RESUMO
The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90ß-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.
Assuntos
Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Nucleotídeos/farmacologia , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Calorimetria , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Cinética , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/farmacologia , TermodinâmicaRESUMO
Benign familial infantile seizure (BFIS) is an autosomal dominant infantile-onset epilepsy syndrome with a typically benign prognosis. It is commonly associated with heterozygous mutations of the PRRT2 gene located on chromosome 16p11.2. The frameshift heterozygous mutation (c.649dupC, p.Arg217Profs∗8) in PRRT2 is responsible for the majority of BFIS cases. In this report, we present a rare case of a girl with a confirmed clinical and genetic diagnosis of BFIS due to a frameshift heterozygous mutation in PRRT2 (c.649dupC). She exhibited typical neurodevelopment until 15 months of age, followed by an unexpected severe autistic regression. In addition to BFIS, PRRT2 mutations are also associated with paroxysmal kinesigenic dyskinesia (PKD) and infantile convulsions and paroxysmal choreoathetosis (ICCA), indicating a complex genotype-phenotype heterogeneity in PRRT2 mutations. This clinical observation highlights the possibility that BFIS patients with PRRT2 mutations may not always have a benign neurodevelopmental prognosis, emphasizing the need for long-term clinical follow-up.
RESUMO
BACKGROUND: Limited research has explored the associations of gestational age (GA) and breastfeeding practices with growth and nutrition in term infants. METHODS: This multicenter cross-sectional study recruited 7299 singleton term infants from well-child visits in Shandong, China, between March 2021 and November 2022. Data on GA, gender, ethnicity, birth weight, parental heights, gestational diabetes and hypertension, age at visit, breastfeeding practices (point-in-time data at visit for infants < 6 months and retrospective data at 6 months for infants ≥ 6 months), complementary foods introduction, infant length and weight, were collected. 7270 infants were included in the analysis after excluding outliers with Z-scores of length (LAZ), weight or weight for length (WLZ) <-4 or > 4. Linear regression models adjused for covariates explored the impact of GA and breastfeeding practices on LAZ and WLZ, while logistic regression models evaluated their effect on the likelihood of moderate and severe stunting (MSS, LAZ<-2), moderate and severe acute malnutrition (MSAM, WLZ<-2) and overweight/obesity (WLZ > 2). Sensitivity analysis was conducted on normal birth weight infants (2.5-4.0 kg). RESULTS: Infants born early-term and exclusively breastfed accounted for 31.1% and 66.4% of the sample, respectively. Early-term birth related to higher WLZ (< 6 months: ß = 0.23, 95% confidence interval (CI): 0.16, 0.29; ≥6 months: ß = 0.12, 95% CI: 0.04, 0.20) and an increased risk of overweight/obesity throughout infancy (< 6 months: OR: 1.41, 95% CI 1.08, 1.84; ≥6 months: OR: 1.35, 95% CI 1.03, 1.79). Before 6 months, early-term birth correlated with lower LAZ (ß=-0.16, 95% CI: -0.21, -0.11) and an increased risk of MSS (OR: 1.01, 95%CI 1.00, 1.02); Compared to exclusive breastfeeding, exclusive formula-feeding and mixed feeding linked to lower WLZ (ß=-0.15, 95%CI -0.30, 0.00 and ß=-0.12, 95%CI -0.19, -0.05, respectively) and increased risks of MSAM (OR: 5.57, 95%CI 1.95, 15.88 and OR: 3.19, 95%CI 1.64, 6.19, respectively). Sensitivity analyses confirmed these findings. CONCLUSIONS: The findings emphasize the health risks of early-term birth and the protective effect of exclusive breastfeeding in singleton term infants, underscoring the avoidance of nonmedically indicated delivery before 39 weeks and promoting exclusive breastfeeding before 6 months.
Assuntos
Aleitamento Materno , Humanos , Aleitamento Materno/estatística & dados numéricos , Estudos Transversais , Feminino , Masculino , Recém-Nascido , Lactente , China/epidemiologia , Idade Gestacional , Fenômenos Fisiológicos da Nutrição do Lactente , Nascimento a Termo , Estudos Retrospectivos , Adulto , Estado NutricionalRESUMO
The enzyme MurA has been an established antibiotic target since the discovery of fosfomycin, which specifically inhibits MurA by covalent modification of the active site residue Cys-115. Early biochemical studies established that Cys-115 also covalently reacts with substrate phosphoenolpyruvate (PEP) to yield a phospholactoyl adduct, but the structural and functional consequences of this reaction remained obscure. We captured and depicted the Cys-115-PEP adduct of Enterobacter cloacae MurA in various reaction states by X-ray crystallography. The data suggest that cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM), the product of the MurB reaction, with PEP covalently attached to Cys-115. The uniqueness and rigidity of this "dormant" complex was previously not recognized and presumably accounts for the failure of drug discovery efforts toward the identification of novel and effective MurA inhibitors. We demonstrate that recently published crystal structures of MurA from various organisms determined by different laboratories were indeed misinterpreted and actually contain UNAM and covalently bound PEP. The Cys-115-PEP adduct was also captured in vitro during the reaction of free MurA and substrate UDP-N-acetylglucosamine or isomer UDP-N-acetylgalactosamine. The now available series of crystal structures allows a comprehensive view of the reaction cycle of MurA. It appears that the covalent reaction of MurA with PEP fulfills dual functions by tightening the complex with UNAM for the efficient feedback regulation of murein biosynthesis and by priming the PEP molecule for instantaneous reaction with substrate UDP-N-acetylglucosamine.
Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Enterobacter cloacae/enzimologia , Fosfoenolpiruvato/metabolismo , Alquil e Aril Transferases/genética , Cristalografia por Raios X , Adutos de DNA/metabolismo , Enterobacter cloacae/genética , Ativação Enzimática/fisiologia , Escherichia coli/genética , Ácidos Murâmicos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/fisiologiaRESUMO
The crystal structure of the 11.14 kDa orphan ORF 1382 from Archaeoglobus fulgidus (AF1382) has been determined by sulfur SAD phasing using a moderately diffracting crystal and 1.9 Å wavelength synchrotron X-rays. AF1382 was selected as a structural genomics target by the Southeast Collaboratory for Structural Genomics (SECSG) since sequence analyses showed that it did not belong to the Pfam-A database and thus could represent a novel fold. The structure was determined by exploiting longer wavelength X-rays and data redundancy to increase the anomalous signal in the data. AF1382 is a 95-residue protein containing five S atoms associated with four methionine residues and a single cysteine residue that yields a calculated Bijvoet ratio (ΔF(anom)/F) of 1.39% for 1.9 Å wavelength X-rays. Coupled with an average Bijvoet redundancy of 25 (two 360° data sets), this produced an excellent electron-density map that allowed 69 of the 95 residues to be automatically fitted. The S-SAD model was then manually completed and refined (R = 23.2%, R(free) = 26.8%) to 2.3 Å resolution (PDB entry 3o3k). High-resolution data were subsequently collected from a better diffracting crystal using 0.97 Å wavelength synchrotron X-rays and the S-SAD model was refined (R = 17.9%, R(free) = 21.4%) to 1.85 Å resolution (PDB entry 3ov8). AF1382 has a winged-helix-turn-helix structure common to many DNA-binding proteins and most closely resembles the N-terminal domain (residues 1-82) of the Rio2 kinase from A. fulgidus, which has been shown to bind DNA, and a number of MarR-family transcriptional regulators, suggesting a similar DNA-binding function for AF1382. The analysis also points out the advantage gained from carrying out data reduction and structure determination on-site while the crystal is still available for further data collection.
Assuntos
Proteínas Arqueais/química , Archaeoglobus fulgidus/química , Enxofre/química , Cristalografia por Raios X , Modelos Moleculares , Fases de Leitura Aberta , Estrutura Terciária de ProteínaRESUMO
In an attempt to identify novel small-molecule ligands of cyclin-dependent kinase 2 (CDK2) with potential as allosteric inhibitors, we have devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds that displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC(50) values) ranging from 6 to 44 µM, all of which were classified as ATP-site-directed ligands. Four new type I inhibitor scaffolds were confirmed by X-ray crystallography. Although this small compound library contained only ATP-site-directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action.
Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Regulação Alostérica , Naftalenossulfonato de Anilina/química , Sítios de Ligação , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Eight- and four-membered analogues of N-butyldeoxynojirimycin (NB-DNJ), a reversible male contraceptive in mice, were prepared and tested. A chiral pool approach was used for the synthesis of the target compounds. Key steps for the synthesis of the eight-membered analogues involve ring-closing metathesis and Sharpless asymmetric dihydroxylation and for the four-membered analogues Sharpless epoxidation, epoxide ring-opening (azide), and Mitsunobu reaction to form the four-membered ring. (3S,4R,5S,6R,7R)-1-Nonylazocane-3,4,5,6,7-pentaol (6) was moderately active against rat-derived ceramide-specific glucosyltransferase, and four of the other eight-membered analogues were weakly active against rat-derived ß-glucosidase 2. Among the four-membered analogues, ((2R,3S,4S)-3-hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (25) displayed selective inhibitory activity against mouse-derived ceramide-specific glucosyltransferase and was about half as potent as NB-DNJ against the rat-derived enzyme. ((2S,4S)-3-Hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (27) was found to be a selective inhibitor of ß-glucosidase 2, with potency similar to NB-DNJ. Additional glycosidase assays were performed to identify potential other therapeutic applications. The eight-membered iminosugars exhibited specificity for almond-derived ß-glucosidase, and the 1-nonylazetidine 25 inhibited α-glucosidase (Saccharomyces cerevisiae) with an IC(50) of 600 nM and ß-glucosidase (almond) with an IC(50) of 20 µM. Only N-nonyl derivatives were active, emphasizing the importance of a long lipophilic side chain for inhibitory activity of the analogues studied.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Açúcares/síntese química , Imino Açúcares/farmacologia , beta-Glucosidase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/química , Glucosiltransferases/química , Glicosídeo Hidrolases/química , Imino Açúcares/química , Concentração Inibidora 50 , Masculino , Estrutura Molecular , Ratos , beta-Glucosidase/químicaRESUMO
BRD4 and other members of the bromodomain and extraterminal (BET) family of proteins are promising epigenetic targets for the development of novel therapeutics. Among the reported BRD4 inhibitors are dihydropteridinones and benzopyrimidodiazepinones originally designed to target the kinases PLK1, ERK5, and LRRK2. While these kinase inhibitors were identified as BRD4 inhibitors, little is known about their binding potential and structural details of interaction with the other BET bromodomains. We comprehensively characterized a series of known and newly identified dual BRD4-kinase inhibitors against all eight individual BET bromodomains. A detailed analysis of 23 novel cocrystal structures of BET-kinase inhibitor complexes in combination with direct binding assays and cell signaling studies revealed significant differences in molecular shape complementarity and inhibitory potential. Collectively, the data offer new insights into the action of kinase inhibitors across BET bromodomains, which may aid the development of drugs to inhibit certain BET proteins and kinases differentially.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Fatores de Transcrição/químicaRESUMO
Inhibition of the bromodomain containing protein 9 (BRD9) by small molecules is an attractive strategy to target mutated SWI/SNF chromatin-remodeling complexes in cancer. However, reported BRD9 inhibitors also inhibit the closely related bromodomain-containing protein 7 (BRD7), which has different biological functions. The structural basis for differential potency and selectivity of BRD9 inhibitors is largely unknown because of the lack of structural information on BRD7. Here, we biochemically and structurally characterized diverse inhibitors with varying degrees of potency and selectivity for BRD9 over BRD7. Novel cocrystal structures of BRD7 liganded with new and previously reported inhibitors of five different chemical scaffolds were determined alongside BRD9 and BRD4. We also report the discovery of first-in-class dual bromodomain-kinase inhibitors outside the bromodomain and extraterminal family targeting BRD7 and BRD9. Combined, the data provide a new framework for the development of BRD7/9 inhibitors with improved selectivity or additional polypharmacologic properties.
Assuntos
Proteínas Cromossômicas não Histona/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/química , Domínios Proteicos/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Sítios de Ligação , Calorimetria/métodos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Fluorometria/métodos , Compostos Heterocíclicos com 2 Anéis/metabolismo , Humanos , Ligantes , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Enzymes of the ALDH1A subfamily of aldehyde dehydrogenases are crucial in regulating retinoic acid (RA) signaling and have received attention as potential drug targets. ALDH1A2 is the primary RA-synthesizing enzyme in mammalian spermatogenesis and is therefore considered a viable drug target for male contraceptive development. However, only a small number of ALDH1A2 inhibitors have been reported, and information on the structure of ALDH1A2 was limited to the NAD-liganded enzyme void of substrate or inhibitors. Herein, we describe the mechanism of action of structurally unrelated reversible and irreversible inhibitors of human ALDH1A2 using direct binding studies and X-ray crystallography. All inhibitors bind to the active sites of tetrameric ALDH1A2. Compound WIN18,446 covalently reacts with the side chain of the catalytic residue Cys320, resulting in a chiral adduct in ( R) configuration. The covalent adduct directly affects the neighboring NAD molecule, which assumes a contracted conformation suboptimal for the dehydrogenase reaction. The reversible inhibitors interact predominantly through direct hydrogen bonding interactions with residues in the vicinity of Cys320 without affecting NAD. Upon interaction with inhibitors, a large flexible loop assumes regular structure, thereby shielding the active site from solvent. The precise knowledge of the binding modes provides a new framework for the rational design of novel inhibitors of ALDH1A2 with improved potency and selectivity profiles.
Assuntos
Anticoncepcionais Masculinos/química , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Retinal Desidrogenase/antagonistas & inibidores , Família Aldeído Desidrogenase 1 , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Ligação de Hidrogênio , Conformação Proteica , Retinal Desidrogenase/química , Transdução de Sinais , Tretinoína/metabolismoRESUMO
As regulators of transcription, epigenetic proteins that interpret post-translational modifications to N-terminal histone tails are essential for maintaining cellular homeostasis. When dysregulated, "reader" proteins become drivers of disease. In the case of bromodomains, which recognize N-ε-acetylated lysine, selective inhibition of individual bromodomain-and-extra-terminal (BET)-family bromodomains has proven challenging. We describe the >55-fold N-terminal-BET bromodomain selectivity of 1,4,5-trisubstituted-imidazole dual kinase-bromodomain inhibitors. Selectivity for the BRD4 N-terminal bromodomain (BRD4(1)) over its second bromodomain (BRD4(2)) arises from the displacement of ordered waters and the conformational flexibility of lysine-141 in BRD4(1). Cellular efficacy was demonstrated via reduction of c-Myc expression, inhibition of NF-κB signaling, and suppression of IL-8 production through potential synergistic inhibition of BRD4(1) and p38α. These dual inhibitors provide a new scaffold for domain-selective inhibition of BRD4, the aberrant function of which plays a key role in cancer and inflammatory signaling.
Assuntos
Imidazóis/química , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Células A549 , Humanos , Domínios Proteicos , Água/química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/químicaRESUMO
Members of the Wee family of kinases negatively regulate the cell cycle via phosphorylation of CDK1 and are considered potential drug targets. Herein, we investigated the structure-function relationship of human Wee1, Wee2, and Myt1 (PKMYT1). Purified recombinant full-length proteins and kinase domain constructs differed substantially in phosphorylation states and catalytic competency, suggesting complex mechanisms of activation. A series of crystal structures reveal unique features that distinguish Wee1 and Wee2 from Myt1 and establish the structural basis of differential inhibition by the widely used Wee1 inhibitor MK-1775. Kinome profiling and cellular studies demonstrate that, in addition to Wee1 and Wee2, MK-1775 is an equally potent inhibitor of the polo-like kinase PLK1. Several previously unrecognized inhibitors of Wee kinases were discovered and characterized. Combined, the data provide a comprehensive view on the catalytic and structural properties of Wee kinases and a framework for the rational design of novel inhibitors thereof.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , PirimidinonasRESUMO
Analogues of N-butyl-1-deoxynojirimycin (NB-DNJ) were prepared and assayed for inhibition of ceramide-specific glucosyltransferase (CGT), non-lysosomal ß-glucosidaseâ 2 (GBA2) and the lysosomal ß-glucosidaseâ 1 (GBA1). Compounds 5 a-6 f, which carry sterically demanding nitrogen substituents, and compound 13, devoid of the C3 and C5 hydroxy groups present in DNJ/NB-DGJ (N-butyldeoxygalactojirimycin) showed no inhibitory activity for CGT or GBA2. Inversion of stereochemistry at C4 of N-(n-butyl)- and N-(n-nonyl)-DGJ (compounds 24) also led to a loss of activity in these assays. The aminocyclopentitols N-(n-butyl)- (35 a), N-(n-nonyl)-4-amino-5-(hydroxymethyl)cyclopentane- (35 b), and N-(1-(pentyloxy)methyl)adamantan-1-yl)-1,2,3-triol (35 f), were found to be selective inhibitors of GBA1 and GBA2 that did not inhibit CGT (>1â mm), with the exception of 35 f, which inhibited CGT with an IC50 value of 1â mm. The N-butyl analogue 35 a was 100-fold selective for inhibiting GBA1 over GBA2 (Ki values of 32â nm and 3.3â µm for GBA1 and GBA2, respectively). The N-nonyl analogue 35 b displayed a Ki value of âª14â nm for GBA1 inhibition and a Ki of 43â nm for GBA2. The N-(1-(pentyloxy)methyl)adamantan-1-yl) derivative 35 f had Ki values of ≈16 and 14â nm for GBA1 and GBA2, respectively. The related N-bis-substituted aminocyclopentitols were found to be significantly less potent inhibitors than their mono-substituted analogues. The aminocyclopentitol scaffold should hold promise for further inhibitor development.
Assuntos
1-Desoxinojirimicina/farmacologia , Amino Álcoois/farmacologia , Ciclopentanos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , beta-Glucosidase/antagonistas & inibidores , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/química , Amino Álcoois/química , Ciclopentanos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Conformação Molecular , Relação Estrutura-Atividade , beta-Glucosidase/metabolismoRESUMO
Computational screening is a method to prioritize small-molecule compounds based on the structural and biochemical attributes built from ligand and target information. Previously, we have developed a scalable virtual screening workflow to identify novel multitarget kinase/bromodomain inhibitors. In the current study, we identified several novel N-[3-(2-oxo-pyrrolidinyl)phenyl]-benzenesulfonamide derivatives that scored highly in our ensemble docking protocol. We quantified the binding affinity of these compounds for BRD4(BD1) biochemically and generated cocrystal structures, which were deposited in the Protein Data Bank. As the docking poses obtained in the virtual screening pipeline did not align with the experimental cocrystal structures, we evaluated the predictions of their precise binding modes by performing molecular dynamics (MD) simulations. The MD simulations closely reproduced the experimentally observed protein-ligand cocrystal binding conformations and interactions for all compounds. These results suggest a computational workflow to generate experimental-quality protein-ligand binding models, overcoming limitations of docking results due to receptor flexibility and incomplete sampling, as a useful starting point for the structure-based lead optimization of novel BRD4(BD1) inhibitors.
RESUMO
Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure-activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure-activity data. An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular , Cristalografia por Raios X , Polarização de Fluorescência , Fluorometria/métodos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Nucleares/metabolismo , Domínios Proteicos , Pirimidinas/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Interface Usuário-ComputadorRESUMO
Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054-67. ©2017 AACR.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Inibidores de Proteínas Quinases/química , Proteínas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Increasing evidence suggests key roles for members of the mammalian Sterile20-like (MST) family of kinases in many aspects of biology. MST3 is a member of the STRIPAK complex, the deregulation of which has recently been associated with cancer cell migration and metastasis. Targeting MST3 with small-molecule inhibitors may be beneficial for the treatment of certain cancers, but little information exists on the potential of kinase inhibitor scaffolds to engage with MST3. In this study we screened MST3 against a library of 277 kinase inhibitors using differential scanning fluorimetry and confirmed 14 previously unknown MST3 inhibitors by X-ray crystallography. These compounds, of which eight are in clinical trials or FDA approved, comprise nine distinct chemical scaffolds that inhibit MST3 enzymatic activity with IC50 values between 0.003 and 23â µm. The structure-activity relationships explain the differential inhibitory activity of these compounds against MST3 and the structural basis for high binding potential, the information of which may serve as a framework for the rational design of MST3-selective inhibitors as potential therapeutics and to interrogate the function of this enzyme in diseased cells.
Assuntos
Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Fluorometria , Humanos , Ligantes , Células MCF-7 , Simulação de Dinâmica Molecular , Domínios Proteicos , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-AtividadeRESUMO
Evaluating the engagement of a small molecule ligand with a protein target in cells provides useful information for chemical probe optimization and pharmaceutical development. While several techniques exist that can be performed in a low-throughput manner, systematic evaluation of large compound libraries remains a challenge. In-cell engagement measurements are especially useful when evaluating compound classes suspected to target multiple cellular factors. In this study we used a bioluminescent resonant energy transfer assay to assess bromodomain engagement by a compound series containing bromodomain- and kinase-biasing polypharmacophores based on the known dual BRD4 bromodomain/PLK1 kinase inhibitor BI2536. With this assay, we discovered several novel agents with bromodomain-selective specificity profiles and cellular activity. Thus, this platform aids in distinguishing molecules whose cellular activity is difficult to assess due to polypharmacologic effects.
Assuntos
Proteínas Nucleares/metabolismo , Pteridinas/química , Fatores de Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Humanos , Medições Luminescentes , Proteínas Nucleares/antagonistas & inibidores , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/metabolismo , Pteridinas/toxicidade , Fatores de Transcrição/antagonistas & inibidores , Quinase 1 Polo-LikeRESUMO
Members of the bromodomain and extra terminal (BET) family of proteins are essential for the recognition of acetylated lysine (KAc) residues in histones and have emerged as promising drug targets in cancer, inflammation, and contraception research. In co-crystallization screening campaigns using the first bromodomain of BRD4 (BRD4-1) against kinase inhibitor libraries, we identified and characterized 14 kinase inhibitors (10 distinct chemical scaffolds) as ligands of the KAc binding site. Among these, the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 displayed strongest inhibitory potential against BRD4 (IC50=25 nM and 130 nM, respectively) and high selectivity for BET bromodomains. Comparative structural analysis revealed markedly different binding modes of kinase hinge-binding scaffolds in the KAc binding site, suggesting that BET proteins are potential off-targets of diverse kinase inhibitors. Combined, these findings provide a new structural framework for the rational design of next-generation BET-selective and dual-activity BET-kinase inhibitors.