RESUMEN
The porcine reproductive and respiratory syndrome viruses (PRRSV) led to a global panzootic and huge economical losses to the pork industry. PRRSV targets the scavenger receptor CD163 for productive infection. However, currently no effective treatment is available to control the spread of this disease. Using bimolecular fluorescence complementation (BiFC) assays, we screened a set of small molecules potentially targeting the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. We found that the assay examining protein-protein interactions (PPI) between PRRSV glycoprotein 4 (GP4) and the CD163-SRCR5 domain mainly identifies compounds that potently inhibit PRRSV infection, while examining the PPI between PRRSV-GP2a and the SRCR5 domain maximized the identification of positive compounds, including additional ones with various antiviral capabilities. These positive compounds significantly inhibited both types 1 and 2 PRRSV infection of porcine alveolar macrophages. We confirmed that the highly active compounds physically bind to the CD163-SRCR5 protein, with dissociation constant (KD) values ranging from 28 to 39 µM. Structure-activity-relationship (SAR) analysis revealed that although both the 3-(morpholinosulfonyl)anilino and benzenesulfonamide moieties in these compounds are critical for the potency to inhibit PRRSV infection, the morpholinosulfonyl group can be replaced by chlorine substituents without significant loss of antiviral potency. Our study established a system for throughput screening of natural or synthetic compounds highly effective on blocking of PRRSV infection and shed light on further SAR modification of these compounds. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Current vaccines cannot provide cross protection against different strains, and there are no effective treatments available to hamper the spread of this disease. In this study, we identified a group of new small molecules that can inhibit the PRRSV interaction with its specific receptor CD163 and dramatically block the infection of both types 1 and type 2 PRRSVs to host cells. We also demonstrated the physical association of these compounds with the SRCR5 domain of CD163. In addition, molecular docking and structure-activity relationship analyses provided new insights for the CD163/PRRSV glycoprotein interaction and further improvement of these compounds against PRRSV infection.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Porcinos , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Receptores DepuradoresRESUMEN
Here, we describe the use of artificial intelligence to identify novel agonists of the SH2-containing 5' inositol phosphatase 1 (SHIP1). One of the compounds, K306, represents the most potent agonist identified to date. We find that K306 exhibits selectivity for SHIP1 vs. the paralog enzyme SHIP2, and this activation does not require the C2 domain of SHIP1 which other known SHIP1 agonists require. Thus, K306 represents a new class of SHIP1 agonists with a novel mode of agonism. Importantly, we find that K306 can suppress induction of inflammatory cytokines and iNOS in macrophages or microglia, but not by their SHIP1-deficient counterparts. K306 also reduces TNF-α production in vivo in an LPS-induced endotoxemia assay. Finally, we show that K306 enhances phagolysosomal degradation of synaptosomes and dead neurons by microglia revealing a novel function for SHIP1 that might be exploited therapeutically in dementia.
RESUMEN
Chlorophyll and heme are among the "pigments of life", tetrapyrrolic structures, without which life on Earth would not be possible. Their catabolites, the phyllobilins and the bilins, respectively, share not only structural features, but also a similar story: Long considered waste products of detoxification processes, important bioactivities for both classes have now been demonstrated. For phyllobilins, however, research on physiological roles is sparse. Here, we introduce actin, the major component of the cytoskeleton, as the first discovered target of phyllobilins and as a novel target of bilins. We demonstrate the inhibition of actin dynamics in vitro and effects on actin and related processes in cancer cells. A direct interaction with G-actin is shown by in silico studies and confirmed by affinity chromatography. Our findings open a new chapter in bioactivities of tetrapyrroles-especially phyllobilins-for which they form the basis for broad implications in plant science, ecology, and physiology.
Asunto(s)
Actinas/antagonistas & inhibidores , Clorofila/química , Hemo/química , Pigmentos Biológicos/farmacología , Tetrapirroles/farmacología , Actinas/metabolismo , Humanos , Pigmentos Biológicos/química , Tetrapirroles/químicaRESUMEN
Cullin-RING E3 ligases (CRLs) regulate the turnover of approximately 20% of mammalian cellular proteins. Neddylation of individual cullin proteins is essential for the activation of each CRL. We report herein the discovery of DI-1548 and DI-1859 as two potent, selective and covalent DCN1 inhibitors. These inhibitors selectively inhibit neddylation of cullin 3 in cells at low nanomolar concentrations and are 2-3 orders of magnitude more potent than our previously reported reversible DCN1 inhibitor. Mass spectrometric analysis and co-crystal structures reveal that these compounds employ a unique mechanism of covalent bond formation with DCN1. DI-1859 induces a robust increase of NRF2 protein, a CRL3 substrate, in mouse liver and effectively protects mice from acetaminophen-induced liver damage. Taken together, this study demonstrates the therapeutic potential of selective inhibition of cullin neddylation.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Proteínas Cullin/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Sustancias Protectoras/farmacología , Acetaminofén/administración & dosificación , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones , Proteína NEDD8/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Sustancias Protectoras/química , Sustancias Protectoras/uso terapéutico , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
PURPOSE: On the basis of the recent discovery of mutations in Bruton tyrosine kinase (BTK) in follicular lymphoma, we studied their functional properties. EXPERIMENTAL DESIGN: We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells. RESULTS: We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLCγ2) is a substrate of BTK but the BTK mutants did not alter PLCγ2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK, we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK-mutant follicular lymphoma. The augmented AKT phosphorylation following BCR crosslinking could be abrogated by pretreatment with a PI3Kδ inhibitor. CONCLUSIONS: Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma.See related commentary by Afaghani and Taylor, p. 2123.
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Agammaglobulinemia Tirosina Quinasa/genética , Linfoma Folicular/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Agammaglobulinemia Tirosina Quinasa/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Análisis Mutacional de ADN , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mutación con Pérdida de Función , Linfoma Folicular/patología , Mutagénesis Sitio-Dirigida , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Cultivo Primario de Células , Estabilidad ProteicaRESUMEN
BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the pork industry globally. PRRS is caused by PRRS virus (PRRSV). Currently there are no effective treatments against this swine disease. METHODS: Through artificial intelligence molecular screening, we obtained a set of small molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163, which is a cell surface receptor specific for PRRSV infection. These compounds were screened using a cell-based bimolecular fluorescence complementation (BiFC) assay, and the function of positive hit was further evaluated and validated by PRRSV-infection assay using porcine alveolar macrophages (PAMs). RESULTS: Using the BiFC assay, we identified one compound with previously unverified function, 4-Fluoro-2-methyl-N-[3-(3-morpholin-4-ylsulfonylanilino)quinoxalin-2-yl]benzenesulfonamide (designated here as B7), that significantly inhibits the interaction between the PRRSV glycoprotein (GP2a or GP4) and the CD163-SRCR5 domain. We further demonstrated that compound B7 inhibits PRRSV infection of PAMs, the primary target of PRRSV in a dose-dependent manner. B7 significantly inhibited the infection caused by both type I and type II PRRSV strains. Further comparison and functional evaluation of chemical compounds structurally related to B7 revealed that the 3-(morpholinosulfonyl)aniline moiety of B7 or the 3-(piperidinylsulfonyl)aniline moiety in a B7 analogue is important for the inhibitory function against PRRSV infection. CONCLUSIONS: Our study identified a novel strategy to potentially prevent PRRSV infection in pigs by blocking the PRRSV-CD163 interaction with small molecules.
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Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Receptores de Superficie Celular/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Inteligencia Artificial , Línea Celular , Células HEK293 , Humanos , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Dominios Proteicos , PorcinosRESUMEN
The mixed-lineage leukemia (MLL) protein, also known as MLL1, is a lysine methyltransferase specifically responsible for methylation of histone 3 lysine 4. MLL has been pursued as an attractive therapeutic target for the treatment of acute leukemia carrying the MLL fusion gene or MLL leukemia. Herein, we report the design, synthesis, and evaluation of an S-adenosylmethionine-based focused chemical library which led to the discovery of potent small-molecule inhibitors directly targeting the MLL SET domain. Determination of cocrystal structures for a number of these MLL inhibitors reveals that they adopt a unique binding mode that locks the MLL SET domain in an open, inactive conformation.
RESUMEN
Inhibition of the menin-mixed lineage leukemia (MLL) protein-protein interaction is a promising new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein our structure-based design, synthesis, and evaluation of a new class of small-molecule inhibitors of the menin-MLL interaction (hereafter called menin inhibitors). Our efforts have resulted in the discovery of highly potent menin inhibitors, as exemplified by compound 42 (M-89). M-89 binds to menin with a Kd value of 1.4 nM and effectively engages cellular menin protein at low nanomolar concentrations. M-89 inhibits cell growth in the MV4;11 and MOLM-13 leukemia cell lines carrying MLL fusion with IC50 values of 25 and 55 nM, respectively, and demonstrates >100-fold selectivity over the HL-60 leukemia cell line lacking MLL fusion. The determination of a co-crystal structure of M-89 in a complex with menin provides the structural basis for their high-affinity interaction. Further optimization of M-89 may lead to a new class of therapy for the treatment of MLL leukemia.
Asunto(s)
Descubrimiento de Drogas/métodos , Leucemia Mieloide/tratamiento farmacológico , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Enfermedad Aguda , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Células HL-60 , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Modelos Químicos , Estructura Molecular , Proteína de la Leucemia Mieloide-Linfoide/química , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The discovery of recurrent mutations in subunits of the vacuolar-type H+-translocating ATPase (v-ATPase) in follicular lymphoma (FL) highlights a role for the amino acid- and energy-sensing pathway to mTOR in the pathogenesis of this disease. Here, through the use of complementary experimental approaches involving mammalian cells and Saccharomyces cerevisiae, we have demonstrated that mutations in the human v-ATPase subunit ATP6V1B2 (also known as Vma2 in yeast) activate autophagic flux and maintain mTOR/TOR in an active state. Engineered lymphoma cell lines and primary FL B cells carrying mutated ATP6V1B2 demonstrated a remarkable ability to survive low leucine concentrations. The treatment of primary FL B cells with inhibitors of autophagy uncovered an addiction for survival for FL B cells harboring ATP6V1B2 mutations. These data support the idea of mutational activation of autophagic flux by recurrent hotspot mutations in ATP6V1B2 as an adaptive mechanism in FL pathogenesis and as a possible new therapeutically targetable pathway.
Asunto(s)
Muerte Celular Autofágica , Linfoma Folicular/enzimología , Mutación , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Humanos , Linfoma Folicular/genética , Linfoma Folicular/patología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinasas TOR/genética , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
The Cullin-RING ligases (CRLs) regulate the turnover of approximately 20% of the proteins in mammalian cells and are emerging therapeutic targets in human diseases. The activation of CRLs requires the neddylation of their cullin subunit, which is controlled by an activation complex consisting of Cullin-RBX1-UBC12-NEDD8-DCN1. Herein, we describe the design, synthesis, and evaluation of peptidomimetics targeting the DCN1-UBC12 protein-protein interaction. Starting from a 12-residue UBC12 peptide, we have successfully obtained a series of peptidomimetic compounds that bind to DCN1 protein with KD values of <10 nM. Determination of a cocrystal structure of a potent peptidomimetic inhibitor complexed with DCN1 provides the structural basis for their high-affinity interaction. Cellular investigation of one potent DCN1 inhibitor, compound 36 (DI-404), reveals that it effectively and selectively inhibits the neddylation of cullin 3 over other cullin members. Further optimization of DI-404 may yield a new class of therapeutics for the treatment of human diseases in which cullin 3 CRL plays a key role.
Asunto(s)
Peptidomiméticos/farmacología , Unión Proteica/efectos de los fármacos , Proteínas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Cristalografía por Rayos X , Proteínas Cullin/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Peptidomiméticos/química , Peptidomiméticos/uso terapéutico , Proteínas/antagonistas & inhibidores , Proteínas/química , Enzimas Ubiquitina-Conjugadoras/químicaRESUMEN
The structure-based design of M-525 as the first-in-class, highly potent, irreversible small-molecule inhibitor of the menin-MLL interaction is presented. M-525 targets cellular menin protein at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL-regulated gene expression in MLL leukemia cells. M-525 demonstrates high cellular specificity over non-MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co-crystal structure of M-525 in complex with menin firmly establish its mode of action. A single administration of M-525 effectively suppresses MLL-regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M-525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.
Asunto(s)
Antineoplásicos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Antineoplásicos/química , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Simulación de Dinámica Molecular , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
The Cullin-RING E3 ubiquitin ligases (CRLs) regulate homeostasis of ~20% of cellular proteins and their activation require neddylation of their cullin subunit. Cullin neddylation is modulated by a scaffolding DCN protein through interactions with both the cullin protein and an E2 enzyme such as UBC12. Here we report the development of DI-591 as a high-affinity, cell-permeable small-molecule inhibitor of the DCN1-UBC12 interaction. DI-591 binds to purified recombinant human DCN1 and DCN2 proteins with K i values of 10-12 nM, and disrupts the DCN1-UBC12 interaction in cells. Treatment with DI-591 selectively converts cellular cullin 3 into an un-neddylated inactive form with no or minimum effect on other cullin members. Our data firmly establish a previously unrecognized specific role of the DCN1-UBC12 interaction for cellular neddylation of cullin 3. DI-591 is an excellent probe compound to investigate the role of the cullin 3 CRL ligase in biological processes and human diseases.
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Proteínas Cullin/metabolismo , Morfolinas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Tiazoles/farmacología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Química Farmacéutica , Clonación Molecular , Biología Computacional , Cristalografía por Rayos X , Diseño de Fármacos , Células HeLa , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Péptidos y Proteínas de Señalización Intracelular , Cinética , Permeabilidad , Unión Proteica , Dominios Proteicos , Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
We report herein the design, synthesis, and evaluation of macrocyclic peptidomimetics that bind to WD repeat domain 5 (WDR5) and block the WDR5-mixed lineage leukemia (MLL) protein-protein interaction. Compound 18 (MM-589) binds to WDR5 with an IC50 value of 0.90 nM (Ki value <1 nM) and inhibits the MLL H3K4 methyltransferase (HMT) activity with an IC50 value of 12.7 nM. Compound 18 potently and selectively inhibits cell growth in human leukemia cell lines harboring MLL translocations and is >40 times better than the previously reported compound MM-401. Cocrystal structures of 16 and 18 complexed with WDR5 provide structural basis for their high affinity binding to WDR5. Additionally, we have developed and optimized a new AlphaLISA-based MLL HMT functional assay to facilitate the functional evaluation of these designed compounds. Compound 18 represents the most potent inhibitor of the WDR5-MLL interaction reported to date, and further optimization of 18 may yield a new therapy for acute leukemia.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Péptidos Cíclicos/farmacología , Peptidomiméticos/química , Peptidomiméticos/farmacología , Animales , Unión Competitiva , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Técnicas de Química Sintética , Descubrimiento de Drogas , Estabilidad de Medicamentos , Ensayos Analíticos de Alto Rendimiento/métodos , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Leucemia/tratamiento farmacológico , Leucemia/patología , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Espectroscopía de Resonancia Magnética , Ratones , Microsomas/efectos de los fármacos , Simulación del Acoplamiento Molecular , Proteína de la Leucemia Mieloide-Linfoide/genética , Péptidos Cíclicos/química , Peptidomiméticos/metabolismo , Dominios y Motivos de Interacción de Proteínas , RatasRESUMEN
MDM2 is a primary cellular inhibitor of p53. It inhibits p53 function by multiple mechanisms, each of which, however, is mediated by their direct interaction. It has been proposed that small-molecule inhibitors designed to block the MDM2-p53 interaction may be effective in the treatment of human cancer retaining wild-type p53 by reactivating the p53 tumor suppressor function. Through nearly two decades of intense efforts, a number of structurally distinct, highly potent, nonpeptide, small-molecule inhibitors of the MDM2-p53 interaction (MDM2 inhibitors) have been successfully designed and developed, and at least seven such compounds have now been advanced into human clinical trials as new anticancer drugs. This review offers a perspective on the design and development of MDM2 small-molecule inhibitors and discusses early clinical data for some of the MDM2 small-molecule inhibitors and future challenges for the successful clinical development of MDM2 inhibitors for cancer treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Antineoplásicos/química , Humanos , Imidazolinas/farmacología , Indoles/farmacología , Unión Proteica , Compuestos de Espiro/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
We previously reported the design of spirooxindoles with two identical substituents at the carbon-2 of the pyrrolidine core as potent MDM2 inhibitors. In this paper we describe an extensive structure-activity relationship study of this class of MDM2 inhibitors, which led to the discovery of 60 (AA-115/APG-115). Compound 60 has a very high affinity to MDM2 (Ki < 1 nM), potent cellular activity, and an excellent oral pharmacokinetic profile. Compound 60 is capable of achieving complete and long-lasting tumor regression in vivo and is currently in phase I clinical trials for cancer treatment.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Pirrolidinas/química , Pirrolidinas/uso terapéutico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/farmacocinética , Compuestos Bicíclicos con Puentes/farmacología , Compuestos Bicíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Halogenación , Humanos , Indoles/química , Indoles/farmacocinética , Indoles/farmacología , Indoles/uso terapéutico , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Leucemia/patología , Ratones , Simulación del Acoplamiento Molecular , Neoplasias/metabolismo , Neoplasias/patología , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirrolidinas/farmacocinética , Pirrolidinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
PURPOSE: This study was performed to further our understanding of the biological and genetic basis of follicular lymphoma and to identify potential novel therapy targets. EXPERIMENTAL DESIGN: We analyzed previously generated whole exome sequencing data of 23 follicular lymphoma cases and one transformed follicular lymphoma case and expanded findings to a combined total of 125 follicular lymphoma/3 transformed follicular lymphoma. We modeled the three-dimensional location of RRAGC-associated hotspot mutations. We performed functional studies on novel RRAGC mutants in stable retrovirally transduced HEK293T cells, stable lentivirally transduced lymphoma cell lines, and in Saccharomyces cerevisiae RESULTS: We report recurrent mutations, including multiple amino acid hotspots, in the small G-protein RRAGC, which is part of a protein complex that signals intracellular amino acid concentrations to MTOR, in 9.4% of follicular lymphoma cases. Mutations in RRAGC distinctly clustered on one protein surface area surrounding the GTP/GDP-binding sites. Mutated RRAGC proteins demonstrated increased binding to RPTOR (raptor) and substantially decreased interactions with the product of the tumor suppressor gene FLCN (folliculin). In stable retrovirally transfected 293T cells, cultured in the presence or absence of leucine, multiple RRAGC mutations demonstrated elevated MTOR activation as evidenced by increased RPS6KB/S6-kinase phosphorylation. Similar activation phenotypes were uncovered in yeast engineered to express mutations in the RRAGC homolog Gtr2 and in multiple lymphoma cell lines expressing HA-tagged RRAGC-mutant proteins. CONCLUSIONS: Our discovery of activating mutations in RRAGC in approximately 10% of follicular lymphoma provides the mechanistic rationale to study mutational MTOR activation and MTOR inhibition as a potential novel actionable therapeutic target in follicular lymphoma. Clin Cancer Res; 22(21); 5383-93. ©2016 AACR.
Asunto(s)
Linfoma Folicular/genética , Proteínas de Unión al GTP Monoméricas/genética , Mutación/genética , Recurrencia Local de Neoplasia/genética , Serina-Treonina Quinasas TOR/genética , Aminoácidos/genética , Sitios de Unión/genética , Línea Celular , Genes Supresores de Tumor/fisiología , Guanosina Difosfato/genética , Guanosina Trifosfato/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Fosforilación/genética , Proteína Reguladora Asociada a mTOR/genética , Transducción de Señal/genéticaRESUMEN
The p53 protein, a tumor suppressor, is inactivated in many human cancers through mutations or by its interaction with an oncoprotein, MDM2. Blocking the MDM2-p53 protein-protein interaction has the effect of activating wild-type p53 and has been pursued as a novel anticancer strategy. Small-molecule inhibitors of the MDM2-p53 interaction have been discovered through various approaches, and a number of them have progressed into clinical trials for cancer treatment. Here, we describe the methods and techniques used in the discovery of small-molecule inhibitors of the MDM2-p53 interaction.
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Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Humanos , Proteínas de Unión al ARN/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína p53 Supresora de Tumor/antagonistas & inhibidoresRESUMEN
Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world. FL cell-intrinsic and cell-extrinsic factors influence FL biology and clinical outcome. To further our understanding of the genetic basis of FL, we performed whole-exome sequencing of 23 highly purified FL cases and 1 transformed FL case and expanded findings to a combined total of 114 FLs. We report recurrent mutations in the transcription factor STAT6 in 11% of FLs and identified the STAT6 amino acid residue 419 as a novel STAT6 mutation hotspot (p.419D/G, p.419D/A, and p.419D/H). FL-associated STAT6 mutations were activating, as evidenced by increased transactivation in HEK293T cell-based transfection/luciferase reporter assays, heightened interleukin-4 (IL-4) -induced activation of target genes in stable STAT6 transfected lymphoma cell lines, and elevated baseline expression levels of STAT6 target genes in primary FL B cells harboring mutant STAT6. Mechanistically, FL-associated STAT6 mutations facilitated nuclear residency of STAT6, independent of IL-4-induced STAT6-Y641 phosphorylation. Structural modeling of STAT6 based on the structure of the STAT1-DNA complex revealed that most FL-associated STAT6 mutants locate to the STAT6-DNA interface, potentially facilitating heightened interactions. The genetic and functional data combined strengthen the recognition of the IL-4/JAK/STAT6 axis as a driver of FL pathogenesis.
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Núcleo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Linfoma Folicular/metabolismo , Mutación Missense , Proteínas de Neoplasias/metabolismo , Factor de Transcripción STAT6/metabolismo , Transporte Activo de Núcleo Celular/genética , Línea Celular Tumoral , Núcleo Celular/genética , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Interleucina-4/genética , Interleucina-4/metabolismo , Quinasas Janus/genética , Quinasas Janus/metabolismo , Linfoma Folicular/genética , Linfoma Folicular/patología , Proteínas de Neoplasias/genética , Fosforilación/genética , Factor de Transcripción STAT6/genética , Activación Transcripcional/genéticaRESUMEN
Design of small-molecule inhibitors (MDM2 inhibitors) to block the MDM2-p53 protein-protein interaction has been pursued as a new cancer therapeutic strategy. In recent years, potent, selective, and efficacious MDM2 inhibitors have been successfully obtained and seven such compounds have been advanced into early phase clinical trials for the treatment of human cancers. Here, we review the design, synthesis, properties, preclinical, and clinical studies of these clinical-stage MDM2 inhibitors.
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Ensayos Clínicos como Asunto , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Neoplasias/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Inhibition of the MDM2-p53 protein-protein interaction is being actively pursued as a new anticancer therapeutic strategy, and spiro-oxindoles have been designed as a class of potent and efficacious small-molecule inhibitors of this interaction (MDM2 inhibitors). Our previous study showed that some of our first-generation spiro-oxindoles undergo a reversible ring-opening-cyclization reaction that, from a single compound in protic solution, results in an equilibrium mixture of four diastereoisomers. By exploiting the ring-opening-cyclization reaction mechanism, we have designed and synthesized a series of second-generation spiro-oxindoles with symmetrical pyrrolidine C2 substitution. These compounds undergo a rapid and irreversible conversion to a single, stable diastereoisomer. Our study has yielded compound 31 (MI-1061), which binds to MDM2 with Ki = 0.16 nM, shows excellent chemical stability, and achieves tumor regression in the SJSA-1 xenograft tumor model in mice.