RESUMEN
The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15N-NMR-based binding affinity assays and 19F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Humanos , Ligandos , Unión Proteica , Sitios de Unión , Dominios Proteicos , Modelos Moleculares , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Cristalografía por Rayos X , Descubrimiento de Drogas/métodosRESUMEN
Identifying promising chemical starting points for small molecule inhibitors of active, GTP-loaded KRAS "on" remains of great importance to clinical oncology and represents a significant challenge in medicinal chemistry. Here, we describe broadly applicable learnings from a KRAS hit finding campaign: While we initially identified KRAS inhibitors in a biochemical high-throughput screen, we later discovered that compound potencies were all but assay artifacts linked to metal salts interfering with KRAS AlphaScreen assay technology. The source of the apparent biochemical KRAS inhibition was ultimately traced to unavoidable palladium impurities from chemical synthesis. This discovery led to the development of a Metal Ion Interference Set (MIIS) for up-front assay development and testing. Profiling of the MIIS across 74 assays revealed a reduced interference liability of label-free biophysical assays and, as a result, provided general estimates for luminescence- and fluorescence-based assay susceptibility to metal salt interference.
Asunto(s)
Paladio , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Paladio/química , Ensayos Analíticos de Alto Rendimiento/métodos , Sales (Química)/químicaRESUMEN
Import of proteins into peroxisomes depends on PEX5, PEX13 and PEX14. By combining biochemical methods and structural biology, we show that the C-terminal SH3 domain of PEX13 mediates intramolecular interactions with a proximal FxxxF motif. The SH3 domain also binds WxxxF peptide motifs in the import receptor PEX5, demonstrating evolutionary conservation of such interactions from yeast to human. Strikingly, intramolecular interaction of the PEX13 FxxxF motif regulates binding of PEX5 WxxxF/Y motifs to the PEX13 SH3 domain. Crystal structures reveal how FxxxF and WxxxF/Y motifs are recognized by a non-canonical surface on the SH3 domain. The PEX13 FxxxF motif also mediates binding to PEX14. Surprisingly, the potential PxxP binding surface of the SH3 domain does not recognize PEX14 PxxP motifs, distinct from its yeast ortholog. Our data show that the dynamic network of PEX13 interactions with PEX5 and PEX14, mediated by diaromatic peptide motifs, modulates peroxisomal matrix import.
Asunto(s)
Proteínas de la Membrana , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae , Dominios Homologos src , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Péptidos/química , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/metabolismo , Peroxisomas/metabolismo , Unión Proteica , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dominios Homologos src/genética , Dominios Homologos src/fisiologíaRESUMEN
Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities.
Asunto(s)
Factores de Transcripción NFATC , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/química , Unión Proteica , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
Hypusination is a unique post-translational modification of the eukaryotic translation factor 5A (eIF5A) that is essential for overcoming ribosome stalling at polyproline sequence stretches. The initial step of hypusination, the formation of deoxyhypusine, is catalyzed by deoxyhypusine synthase (DHS), however, the molecular details of the DHS-mediated reaction remained elusive. Recently, patient-derived variants of DHS and eIF5A have been linked to rare neurodevelopmental disorders. Here, we present the cryo-EM structure of the human eIF5A-DHS complex at 2.8 Å resolution and a crystal structure of DHS trapped in the key reaction transition state. Furthermore, we show that disease-associated DHS variants influence the complex formation and hypusination efficiency. Hence, our work dissects the molecular details of the deoxyhypusine synthesis reaction and reveals how clinically-relevant mutations affect this crucial cellular process.
Asunto(s)
Enfermedades Neurodegenerativas , Trastornos del Neurodesarrollo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Factores de Iniciación de Péptidos , Humanos , Microscopía por Crioelectrón , Factores de Iniciación de Péptidos/química , Procesamiento Proteico-Postraduccional , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Nucleares/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/genética , Supervivencia Celular , Células HEK293 , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Células K562 , Cinética , Simulación del Acoplamiento Molecular , Proteínas Nucleares/genética , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In Wilson disease, excessive copper accumulates in patients' livers and may, upon serum leakage, severely affect the brain according to current viewpoints. Present remedies aim at avoiding copper toxicity by chelation, for example, by D-penicillamine (DPA) or bis-choline tetrathiomolybdate (ALXN1840), the latter with a very high copper affinity. Hence, ALXN1840 may potentially avoid neurological deterioration that frequently occurs upon DPA treatment. As the etiology of such worsening is unclear, we reasoned that copper loosely bound to albumin, that is, mimicking a potential liver copper leakage into blood, may damage cells that constitute the blood-brain barrier, which was found to be the case in an in vitro model using primary porcine brain capillary endothelial cells. Such blood-brain barrier damage was avoided by ALXN1840, plausibly due to firm protein embedding of the chelator bound copper, but not by DPA. Mitochondrial protection was observed, a prerequisite for blood-brain barrier integrity. Thus, high-affinity copper chelators may minimize such deterioration in the treatment of neurologic Wilson disease.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Cobre/metabolismo , Molibdeno/farmacología , Penicilamina/farmacología , Animales , Transporte Biológico , Biomarcadores , Barrera Hematoencefálica/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Supervivencia Celular , Quelantes/farmacología , Cobre/efectos adversos , Cobre/química , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Ratones Transgénicos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Modelos Moleculares , Tomografía de Emisión de Positrones , Unión Proteica , Ratas , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Relación Estructura-ActividadRESUMEN
Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.
Asunto(s)
Péptido Hidrolasas/metabolismo , Periodontitis/microbiología , Inhibidores de Proteasas/farmacología , Tannerella forsythia/enzimología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Descubrimiento de Drogas , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Humanos , Espectroscopía de Resonancia Magnética/métodos , Simulación del Acoplamiento Molecular , Estructura Molecular , Péptido Hidrolasas/efectos de los fármacos , Inhibidores de Proteasas/química , Tannerella forsythia/aislamiento & purificación , TemperaturaRESUMEN
Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-ß-lactamases (MBLs) target the most widely used antibiotic class, the ß-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-ß-lactamase inhibitors, essential in the fight against antibiotic resistance.
Asunto(s)
beta-Lactamasas , Antibacterianos/farmacología , Inhibidores de beta-Lactamasas , beta-Lactamasas/genética , beta-Lactamas , CarbapenémicosRESUMEN
Glucose phosphorylating enzymes are crucial in the regulation of basic cellular processes, including metabolism and gene expression. Glucokinases and hexokinases provide a pool of phosphorylated glucose in an adenosine diphosphate (ADP)- and ATP-dependent manner to shape the cell metabolism. The glucose processing enzymes from Kluyveromyces lactis are poorly characterized despite the emerging contribution of this yeast strain to industrial and laboratory scale biotechnology. The first reports on K. lactis glucokinase (KlGlk1) positioned the enzyme as an essential component required for glucose signaling. Nevertheless, no biochemical and structural information was available until now. Here, we present the first crystal structure of KlGlk1 together with biochemical characterization, including substrate specificity and enzyme kinetics. Additionally, comparative analysis of the presented structure and the prior structures of lactis hexokinase (KlHxk1) demonstrates the potential transitions between open and closed enzyme conformations upon ligand binding.
Asunto(s)
Glucoquinasa/química , Kluyveromyces/enzimología , Modelos Moleculares , Conformación Proteica , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosa/metabolismo , Cinética , Kluyveromyces/genética , Kluyveromyces/metabolismo , Especificidad por SustratoRESUMEN
Fungal lipases are efficient and environment-friendly biocatalysts for many industrially relevant processes. One of the most widely applied lipases in the manufacturing industry is Candida antarctica lipase B (CALB). Here, we report the biochemical and structural characterization of a novel CALB-like lipase from an important human pathogen-Aspergillus fumigatus (AFLB), which has high sn-1,3-specificity toward triolein. AFLB crystal structure displays a CALB-like catalytic domain and hosts a unique tightly closed 'lid' domain that contains a disulfide bridge, as well as an extra N-terminal subdomain composed of residues 1-128 (including the helix α1-α5 located above the active site). To gain insight into the function of this novel lid and N-terminal subdomain, we constructed and characterized a series of mutants in these two domains. Deleting the protruding bulk lid's residues, replacing the bulk and tight lid with a small and loose lid from CALB, or breaking the disulfide bridge increased the affinity of CALB for glyceride substrates and improved its catalytic activity, along with the loss of enzyme fold stability and thermostability. N-terminal truncation mutants revealed that the N-terminal peptide (residues 1-59) is a strong inhibitor of AFLB binding to lipid films. This peptide thus limits AFLB's penetration power and specific activity, revealing a unique enzyme activity regulatory mechanism. Our findings on the functional and structural properties of AFLB provide a better understanding of the functions of the CALB-like lipases and pave the way for its future protein engineering. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6IDY.
Asunto(s)
Aspergilosis/enzimología , Aspergillus fumigatus/enzimología , Lipasa/química , Conformación Proteica , Aspergilosis/genética , Aspergilosis/microbiología , Aspergillus fumigatus/química , Dominio Catalítico/genética , Estabilidad de Enzimas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Lipasa/genética , Simulación de Dinámica Molecular , Ingeniería de Proteínas , Relación Estructura-ActividadRESUMEN
Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition.
RESUMEN
Blockade of the immunoinhibitory PD-1/PD-L1 pathway using monoclonal antibodies has shown impressive results with durable clinical antitumor responses. Anti-PD-1 and anti-PD-L1 antibodies have now been approved for the treatment of a number of tumor types, whereas the development of small molecules targeting immune checkpoints lags far behind. We characterized two classes of macrocyclic-peptide inhibitors directed at the PD-1/PD-L1 pathway. We show that these macrocyclic compounds act by directly binding to PD-L1 and that they are capable of antagonizing PD-L1 signaling and, similarly to antibodies, can restore the function of T-cells. We also provide the crystal structures of two of these small-molecule inhibitors bound to PD-L1. The structures provide a rationale for the checkpoint inhibition by these small molecules, and a description of their small molecule/PD-L1 interfaces provides a blueprint for the design of small-molecule inhibitors of the PD-1/PD-L1 pathway.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Antígeno B7-H1/inmunología , Descubrimiento de Drogas , Humanos , Células Jurkat , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Simulación del Acoplamiento Molecular , Receptor de Muerte Celular Programada 1/inmunología , Mapas de Interacción de Proteínas/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Cancer cells can avoid and suppress immune responses through activation of inhibitory immune checkpoint proteins, such as PD-1, PD-L1, and CTLA-4. Blocking the activities of these proteins with monoclonal antibodies, and thus restoring T cell function, has delivered breakthrough therapies against cancer. In this review, we describe the latest work on structural characterization of the checkpoint proteins, their interactions with cognate ligands and with therapeutic antibodies. Structures of the extracellular portions of these proteins reveal that they all have a similar modular structure, composed of small domains similar in topology to the domains found in antibodies. Structural basis for blocking the PD-1/PD-L1 interaction by small molecules is illustrated with the compound BMS-202 that binds to and induces dimerization of PD-L1.
Asunto(s)
Antineoplásicos Inmunológicos/química , Antígeno B7-H1/química , Proteína 2 Ligando de Muerte Celular Programada 1/química , Receptor de Muerte Celular Programada 1/química , Animales , Antineoplásicos Inmunológicos/metabolismo , Antígeno B7-H1/metabolismo , Sitios de Unión , Humanos , Simulación del Acoplamiento Molecular , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Unión ProteicaRESUMEN
Blockade of the PD-1/PD-L1 immune checkpoint pathway with monoclonal antibodies has provided significant advances in cancer treatment. The antibody-based immunotherapies carry a number of disadvantages such as the high cost of the antibodies, their limited half-life, and immunogenicity. Development of small-molecule PD-1/PD-L1 inhibitors that could overcome these drawbacks is slow because of the incomplete structural information for this pathway. The first chemical PD-1/PD-L1 inhibitors have been recently disclosed by Bristol-Myers Squibb. Here we present NMR and X-ray characterization for the two classes of these inhibitors. The X-ray structures of the PD-L1/inhibitor complexes reveal one inhibitor molecule located at the center of the PD-L1 homodimer, filling a deep hydrophobic channel-like pocket between two PD-L1 molecules. Derivatives of (2-methyl-3-biphenylyl)methanol exhibit the structures capped on one side of the channel, whereas the compounds based on [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol induce an enlarged interaction interface that results in the open "face-back" tunnel through the PD-L1 dimer.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Mapas de Interacción de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Antígeno B7-H1/química , Antígeno B7-H1/metabolismo , Cristalografía por Rayos X , Humanos , Simulación del Acoplamiento Molecular , Receptor de Muerte Celular Programada 1/química , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
The p53 pathway is inactivated in almost all types of cancer by mutations in the p53 encoding gene or overexpression of the p53 negative regulators, Mdm2 and/or Mdmx. Restoration of the p53 function by inhibition of the p53-Mdm2/Mdmx interaction opens up a prospect for a nongenotoxic anticancer therapy. Here, we present the syntheses, activities, and crystal structures of two novel classes of Mdm2-p53 inhibitors that are based on the 3-pyrrolin-2-one and 2-furanone scaffolds. The structures of the complexes formed by these inhibitors and Mdm2 reveal the dimeric protein molecular organization that has not been observed in the small-molecule/Mdm2 complexes described until now. In particular, the 6-chloroindole group does not occupy the usual Trp-23 pocket of Mdm2 but instead is engaged in dimerization. This entirely unique binding mode of the compounds opens new possibilities for optimization of the Mdm2-p53 interaction inhibitors.
Asunto(s)
4-Butirolactona/análogos & derivados , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Pirroles/química , Pirroles/farmacología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , 4-Butirolactona/química , 4-Butirolactona/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first nonpeptidic chemical inhibitors that target the PD-1/PD-L1 interaction have only been recently disclosed by Bristol-Myers Squibb. Here, we show that these small-molecule compounds bind directly to PD-L1 and that they potently block PD-1 binding. Structural studies reveal a dimeric protein complex with a single small molecule which stabilizes the dimer thus occluding the PD-1 interaction surface of PD-L1s. The small-molecule interaction "hot spots" on PD-L1 surfaces suggest approaches for the PD-1/PD-L1 antagonist drug discovery.
Asunto(s)
Antígeno B7-H1/química , Receptor de Muerte Celular Programada 1/química , Dominios Proteicos , Bibliotecas de Moléculas Pequeñas/química , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Cristalografía por Rayos X , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The proteinprotein interaction of p53 and MDM2/X is a promising non genotoxic anticancer target. A rapid and efficient methodology was developed to synthesize the 2,30-bis(10H-indole) heterocyclic scaffold 2 as ester, acid and amide derivatives. Their binding affinity with MDM2 was evaluated using both fluorescence polarization (FP) assay and HSQC experiments, indicating good inhibition and a perfect starting point for further optimizations.
Asunto(s)
Compuestos Heterocíclicos/química , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Sitios de Unión , Polarización de Fluorescencia , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/metabolismo , Indoles/química , Simulación del Acoplamiento Molecular , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has recently provided breakthrough progress in the treatment of melanoma, non-small cell lung cancer, and other types of cancer. Small-molecule drugs interfering with this pathway are highly awaited, but their development is hindered by insufficient structural information. This study reveals the molecular details of the human PD-1/PD-L1 interaction based on an X-ray structure of the complex. First, it is shown that the ligand binding to human PD-1 is associated with significant plasticity within the receptor. Second, a detailed molecular map of the interaction surface is provided, allowing definition of the regions within both interacting partners that may likely be targeted by small molecules.