RESUMEN
Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure-activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.
Asunto(s)
Receptores de Activinas Tipo I/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Simulación por Computador , Cristalografía por Rayos X , Semivida , Humanos , Microsomas Hepáticos/efectos de los fármacos , Estructura Molecular , Miositis Osificante/enzimología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Espectroscopía de Protones por Resonancia Magnética , Pirazoles/administración & dosificación , Pirazoles/química , Pirazoles/farmacocinética , Solubilidad , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Imidazoles/farmacología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/ultraestructura , Estaurosporina/farmacología , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Ensayo de Cambio de Movilidad Electroforética , Humanos , Imidazoles/química , Modelos Moleculares , Piperazinas/química , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Estaurosporina/químicaRESUMEN
The small kinase inhibitor SKF86002 lacks intrinsic fluorescence but becomes fluorescent upon binding to the ATP-binding sites of p38 mitogen-activated protein kinase (p38α). It was found that co-crystals of this compound with various kinases were distinguishable by their strong fluorescence. The co-crystals of SKF86002 with p38α, Pim1, ASK1, HCK and AMPK were fluorescent. Addition of SKF86002, which binds to the ATP site, to the co-crystallization solution of HCK promoted protein stability and thus facilitated the production of crystals that otherwise would not grow in the apo form. It was further demonstrated that the fluorescence of SKF86002 co-crystals can be applied to screen for candidate kinase inhibitors. When a compound binds competitively to the ATP-binding site of a kinase crystallized with SKF86002, it displaces the fluorescent SKF86002 and the crystal loses its fluorescence. Lower fluorescent signals were reported after soaking SKF86002-Pim1 and SKF86002-HCK co-crystals with the inhibitors quercetin, a quinazoline derivative and A-419259. Determination of the SKF86002-Pim1 and SKF86002-HCK co-crystal structures confirmed that SKF86002 interacts with the ATP-binding sites of Pim1 and HCK. The structures of Pim1-SKF86002 crystals soaked with the inhibitors quercetin and a quinazoline derivative and of HCK-SKF86002 crystals soaked with A-419259 were determined. These structures were virtually identical to the deposited crystal structures of the same complexes. A KINOMEscan assay revealed that SKF86002 binds a wide variety of kinases. Thus, for a broad range of kinases, SKF86002 is useful as a crystal marker, a crystal stabilizer and a marker to identify ligand co-crystals for structural analysis.
Asunto(s)
Adenosina Trifosfato/química , Colorantes Fluorescentes/química , Imidazoles/química , Sondas Moleculares/química , Inhibidores de Proteínas Quinasas/química , Tiazoles/química , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Unión Competitiva , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , MAP Quinasa Quinasa Quinasa 5/química , MAP Quinasa Quinasa Quinasa 5/genética , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/química , Proteína Quinasa 14 Activada por Mitógenos/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-hck/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-hck/química , Proteínas Proto-Oncogénicas c-hck/genética , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/química , Proteínas Proto-Oncogénicas c-pim-1/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
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.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/química , Sustancias Macromoleculares/química , S-Adenosilmetionina/química , Cristalografía por Rayos X , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/aislamiento & purificación , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Sustancias Macromoleculares/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , S-Adenosilmetionina/metabolismoRESUMEN
Discs large homolog 1 (DLG1/SAP97) is involved in the development and regulation of neuronal and immunological synapses. DLG1 is a member of the membrane associated guanylate kinase (MAGUK) family of proteins, which function as molecular scaffolds. The C-terminal guanylate kinase (GK) domain of DLG1 binds peptides with a phosphorylated serine residue. In this study, we solved the crystal structure of the GK domain of human DLG1. The C-terminal tail of DLG1 is bound to the peptide-binding site of an adjacent symmetry-related DLG1 GK molecule. The binding direction of the C-terminal tail to the peptide-binding site is opposite to that of the phosphorylated LGN peptide in complex with the rat DLG1 GK domain. The C-terminal tail forms a 310 helix, which is also different from the conformation of the phosphorylated LGN peptide. Nevertheless, the side chain interactions of the C-terminal tail with the DLG1 GK domain are similar to those of the phosphorylated LGN peptide.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de la Membrana/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Homólogo 1 de la Proteína Discs Large , Guanilato-Quinasas/química , Humanos , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas , Proteínas Recombinantes/química , Especificidad de la EspecieRESUMEN
Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cells We have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, ß-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to ß-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.
Asunto(s)
Proteínas Portadoras/fisiología , Adhesiones Focales/patología , Metástasis de la Neoplasia , Neoplasias/patología , Secuencias de Aminoácidos , Animales , Calpaína/metabolismo , Proteínas Portadoras/química , Línea Celular , Movimiento Celular , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Ratones , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012), J. Med. Chem. 55, 5151-5156]. The report described the process of optimization of the structure-activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the ß3 strand located above the binding site, is not usually observed in Pim-1 structures.
Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-pim-1/química , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Estructura Cuaternaria de Proteína , Homología Estructural de ProteínaRESUMEN
AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor to maintain energy balance at both the cellular and the whole-body levels and is therefore a potential target for drug design against metabolic syndrome, obesity and type 2 diabetes. Here, the crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C. The AMPK α2 kinase domain exhibits a typical bilobal kinase fold and exists as a monomer in the crystal. Like the wild-type apo form, the T172D mutant apo form adopts the autoinhibited structure of the `DFG-out' conformation, with the Phe residue of the DFG motif anchored within the putative ATP-binding pocket. Compound C binding dramatically alters the conformation of the activation loop, which adopts an intermediate conformation between DFG-out and DFG-in. This induced fit forms a compound-C binding pocket composed of the N-lobe, the C-lobe and the hinge of the kinase domain. The pocket partially overlaps with the putative ATP-binding pocket. These three-dimensional structures will be useful to guide drug discovery.
Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas Activadas por AMP/genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/enzimología , Humanos , Síndrome Metabólico/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Obesidad/enzimología , Estructura Terciaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/genética , Alineación de SecuenciaRESUMEN
The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study.
RESUMEN
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.
Asunto(s)
Desarrollo de Medicamentos , Inhibidores Enzimáticos/farmacología , Histona Demetilasas/antagonistas & inhibidores , Tranilcipromina/farmacología , Alquilación , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Histona Demetilasas/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Tranilcipromina/síntesis química , Tranilcipromina/químicaRESUMEN
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.
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Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Diseño de Fármacos , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Cristalografía por Rayos X , Humanos , Ligandos , Masculino , FosforilaciónRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a rare but severe genetic disorder in which acute inflammation elicits progressive heterotopic ossification in the muscles, tendons, and ligaments. Classic FOP is caused by the R206H mutation in ALK2/ACVR1. While several activin receptor-like kinase 2 (ALK2) inhibitors were found to be efficacious in animal models of FOP, most of the ALK2 (R206H) inhibitors lacked sufficient oral bioavailability for efficacy. Previously, the synthesis of a series of novel bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitors that achieved both substantial potency and an improved ADMET profile was reported. In the present study, the detailed procedure of the in silico approach employed to identify the initial bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitor RK-59638 and the analysis of the ALK2 (R206H) RK-59638 complex structure to guide the synthetic optimization of the chemical series, obtaining RK-71807 showing improved potency and metabolic stability, were described. According to the initial in silico screening, the screening efficiencies and chemical diversity of the hit compounds of both ligand-based and structure-based methods were evaluated. Then, X-ray structures of ALK2 (R206H) and the inhibitors were analyzed to assess the structure-activity relationships of the synthesized compounds. The 3D-RISM analysis indicated the existence of the additional hydrogen bond via water molecules restricting the attachment point in the pyrazole scaffold. The quantum mechanics calculation of the newly determined ALK2 (R206H) RK-71807 complex structure using a fragment molecular orbital method and pair interaction energy decomposition analysis was employed to evaluate the interaction energies between the inhibitor and each of the amino acid residues and decompose them to electrostatic, exchange-repulsion, and charge transfer energies. The pattern of decomposed interaction energies was then compared to that formed by RK-59638 and LDN-193189 to investigate the structural basis of ALK2 (R206H) inhibition.
RESUMEN
BACKGROUND: Induced pluripotent stem cell (iPSC)âbased regenerative therapy is a promising strategy for cardiovascular disease treatment; however, the method is limited by the myocardial retention of grafted iPSCs. Thus, an injection protocol that efficiently introduces and retains human iPSC-derived cardiomyocytes (hiPSC-CMs) within the myocardium is urgently needed. The objective of the present study was to develop a method to improve the retention of hiPSCs in the myocardium for cardiac therapy. METHODS: We efficiently produced hiPSC-CM spheroids in 3-dimensional (3D) culture using microwell plates, and developed an injection device for optimal 3D distribution of the spheroids in the myocardial layer. Device biocompatibility was assessed with purified hiPSC-CM spheroids. Device effectiveness was evaluated in 10- to 15-month-old farm pigs (nâ¯=â¯15) and 5- to 24-month-old micro-minipigs (nâ¯=â¯20). The pigs were euthanized after injection, and tissues were harvested for retention and histologic analysis. RESULTS: We demonstrated an injection device for direct intramyocardial transplantation of hiPSC-CM spheroids from large-scale culture. The device had no detrimental effects on cell viability, spheroid shape, or size. Direct epicardial injection of spheroids mixed with gelatin hydrogel into beating porcine hearts using this device resulted in better distribution and retention of transplanted spheroids in a layer within the myocardium than did conventional needle injection procedures. CONCLUSIONS: The combination of the newly developed transplant device and spheroid formation promotes the retention of transplanted CMs. These findings support the clinical application of hiPSC-CM spheroidâbased cardiac regenerative therapy in patients with heart failure.
Asunto(s)
Insuficiencia Cardíaca/terapia , Células Madre Pluripotentes Inducidas/trasplante , Miocitos Cardíacos/citología , Trasplante de Células Madre/instrumentación , Animales , Materiales Biocompatibles , Diferenciación Celular , Modelos Animales de Enfermedad , Diseño de Equipo , Femenino , Insuficiencia Cardíaca/patología , Humanos , Inyecciones/instrumentación , Esferoides Celulares , Porcinos , Porcinos EnanosRESUMEN
The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.
Asunto(s)
Proteínas de Complejo Poro Nuclear/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Dimerización , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.
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Ciproheptadina/farmacología , Inhibidores Enzimáticos/farmacología , Estrógenos/fisiología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Transcripción Genética/fisiología , Cristalografía por Rayos X , Ciproheptadina/química , Inhibidores Enzimáticos/química , Receptor alfa de Estrógeno/química , N-Metiltransferasa de Histona-Lisina/química , Humanos , Células MCF-7 , Estructura MolecularRESUMEN
The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.
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Proteínas Bacterianas/química , Modelos Moleculares , Terpenos/metabolismo , Thermus thermophilus/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Datos de Secuencia Molecular , Quinonas/química , Quinonas/metabolismo , Alineación de Secuencia , Terpenos/químicaRESUMEN
The TT1542 protein from Thermus thermophilus HB8 is annotated as a conserved hypothetical protein, and belongs to the DUF158 family in the Pfam database. A BLAST search revealed that homologs of TT1542 are present in a wide range of organisms. The TT1542 homologs in eukaryotes, PIG-L in mammals, and GPI12 in yeast and protozoa, have N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase activity. Although most of the homologs in prokaryotes are hypothetical and have no known function, Rv1082 and Rv1170 from Mycobacterium tuberculosis are enzymes involved in the mycothiol detoxification pathway. Here we report the crystal structure of the TT1542 protein at 2.0 A resolution, which represents the first structure for this superfamily of proteins. The structure of the TT1542 monomer consists of a twisted beta-sheet composed of six parallel beta-strands and one antiparallel beta-strand (with the strand order 3-2-1-4-5-7-6) sandwiched between six alpha-helices. The N-terminal five beta-strands and four alpha-helices form an incomplete Rossmann fold-like structure. The structure shares some similarity to the sugar-processing enzymes with Rossmann fold-like domains, especially those of the GPGTF (glycogen phosphorylase/glycosyl transferase) superfamily, and also to the NAD(P)-binding Rossmann fold domains. TT1542 is a homohexamer in the crystal and in solution, the six monomers forming a cylindrical structure. Putative active sites are suggested by the structure and conserved amino acid residues.
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Proteínas Bacterianas/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Electricidad Estática , UltracentrifugaciónRESUMEN
Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.
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Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Adulto , Anciano , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Trasplante de Médula Ósea , Cristalografía por Rayos X , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Hematopoyesis/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Ratones , Persona de Mediana Edad , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-hck/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-hck/química , Proteínas Proto-Oncogénicas c-hck/metabolismo , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Células Tumorales Cultivadas , Adulto JovenRESUMEN
A new screening method using fluorescent correlation spectroscopy was developed to select kinase inhibitors that competitively inhibit the binding of a fluorescently labeled substrate peptide. Using the method, among approximately 700 candidate compounds selected by virtual screening, we identified a novel Pim-1 kinase inhibitor targeting its peptide binding residues. X-ray crystal analysis of the complex structure of Pim-1 with the inhibitor indicated that the inhibitor actually binds to the ATP-binding site and also forms direct interactions with residues (Asp128 and Glu171) that bind the substrate peptide. These interactions, which cause small side-chain movements, seem to affect the binding ability of the fluorescently labeled substrate. The compound inhibited Pim-1 kinase in vitro, with an IC(50) value of 150 nM. Treatment of cultured leukemia cells with the compound reduced the amount of p21 and increased the amount of p27, due to Pim-1 inhibition, and then triggered apoptosis after cell-cycle arrest at the G(1)/S phase. This screening method may be widely applicable for the identification of various new Pim-1 kinase inhibitors targeting the residues that bind the substrate peptide.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/química , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Sitios de Unión , Unión Competitiva , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Espectrometría de Fluorescencia/métodosRESUMEN
The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P(i) ). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg(2+) ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P(i) increased the affinity in the presence of Mg(2+) ion, but not in its absence. Thus, P(i) enhances the effect of the Mg(2+) ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg(2+) ion. As compared with the Mg(2+) ion-free structure, the ADP- and Mg(2+) ion-bound NBD contains one Mg(2+) ion, which is coordinated with the ß-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg(2+) ion is also coordinated with one P(i) molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P(i) . Therefore, the Mg(2+) ion-mediated network, including the P(i) and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg(2+) ion-mediated network.