RESUMO
The discovery and development of structurally distinct lysine methyltransferase G9a inhibitors have been the subject of intense research in epigenetics. Structure-based optimization was conducted, starting with the previously reported seed compound 7a and lead to the identification of a highly potent G9a inhibitor, compound 7i (IC50 = 0.024 µM). X-ray crystallography for the ligand-protein interaction and kinetics study, along with surface plasmon resonance (SPR) analysis, revealed that compound 7i interacts with G9a in a unique binding mode. In addition, compound 7i caused attenuation of cellular H3K9me2 levels and induction of γ-globin mRNA expression in HUDEP-2 cells in a dose-dependent manner.
Assuntos
Anemia Falciforme , Inibidores Enzimáticos , Epigênese Genética , Histona-Lisina N-Metiltransferase , Humanos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Relação Estrutura-Atividade , Anemia Falciforme/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Epigênese Genética/efeitos dos fármacos , Estrutura Molecular , Antígenos de Histocompatibilidade/metabolismo , Relação Dose-Resposta a Droga , Cristalografia por Raios XRESUMO
Because lysine-specific demethylase 1 (LSD1) regulates the maintenance of cancer stem cell properties, small-molecule inhibitors of LSD1 are expected to be useful for the treatment of several cancers. Reversible inhibitors of LSD1 with submicromolar inhibitory potency have recently been reported, but their exact binding modes are poorly understood. In this study, we synthesized a recently reported reversible inhibitor, 4-[5-(piperidin-4-ylmethoxy)-2-(p-tolyl)pyridin-3-yl]benzonitrile, which bears a 4-piperidinylmethoxy group, a 4-methylphenyl group, and a 4-cyanophenyl group on a pyridine ring, and determined the crystal structure of LSD1 in complex with this inhibitor at 2.96 Å. We observed strong electron density for the compound, showing that its cyano group forms a hydrogen bond with Lys661, which is a critical residue in the lysine demethylation reaction located deep in the catalytic center of LSD1. The piperidine ring interacts with the side chains of Asp555 and Asn540 in two conformations, and the 4-methylphenyl group is bound in a hydrophobic pocket in the catalytic center. Our elucidation of the binding mode of this compound can be expected to facilitate the rational design of more-potent reversible LSD1 inhibitors.
Assuntos
Histona Desmetilases/química , Nitrilas/química , Piperidinas/química , Piridinas/química , Proteínas Correpressoras/química , Proteínas Correpressoras/metabolismo , Cristalografia por Raios X , Histona Desmetilases/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Especificidade por SubstratoRESUMO
Ribosomal protein S6 kinase 1 (S6K1) is a serine/threonine protein kinase that plays an important role in the PIK3/mTOR signaling pathway, and is implicated in diseases including diabetes, obesity, and cancer. The crystal structures of the S6K1 kinase domain in complexes with staurosporine and the S6K1-specific inhibitor PF-4708671 have been reported. In the present study, five compounds (F108, F109, F176, F177, and F179) were newly identified by in silico screening of a chemical library and kinase assay. The crystal structures of the five inhibitors in complexes with the S6K1 kinase domain were determined at resolutions between 1.85 and 2.10 Å. All of the inhibitors bound to the ATP binding site, lying along the P-loop, while the activation loop stayed in the inactive form. Compound F179, with a carbonyl group in the middle of the molecule, altered the αC helix conformation by interacting with the invariant Lys123. Compounds F176 and F177 bound slightly distant from the hinge region, and their sulfoamide groups formed polar interactions with the protein. The structural features required for the specific binding of inhibitors are discussed.
Assuntos
Imidazóis/farmacologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/ultraestrutura , Estaurosporina/farmacologia , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Imidazóis/química , Modelos Moleculares , Piperazinas/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Estaurosporina/químicaRESUMO
SET7/9 is a protein lysine methyltransferase that methylates histone H3 and nonhistone proteins such as p53, TAF10 and oestrogen receptor α. In previous work, novel inhibitors of SET7/9 that are amine analogues of the coenzyme S-(5'-adenosyl)-L-methionine (AdoMet) have been developed. Here, crystal structures of SET7/9 are reported in complexes with two AdoMet analogues, designated DAAM-3 and AAM-1, in which an n-hexylaminoethyl group or an n-hexyl group is attached to the N atom that replaces the S atom of AdoMet, respectively. In both structures, the inhibitors bind to the coenzyme-binding site and their additional alkyl chain binds in the lysine-access channel. The N atom in the azaalkyl chain of DAAM-3 is located at almost the same position as the N-methyl C atom of the methylated lysine side chain in the substrate-peptide complex structures and stabilizes complex formation by hydrogen bonding to the substrate-binding site residues of SET7/9. On the other hand, the alkyl chain of AAM-1, which is a weaker inhibitor than DAAM-3, binds in the lysine-access channel only through hydrophobic and van der Waals interactions. Unexpectedly, the substrate-binding site of SET7/9 complexed with AAM-1 specifically interacts with the artificial N-terminal sequence of an adjacent symmetry-related molecule, presumably stabilizing the alkyl chain of AAM-1.
Assuntos
Histona-Lisina N-Metiltransferase/química , Substâncias Macromoleculares/química , S-Adenosilmetionina/química , Cristalografia por Raios X , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/isolamento & purificação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Substâncias Macromoleculares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , S-Adenosilmetionina/metabolismoRESUMO
The dynamic regulation of histone methylation and demethylation plays an important role in the regulation of gene expression. Aberrant expression of histone lysine demethylases has been implicated in various diseases including intractable cancers, and thus lysine demethylases serve as promising therapeutic targets. Recent studies in epigenomics and chemical biology have led to the development of a series of small-molecule demethylase inhibitors that are potent, specific, and have in vivo efficacy. In this review, we highlight emerging small-molecule inhibitors targeting the histone lysine demethylases and their progress toward drug discovery.
RESUMO
Identification of structurally novel inhibitors of lysine methyltransferase G9a has been a subject of intense research in cancer epigenetics. Starting with the high-throughput screening (HTS) hit rac-10a obtained from the chemical library of the University of Tokyo Drug Discovery Initiative, the structure-activity relationship of the unique substrate-competitive inhibitors was established with the help of X-ray crystallography and fragment molecular orbital (FMO) calculations for the ligand-protein interaction. Further optimization of the in vitro characteristics and drug metabolism and pharmacokinetics (DMPK) properties led to the identification of 26j (RK-701), which is a structurally distinct potent inhibitor of G9a/GLP (IC50 = 27/53 nM). Compound 26j exhibited remarkable selectivity against other related methyltransferases, dose-dependent attenuation of cellular H3K9me2 levels, and tumor growth inhibition in MOLT-4 cells in vitro. Moreover, compound 26j showed inhibition of tumor initiation and growth in a carcinogen-induced hepatocellular carcinoma (HCC) in vivo mouse model without overt acute toxicity.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase , LisinaRESUMO
Sickle cell disease (SCD) is a heritable disorder caused by ß-globin gene mutations. Induction of fetal γ-globin is an established therapeutic strategy. Recently, epigenetic modulators, including G9a inhibitors, have been proposed as therapeutic agents. However, the molecular mechanisms whereby these small molecules reactivate γ-globin remain unclear. Here we report the development of a highly selective and non-genotoxic G9a inhibitor, RK-701. RK-701 treatment induces fetal globin expression both in human erythroid cells and in mice. Using RK-701, we find that BGLT3 long non-coding RNA plays an essential role in γ-globin induction. RK-701 selectively upregulates BGLT3 by inhibiting the recruitment of two major γ-globin repressors in complex with G9a onto the BGLT3 gene locus through CHD4, a component of the NuRD complex. Remarkably, BGLT3 is indispensable for γ-globin induction by not only RK-701 but also hydroxyurea and other inducers. The universal role of BGLT3 in γ-globin induction suggests its importance in SCD treatment.
Assuntos
Anemia Falciforme , RNA Longo não Codificante , Camundongos , Humanos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , gama-Globinas/genética , Células Eritroides/metabolismo , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Expressão Gênica , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismoRESUMO
Viscumin, a lectin used in anti-cancer therapy, was originally considered as ßGal recognizing protein; later, an ability to bind 6'-sialyl N-acetyllactosamine (6'SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galß1-4GlcNAc, was observed. Unexpectedly, only weak interaction was detected with parent neutral ß-galactosides including LN-LN-LN and branched (LN)2LN oligolactosamines; in the light of these data, one should not confidently classify viscumin as a ß-galactoside-binding lectin. Carrying out PGA in the presence of neutral or sulfated/sialylated glycan, together with sequential elution from lactose-sepharose and consideration of the protein structure, lead to the conclusion that two glycan-binding sites of viscumin have different specificities, one of which prefers charged sulfated and sialylated moieties.
Assuntos
Lactose , Animais , Mamíferos , Polissacarídeos , Proteínas Inativadoras de Ribossomos Tipo 2 , SulfatosRESUMO
Lysine-specific demethylase 1 (LSD1/KDM1A) is a promising therapeutic target for the treatment of cancers. Several derivatives of tranylcypromine (trans-2-phenylcyclopropylamine) have been developed as LSD1 inhibitors. One such derivative is S2157; however, this compound has a high hERG channel inhibitory activity and a low microsomal stability, making it unsuitable as a drug candidate. Here, using an in silico hERG inhibition prediction model, we designed, synthesized, and evaluated a novel series of S2157 derivatives characterized by modifications of the benzyloxy and piperazine groups. Among the synthesized derivatives, a compound possessing 2-fluoropyridine and 2,8-diaza-spiro[4.5]decane groups (compound 10) showed the most desirable activities, and its eutomer, S1427, was isolated by the optical resolution of 10. In addition to potent LSD1 inhibitory activity, S1427 exhibited desirable hERG channel inhibition and microsomal stability profiles.
RESUMO
trans-2-Phenylcycloproylamine (trans-PCPA) has been used as the scaffold to develop covalent-binding inhibitors against lysine-specific demethylase 1 (LSD1/KDM1A), a therapeutic target for several cancers. However, the effects of different structural moieties on the inhibitory activity, selectivity, and reactivity of these derivatives, including the cis isomers, against LSD1 and its paralogue LSD2/KDM1B are not fully understood. Here we synthesized 65 cis- and trans-PCPA derivatives and evaluated their inhibitory activity against LSD1 and LSD2. One of the derivatives, 7c (cis-4-Br-2,5-F2-PCPA; S1024), inhibited LSD1 and LSD2 with K i values of 0.094 µM and 8.4 µM, respectively, and increased the level of dimethylated histone H3 at K4 in CCRF-CEM cells. A machine learning-based regression model (Q 2 = 0.61) to predict LSD1-inhibitory activity was also constructed and showed a good prediction accuracy (R 2 = 0.81) for 12 test-set compounds, including 7c. The present methodology would be useful when designing covalent-binding inhibitors for other enzymes.
RESUMO
OBJECTIVES: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae have become a cause for great concern. Although some studies have reported the prevalence of ESBL-producing bacteria and ESBL-encoding genes in horses worldwide, the genetic structure surrounding the ESBL gene has not been analysed in detail. In the present study, we isolated two ESBL-producing Escherichia coli strains from diseased racehorses in Japan and demonstrated the mechanisms underlying the acquisition of their antimicrobial resistance (AMR) genes. METHODS: Two ESBL-producing E. coli strains (E148 and E189) were isolated from the heart and liver of horses with endocarditis and sepsis in 2014 and 2016, respectively, in Japan. Complete genomic sequences of the two strains were analysed using a PacBio RSII sequencer. Antimicrobial susceptibility testing was performed by the agar dilution method. RESULTS: The two isolates possessed a chromosomal AMR gene cluster containing blaCTX-M-1 that was similar to the pEQ1 plasmid found in E. coli isolated from a racehorse in the Czech Republic. In one of the two strains, tandem duplication of the 16-kb region containing blaCTX-M-1 and a class 1 integron, which occurred via IS26-mediated recombination, increased minimum inhibitory concentrations (MICs) associated with the duplicated AMR genes. CONCLUSION: Chromosomal blaCTX-M-1 possibly derived from the pEQ1 or pEQ1-like plasmid was found in Japanese equine E. coli isolates. In Japanese strains, many AMR genes containing blaCTX-M-1 and the class 1 integron are highly accumulated in one region on the chromosome, and the AMR of E. coli was enhanced via the IS26-mediated duplication of the AMR gene cluster.
Assuntos
Anti-Infecciosos , Escherichia coli , Animais , Antibacterianos/farmacologia , Cromossomos , Farmacorresistência Bacteriana , Escherichia coli/genética , Cavalos , Integrons , Japão , beta-Lactamases/genéticaRESUMO
The crystal structure of an uncharacterized protein TTHA0061 from Thermus thermophilus HB8, was determined and refined to 1.8 A by a single wavelength anomalous dispersion (SAD) method. The structural analysis and comparison of TTHA0061 with other existing structures in the Protein Data Bank (PDB) revealed a novel fold, suggesting that this protein may belong to a translation initiation factor or ribosomal protein family. Differential scanning calorimetry analysis suggested that the thermostability of TTHA0061 increased at pH ranges of 5.8-6.2, perhaps due to the abundance of glutamic acid residues.
Assuntos
Fatores de Iniciação em Procariotos/química , Proteínas Ribossômicas/química , Thermus thermophilus/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Ácido Glutâmico/química , Dados de Sequência Molecular , Fatores de Iniciação em Procariotos/genética , Prolina/química , Conformação Proteica , Dobramento de Proteína , Proteínas Ribossômicas/genética , Thermus thermophilus/genéticaRESUMO
Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the demethylation of histone H3 and regulates gene expression. Because it is implicated in the regulation of diseases such as acute myeloid leukemia, potent LSD1-specific inhibitors have been pursued. Trans-2-phenylcyclopropylamine (2-PCPA)-based inhibitors featuring substitutions on the amino group have emerged, with sub-micromolar affinities toward LSD1 and high selectivities over monoamine oxidases (MAOs). We synthesized two N-alkylated 2-PCPA-based LSD1 inhibitors, S2116 and S2157, based on the previously developed S2101. S2116 and S2157 exhibited enhanced potency for LSD1 by 2.0- to 2.6-fold, as compared with S2101. In addition, they exhibited improved selectivity over MAOs. Structural analyses of LSD1 co-crystallized with S2101, S2116, S2157, or another N-alkylated inhibitor (FCPA-MPE) confirmed that the N-substituents enhance the potency of a 2-PCPA-based inhibitor of LSD1, without constituting the adduct formed with FAD.
Assuntos
Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Tranilcipromina/farmacologia , Alquilação , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona Desmetilases/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Tranilcipromina/síntese química , Tranilcipromina/químicaRESUMO
CaMKK2 (calcium/calmodulin dependent protein kinase kinase 2) is a serine/threonine protein kinase that regulates phosphorylation of CaM kinases (CaMKs) such as CaMKI, CaMKIV, and AMP-activated protein kinase (AMPK). From a pathological perspective, CaMKK2 plays a role in obesity, diabetes, and prostate cancer. Therefore, CaMKK2 is an attractive target protein for drug design. Here, we tried to find new CaMKK2 inhibitors by using ligand-based and structure-based drug design approaches. From the in silico hit compounds, we identified new inhibitors by using a CaMKK2 kinase assay. We solved X-ray crystallography structures of the CaMKK2-inhibitor complexes and performed Fragment Molecular Orbital (FMO) calculations to analyze the protein-ligand interactions, identify the key residues in inhibitor binding, and quantitatively measure their contribution. We experimentally determined five CaMKK2-inhibitor structures and calculated the binding energies of the inhibitors by the FMO method plus MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) approach. The results showed a high correlation (R = -0.89) between experimentally measured inhibitory activity (pIC50) and the predicted ligand binding energy. We then quantitatively evaluated the contribution of each binding site residue in CaMKK2 by the IFIE (Inter-fragment Interaction Energy)/PIEDA (Pair Interaction Energy Decomposition Analysis) method. The IFIE values indicated that Lys194 and Glu236, which formed hydrogen bonds with the carboxylate groups of the inhibitors, were key residues for ligand binding. PIEDA revealed that the dispersion interaction of inhibitors with hydrophobic residues, such as Ile171, Phe267, and Leu319, contributed highly to ligand binding; we considered that this was due to CH-π interactions with methoxy groups and/or aromatic rings contained in our CaMKK2 inhibitor. These results from the quantitative interaction analysis by the FMO method are useful not only for future CaMMK2 inhibitor development but for application of the FMO method to in silico drug design.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Desenho de Fármacos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cristalografia por Raios X , Humanos , Ligantes , Masculino , FosforilaçãoRESUMO
Tankyrases (TNKS/TNKS2) belong to the poly(ADP-ribose) polymerase family. Inhibition of their enzymatic activities attenuates the Wnt/ß-catenin signaling, which plays an important role in cancer pathogenesis. We previously reported the discovery of RK-287107, a spiroindoline-based, highly selective, potent tankyrase inhibitor. Herein we describe the optimization process of RK-287107 leading to RK-582, which exhibits a markedly improved robust tumor growth inhibition in a COLO-320DM mouse xenograft model when orally administered. In addition to the dose-dependent elevation and attenuation of the levels of biomarkers AXIN2 and ß-catenin, respectively, results of the TCF reporter and cell proliferation studies on COLO-320DM are discussed.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Desenho de Fármacos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/administração & dosagem , Tanquirases/antagonistas & inibidores , Administração Oral , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Inibidores Enzimáticos/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Terciária de Proteína , Ratos , Tanquirases/química , Tanquirases/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Galactose/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/metabolismo , Sambucus nigra/química , Antígenos Glicosídicos Associados a Tumores/química , Sítios de Ligação , Cristalografia por Raios X , Galactose/análise , Galactose/química , Lectinas de Plantas/isolamento & purificação , Polissacarídeos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Inativadoras de Ribossomos/isolamento & purificação , Sambucus nigra/metabolismo , Espalhamento a Baixo Ângulo , Madeira/químicaRESUMO
The canonical WNT pathway plays an important role in cancer pathogenesis. Inhibition of poly(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/ß-catenin signal by preventing poly ADP-ribosylation-dependent degradation of AXIN, a negative regulator of Wnt/ß-catenin signaling. With the goal of investigating the effects of tankyrase and Wnt pathway inhibition on tumor growth, we set out to find small-molecule inhibitors of TNKS/TNKS2 with suitable drug-like properties. Starting from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was discovered as a potent TNKS/TNKS2 inhibitor with >7000-fold selectivity against the PARP1 enzyme, which inhibits WNT-responsive TCF reporter activity and proliferation of human colorectal cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent tumor growth inhibition in a mouse xenograft model. These observations suggest that RK-287107 is a promising lead compound for the development of novel tankyrase inhibitors as anticancer agents.
Assuntos
Inibidores Enzimáticos/farmacologia , Indóis/química , Indóis/farmacologia , Compostos de Espiro/farmacologia , Tanquirases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Compostos de Espiro/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)-1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling.
Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Linhagem Celular , Endocitose , Proteínas de Ligação a Ácido Graxo/metabolismo , Humanos , Ligação Proteica , Mapas de Interação de ProteínasRESUMO
The histone methyltransferase SET7/9 methylates not only histone but also non-histone proteins as substrates, and therefore, SET7/9 inhibitors are considered candidates for the treatment of diseases. Previously, our group identified cyproheptadine, used clinically as a serotonin receptor antagonist and histamine receptor (H1) antagonist, as a novel scaffold of the SET7/9 inhibitor. In this work, we focused on dibenzosuberene as a substructure of cyproheptadine and synthesized derivatives with various functional groups. Among them, the compound bearing a 2-hydroxy group showed the most potent activity. On the other hand, a 3-hydroxy group or another hydrophilic functional group such as acetamide decreased the activity. Structural analysis clarified a rationale for the improved potency only by tightly restricted location and type of the hydrophilic group. In addition, a SET7/9 loop, which was only partially visible in the complex with cyproheptadine, became more clearly visible in the complex with 2-hydroxycyproheptadine. These results are expected to be helpful for further structure-based development of SET7/9 inhibitors.
Assuntos
Ciproeptadina/química , Ciproeptadina/farmacologia , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Conformação ProteicaRESUMO
Until 2004, many researchers believed that protein methylation in eukaryotic cells was an irreversible reaction. However, the discovery of lysine-specific demethylase 1 in 2004 drastically changed this view and the concept of chromatin regulation. Since then, the enzymes responsible for lysine demethylation and their cellular substrates, biological significance, and selective regulation have become major research topics in epigenetics and chromatin biology. Many cell-permeable inhibitors for lysine demethylases have been developed, including both target-specific and nonspecific inhibitors. Structural understanding of how these inhibitors bind to lysine demethylases is crucial both for validation of the inhibitors as chemical probes and for the rational design of more potent, target-specific inhibitors. This review focuses on published small-molecule inhibitors targeted at the two flavin adenine dinucleotide-dependent lysine demethylases, lysine-specific demethylases 1 and 2, and how the inhibitors interact with the tertiary structures of the enzymes.