RESUMEN
Utilizing the already described 3,4-bi-aryl pyridine series as a starting point, incorporation of a second ring system with a hydrogen bond donor and additional hydrophobic contacts yielded the azaindole series which exhibited potent, picomolar RSK2 inhibition and the most potent in vitro target modulation seen thus far for a RSK inhibitor. In the context of the more potent core, several changes at the phenol moiety were assessed to potentially find a tool molecule appropriate for in vivo evaluation.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Cromatografía Liquida , Diseño de Fármacos , Humanos , Espectrometría de Masas , Fenoles/farmacología , Inhibidores de Proteínas Quinasas/química , Espectroscopía de Protones por Resonancia Magnética , Relación Estructura-ActividadRESUMEN
A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAFV600E. Medicinal chemistry techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.
Asunto(s)
Benzamidas/farmacología , Compuestos Heterocíclicos con 2 Anillos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Benzamidas/síntesis química , Benzamidas/química , Relación Dosis-Respuesta a Droga , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/química , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Relación Estructura-ActividadRESUMEN
2-Amino-7-substituted benzoxazole analogs were identified by HTS as inhibitors of RSK2. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of improving in vitro and target modulation potency and physicochemical properties.
Asunto(s)
Benzoxazoles/química , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Benzoxazoles/síntesis química , Benzoxazoles/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Estructura Terciaria de Proteína , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Relación Estructura-ActividadRESUMEN
Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.
Asunto(s)
Neuropilinas/química , Semaforina-3A/química , Factor A de Crecimiento Endotelial Vascular/química , Secuencia de Aminoácidos , Anticuerpos/química , Sitios de Unión , Cristalografía por Rayos X/métodos , Dimerización , Conformación Molecular , Datos de Secuencia Molecular , Neuropilinas/fisiología , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Semaforina-3A/metabolismo , Semaforinas/metabolismo , Homología de Secuencia de Aminoácido , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Dishevelled proteins are key regulators of Wnt signaling pathways that have been implicated in the progression of human cancers. We found that the binding cleft of the Dishevelled PDZ domain is more flexible than those of canonical PDZ domains and enables recognition of both C-terminal and internal peptides. These peptide ligands inhibit Wnt/beta-catenin signaling in cells, showing that Dishevelled PDZ domains are potential targets for small-molecule cancer therapeutics.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Dishevelled , Modelos Moleculares , Biblioteca de Péptidos , Fosfoproteínas/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.
Asunto(s)
Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/genética , Dominios PDZ , Proteoma/análisis , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/clasificación , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Péptidos/análisis , Péptidos/genética , Filogenia , Estructura Secundaria de Proteína , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.
RESUMEN
Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.
Asunto(s)
Antineoplásicos/química , Descubrimiento de Drogas/métodos , Mutación/genética , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Antineoplásicos/farmacología , Diseño de Fármacos , Descubrimiento de Drogas/tendencias , Humanos , Simulación del Acoplamiento Molecular/métodos , Simulación del Acoplamiento Molecular/tendencias , Mutación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Mammalian coronin-1 is preferentially expressed in hematopoietic cells and plays a poorly understood role in the dynamic reorganization of the actin cytoskeleton. Sequence analysis of coronin-1 revealed five WD40 repeats that were predicted to form a beta propeller. They are followed by a 130 residue extension and a 30 residue leucine zipper domain that is responsible for multimerization of the protein. Here, we present the crystal structure of murine coronin-1 without the leucine zipper at 1.75 A resolution. Coronin-1 forms a seven-bladed beta propeller composed of the five predicted WD40 repeats and two additional blades that lack any homology to the canonical WD40 motif. The C-terminal extension adopts an extended conformation, packs tightly against the bottom surface of the propeller, and is likely to be required for the structural stability of the propeller. Analysis of charged and conserved surface residues delineate possible binding sites for F-actin on the beta propeller.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Linfocitos/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/fisiología , Actinas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Citoesqueleto/química , Linfocitos/química , Ratones , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Electricidad Estática , TermodinámicaRESUMEN
In the preceding manuscript [ Moreau et al. 2018 , 10.1021/acs.jmedchem.7b01691 ] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Compuestos Heterocíclicos con 2 Anillos/farmacología , Nucleotidiltransferasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/metabolismo , Bencimidazoles/síntesis química , Bencimidazoles/química , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Sitios de Unión , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mutación , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Unión Proteica , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Triazoles/síntesis química , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Compuestos Heterocíclicos con 2 Anillos/farmacología , Nucleotidiltransferasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/metabolismo , Bencimidazoles/síntesis química , Bencimidazoles/química , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Sitios de Unión , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Unión Proteica , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Triazoles/síntesis química , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.
Asunto(s)
Proteínas Mitocondriales/química , Serina Endopeptidasas/química , Alanina/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Serina Peptidasa A2 que Requiere Temperaturas Altas , Ligandos , Proteínas Mitocondriales/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Estructura Terciaria de Proteína , Serina Endopeptidasas/aislamiento & purificación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.
Asunto(s)
Serina Endopeptidasas/química , Secuencia de Aminoácidos , Chaperoninas/química , Escherichia coli/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Ligandos , Datos de Secuencia Molecular , Dominios PDZ , Péptidos/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.
Asunto(s)
2,2'-Dipiridil/análogos & derivados , Antineoplásicos/farmacología , Benzamidas/farmacología , Quinasas raf/antagonistas & inhibidores , Proteínas ras/genética , 2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Benzamidas/química , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Descubrimiento de Drogas , Estabilidad de Medicamentos , Humanos , Concentración 50 Inhibidora , Ratones , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
While the p90 ribosomal S6 kinase (RSK) family has been implicated in multiple tumor cell functions, the full understanding of this kinase family has been restricted by the lack of highly selective inhibitors. A bis-phenol pyrazole was identified from high-throughput screening as an inhibitor of the N-terminal kinase of RSK2. Structure-based drug design using crystallography, conformational analysis, and scaffold morphing resulted in highly optimized difluorophenol pyridine inhibitors of the RSK kinase family as demonstrated cellularly by the inhibition of YB1 phosphorylation. These compounds provide for the first time in vitro tools with an improved selectivity and potency profile to examine the importance of RSK signaling in cancer cells and to fully evaluate RSK as a therapeutic target.
Asunto(s)
Pirazoles/química , Piridinas/química , Pirimidinas/química , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Animales , Línea Celular , Cristalografía por Rayos X , Humanos , Masculino , Ratones , Modelos Moleculares , Fosforilación , Conformación Proteica , Pirazoles/síntesis química , Pirazoles/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Ratas Sprague-Dawley , Transducción de Señal , Relación Estructura-Actividad , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
Abrogation of errant signaling along the MAPK pathway through the inhibition of B-RAF kinase is a validated approach for the treatment of pathway-dependent cancers. We report the development of imidazo-benzimidazoles as potent B-RAF inhibitors. Robust in vivo efficacy coupled with correlating pharmacokinetic/pharmacodynamic (PKPD) and PD-efficacy relationships led to the identification of RAF265, 1, which has advanced into clinical trials.
RESUMEN
PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended ß-strand conformation by interacting in an antiparallel fashion with a PDZ ß-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.
Asunto(s)
Dominios PDZ , Ingeniería de Proteínas/métodos , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/metabolismo , Humanos , Ligandos , Péptidos/química , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteoma , Proteómica/métodos , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Proteínas de Uniones Estrechas/química , Proteína de la Zonula Occludens-1/químicaRESUMEN
UNLABELLED: The p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases is expressed in a variety of cancers and its substrate phosphorylation has been implicated in direct regulation of cell survival, proliferation, and cell polarity. This study characterizes and presents the most selective and potent RSK inhibitors known to date, LJH685 and LJI308. Structural analysis confirms binding of LJH685 to the RSK2 N-terminal kinase ATP-binding site and reveals that the inhibitor adopts an unusual nonplanar conformation that explains its excellent selectivity for RSK family kinases. LJH685 and LJI308 efficiently inhibit RSK activity in vitro and in cells. Furthermore, cellular inhibition of RSK and its phosphorylation of YB1 on Ser102 correlate closely with inhibition of cell growth, but only in an anchorage-independent growth setting, and in a subset of examined cell lines. Thus, RSK inhibition reveals dynamic functional responses among the inhibitor-sensitive cell lines, underscoring the heterogeneous nature of RSK dependence in cancer. IMPLICATIONS: Two novel potent and selective RSK inhibitors will now allow a full assessment of the potential of RSK as a therapeutic target for oncology.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Secuencia de Aminoácidos , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , FosforilaciónRESUMEN
The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinase MET that could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-MET cells using the novel selective MET inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Resistencia a Antineoplásicos/genética , Mutación Missense , Mutación Puntual , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Quinolinas/farmacología , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Sustitución de Aminoácidos , Aminopiridinas/metabolismo , Aminopiridinas/farmacología , Animales , Antineoplásicos/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Cristalografía por Rayos X , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Activación Enzimática/genética , Humanos , Ratones , Modelos Moleculares , Mutagénesis , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Unión Proteica , Conformación Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/genética , Pirazoles/metabolismo , Pirazoles/farmacología , Quinolinas/metabolismo , Receptores de Factores de Crecimiento/química , Receptores de Factores de Crecimiento/genética , Tirosina/metabolismoRESUMEN
The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. An affinity-improved version of bH1 inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models. Such "two-in-one" antibodies challenge the monoclonal antibody paradigm of one binding site, one antigen. They could also provide new opportunities for antibody-based therapy.