s)
Sitio Alostérico/fisiología , Sistemas de Liberación de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Fragmentos de Péptidos/fisiología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Sitios de Unión/fisiología , Cristalografía por Rayos X , Lípidos , Mycobacterium tuberculosis , Fragmentos de Péptidos/antagonistas & inhibidores , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismo , Relación Estructura-Actividad , Factores de Transcripción/metabolismoAsunto(s)
Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Enlace de Hidrógeno , Ligandos , Unión Proteica , Triazoles/químicaRESUMEN
The members of the cation diffusion facilitator (CDF) family transport heavy metal ions and play an important function in zinc ion homeostasis of the cell. A recent structure of an Escherichia coli CDF transporter protein YiiP has revealed its dimeric nature and autoregulatory zinc transport mechanism. Here, we report the cloning and heterologous production of four different CDF transporters, two each from the pathogenic mesophilic bacterium Salmonella typhimurium and from the hyperthermophilic bacterium Aquifex aeolicus, in E. coli host cells. STM0758 of S. typhimurium was able to restore resistance to zinc ions when tested by complementation assays in the zinc-sensitive GG48 strain. Furthermore, copurification of bicistronically produced STM0758 and cross-linking experiments with the purified protein have revealed its possible oligomeric nature. The interaction between heavy metal ions and Aq_2073 of A. aeolicus was investigated by titration calorimetry. The entropy-driven, high-affinity binding of two Cd2+ and two Zn2+ per protein monomer with Kd values of around 100 nm and 1 µm, respectively, was observed. In addition, at least one more Zn2+ can be bound per monomer with low affinity. This low-affinity site is likely to possess a functional role contributing to Zn2+ transport across membranes.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/metabolismo , Bacterias Gramnegativas/genética , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Clonación Molecular , Biología Computacional , Escherichia coli/citología , Escherichia coli/genética , Metales Pesados/metabolismo , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Cuaternaria de Proteína , TermodinámicaAsunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450 , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Ciclohexanonas/química , Sistema Enzimático del Citocromo P-450/metabolismo , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Ligandos , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Over the past decade, researchers in the pharmaceutical industry and academia have made retrospective analyses of successful drug campaigns in order to establish "rules" to guide the selection of new target proteins. They have identified features that are considered undesirable and some that make targets "unligandable." This review focuses on the factors that make targets difficult: featureless binding sites, the lack of hydrogen-bond donors and acceptors, the presence of metal ions, the need for adaptive changes in conformation, and the lipophilicity of residues at the protein-ligand interface. Protein-protein interfaces of multiprotein assemblies share many of these undesirable features, although those that involve concerted binding and folding in their assembly have better defined pockets or grooves, and these can provide opportunities for identifying hits and for lead optimization. In some protein-protein interfaces conformational changes-often involving rearrangement of large side chains such as those of tyrosine, tryptophan, or arginine-are required to configure an appropriate binding site, and this may require tethering of the ligands until higher affinity is achieved. In many enzymes, larger conformational rearrangements are required to form the binding site, and these can make fragment-based approaches particularly difficult.
Asunto(s)
Descubrimiento de Drogas , Proteínas/química , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Sitios de Unión , Dominio Catalítico , Ligandos , Mapeo de Interacción de Proteínas , Proteínas/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismoRESUMEN
Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.