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1.
Biochem Soc Trans ; 47(4): 1101-1116, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31395755

RESUMO

The SRC, Abelson murine leukemia viral oncogene homolog 1, TEC and C-terminal SRC Kinase families of non-receptor tyrosine kinases (collectively the Src module kinases) mediate an array of cellular signaling processes and are therapeutic targets in many disease states. Crystal structures of Src modules kinases provide valuable insights into the regulatory mechanisms that control activation and generate a framework from which drug discovery can advance. The conformational ensembles visited by these multidomain kinases in solution are also key features of the regulatory machinery controlling catalytic activity. Measurement of dynamic motions within kinases substantially augments information derived from crystal structures. In this review, we focus on a body of work that has transformed our understanding of non-receptor tyrosine kinase regulation from a static view to one that incorporates how fluctuations in conformational ensembles and dynamic motions influence activation status. Regulatory dynamic networks are often shared across and between kinase families while specific dynamic behavior distinguishes unique regulatory mechanisms for select kinases. Moreover, intrinsically dynamic regions of kinases likely play important regulatory roles that have only been partially explored. Since there is clear precedence that kinase inhibitors can exploit specific dynamic features, continued efforts to define conformational ensembles and dynamic allostery will be key to combating drug resistance and devising alternate treatments for kinase-associated diseases.


Assuntos
Proteínas Tirosina Quinases/metabolismo , Animais , Catálise , Domínio Catalítico , Descoberta de Drogas , Ativação Enzimática , Humanos , Fosforilação , Conformação Proteica , Proteínas Tirosina Quinases/química , Transdução de Sinais , Domínios de Homologia de src
2.
Fish Shellfish Immunol ; 93: 313-321, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31351111

RESUMO

The Src family kinases (SFK) are involved in signaling transductions that regulate numerous biological activities including host-virus interaction. These features of SFK have been well explored in vertebrates, however, in shrimp, the invertebrate SFK family member Src64B, has not been characterized and therefore its role in shrimp-virus interaction remains unknown. In this study, two Litopenaeus vannamei Src64B isoforms (designated LvSrc64B1 and LvSrc64B2) were first cloned and their role in white spot syndrome virus (WSSV) infection was explored. Bioinformatics analysis revealed that LvSrc64B1 and LvSrc64B2 were similar to other Src64B family members, with high homology in primary and tertiary structures, and contained the conserved SFK functional domains, as well as the putative myristylation and phosphorylation sites. Tissue distribution analysis showed that both LvSrc64B isoforms were ubiquitously expressed, albeit distinctively in the tested tissues. In addition, transcript levels of LvSrc64B1 and LvSrc64B2 were significantly induced following WSSV challenge and had similar expression patterns. Furthermore, siRNA-mediated knockdown of LvSrc64B1 and LvSrc64B2 followed by WSSV infection resulted in increased expression of viral genes, enhanced viral DNA replication, and elevation of hemocytes apoptosis. Depletion of LvSrc64B1 and LvSrc64B2 also reduced shrimp survival upon WSSV infection. In conclusion, the current data strongly suggest that Src64B is a host factor that inhibits WSSV replication by modulating apoptosis in shrimp.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Biologia Computacional , Perfilação da Expressão Gênica , Filogenia , Proteínas Tirosina Quinases/química , Alinhamento de Sequência , Replicação Viral
3.
Eur J Med Chem ; 175: 247-268, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31121430

RESUMO

As a dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one of the main kinases involved in kinetochore localization and the spindle assembly checkpoint (SAC). Cancer cells often display chromosomal instability, which is a consequence of disfunction of cell cycle checkpoints partially. Mps1 is overexpressed in multiple cancer types to face the pressure from aberrant chromosomes and centrosomes. Therefore, Mps1 is a potential targeting approach to cancer treatment. Several compounds targeting Mps1 have been developed and approved to begin clinical trials for advanced nonhaematologic malignancies treatments, including but not limited to triple negative breast cancer (TNBC) treatment. In this review, we will highlight typical Mps1 inhibitors developed during the last decade and provide a reference for more potential Mps1 inhibitors exploration in the future.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Desenho de Drogas , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Fuso Acromático/efeitos dos fármacos , Antineoplásicos/farmacocinética , Proteínas de Ciclo Celular/química , Resistência a Medicamentos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Conformação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/patologia
4.
Methods Enzymol ; 621: 131-152, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31128775

RESUMO

Protein tyrosine kinases (PTKs) are key signaling molecules and important drug targets. Although the efficient recombinant production of active PTKs is important for both pharmaceutical industry and academic research, most PTKs are still obtained from conventional, expensive and time-consuming insect-cell based expression. Host toxicity, kinase inactivity, insolubility and heterogeneity are among the reasons thought to preclude PTK expression in Escherichia coli. Herein we review these presumed roadblocks and their possible solutions for bacterial expression of PTKs, and give an overview on kinase activity assays. Finally, we report our experiences and observations with the kinases Src, Lyn and FAK as examples to illustrate implementation, effects and pitfalls of E. coli expression and in vitro assaying of PTKs.


Assuntos
Proteínas Tirosina Quinases/genética , Animais , Clonagem Molecular/métodos , Ensaios Enzimáticos/métodos , Escherichia coli/genética , Expressão Gênica , Humanos , Modelos Moleculares , Domínios Proteicos , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
J Biol Chem ; 294(24): 9631-9641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31064840

RESUMO

Serine-arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg-Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic-nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein-protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.


Assuntos
Arginina/metabolismo , Núcleo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Células HeLa , Humanos , Fosforilação , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Homologia de Sequência , Fatores de Processamento de Serina-Arginina/metabolismo , Especificidade por Substrato , beta Carioferinas/metabolismo
6.
BMC Cancer ; 19(1): 301, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30943926

RESUMO

BACKGROUND: Genetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer. METHODS: In 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription-PCR, respectively. RESULTS: Compared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion-positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion-negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion. CONCLUSIONS: This study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion.


Assuntos
Quinase do Linfoma Anaplásico/genética , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas/genética , Regulação para Cima , Quinase do Linfoma Anaplásico/química , Quinase do Linfoma Anaplásico/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret/química , Proteínas Proto-Oncogênicas c-ret/metabolismo
7.
ACS Appl Mater Interfaces ; 11(18): 16336-16346, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-30986026

RESUMO

Gold nanoparticles (AuNPs) have emerged as promising drug delivery candidates that can be leveraged for cancer therapy. Lung cancer (LC) is a heterogeneous disease that imposes a significant burden on society, with an unmet need for new therapies. Chemotherapeutic drugs such as afatinib (Afb), which is clinically approved for the treatment of epidermal growth factor receptor positive LC, is hydrophobic and has low bioavailability leading to spread around the body, causing severe side effects. Herein, we present a novel afatinib-AuNP formulation termed Afb-AuNPs, with the aim of improving drug efficacy and biocompatibility. This was achieved by synthesis of an alkyne-bearing Afb derivative and reaction with azide-functionalized lipoic acid using copper-catalyzed click chemistry, then conjugation to AuNPs via alkylthiol-gold bond formation. The Afb-AuNPs were found to possess up to 3.7-fold increased potency when administered to LC cells in vitro and were capable of significantly inhibiting cancer cell proliferation, as assessed by MTT assay and electric cell-substrate impedance sensing, respectively. Furthermore, when exposed to Afb-AuNPs, human alveolar epithelial type I-like cells, a model of the healthy lung epithelium, maintained viability and were found to release less proinflammatory cytokines when compared to free drug, demonstrating the biocompatibility of our formulation. This study provides a new platform for the development of nontraditional AuNP conjugates which can be applied to other molecules of therapeutic or diagnostic utility, with potential to be combined with photothermal therapy in other cancers.


Assuntos
Afatinib/química , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Nanoconjugados/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Afatinib/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Humanos , Teste de Materiais , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Nanoconjugados/química , Polietilenoglicóis/química , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química
8.
Nucleic Acids Res ; 47(9): 4663-4683, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30916345

RESUMO

Cleavage factor I mammalian (CFIm) complex, composed of cleavage and polyadenylation specificity factor 5 (CPSF5) and serine/arginine-like protein CPSF6, regulates alternative polyadenylation (APA). Loss of CFIm function results in proximal polyadenylation site usage, shortening mRNA 3' untranslated regions (UTRs). Although CPSF6 plays additional roles in human disease, its nuclear translocation mechanism remains unresolved. Two ß-karyopherins, transportin (TNPO) 1 and TNPO3, can bind CPSF6 in vitro, and we demonstrate here that while the TNPO1 binding site is dispensable for CPSF6 nuclear import, the arginine/serine (RS)-like domain (RSLD) that mediates TNPO3 binding is critical. The crystal structure of the RSLD-TNPO3 complex revealed potential CPSF6 interaction residues, which were confirmed to mediate TNPO3 binding and CPSF6 nuclear import. Both binding and nuclear import were independent of RSLD phosphorylation, though a hyperphosphorylated mimetic mutant failed to bind TNPO3 and mislocalized to the cell cytoplasm. Although hypophosphorylated CPSF6 largely supported normal polyadenylation site usage, a significant number of mRNAs harbored unnaturally extended 3' UTRs, similar to what is observed when other APA regulators, such as CFIIm component proteins, are depleted. Our results clarify the mechanism of CPSF6 nuclear import and highlight differential roles for RSLD phosphorylation in nuclear translocation versus regulation of APA.


Assuntos
Poliadenilação/genética , Conformação Proteica , Proteínas de Ligação a RNA/química , beta Carioferinas/química , Transporte Ativo do Núcleo Celular/genética , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Fosforilação/genética , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , RNA Mensageiro , Proteínas de Ligação a RNA/genética , beta Carioferinas/genética , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
9.
Mol Cell ; 74(2): 330-346.e11, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853400

RESUMO

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.


Assuntos
Autofagossomos/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteína Sequestossoma-1/química , Autofagossomos/química , Autofagia/genética , Cristalografia por Raios X , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mapas de Interação de Proteínas/genética , Proteínas Tirosina Quinases/genética , Proteólise , Proteína Sequestossoma-1/genética , Transdução de Sinais/genética , Ubiquitina/química , Ubiquitina/genética
10.
Mol Cell ; 74(2): 347-362.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853401

RESUMO

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células HeLa , Humanos , Mitocôndrias/química , Mitocôndrias/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Peroxissomos/química , Peroxissomos/genética , Fosforilação , Proteínas Quinases/genética , Multimerização Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
11.
Mol Cell ; 74(2): 320-329.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853402

RESUMO

Xenophagy, a selective autophagy pathway that protects the cytosol against bacterial invasion, relies on cargo receptors that juxtapose bacteria and phagophore membranes. Whether phagophores are recruited from a constitutive pool or are generated de novo at prospective cargo remains unknown. Phagophore formation in situ would require recruitment of the upstream autophagy machinery to prospective cargo. Here, we show that, essential for anti-bacterial autophagy, the cargo receptor NDP52 forms a trimeric complex with FIP200 and SINTBAD/NAP1, which are subunits of the autophagy-initiating ULK and the TBK1 kinase complex, respectively. FIP200 and SINTBAD/NAP1 are each recruited independently to bacteria via NDP52, as revealed by selective point mutations in their respective binding sites, but only in their combined presence does xenophagy proceed. Such recruitment of the upstream autophagy machinery by NDP52 reveals how detection of cargo-associated "eat me" signals, induction of autophagy, and juxtaposition of cargo and phagophores are integrated in higher eukaryotes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/genética , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Sítios de Ligação/genética , Citoplasma/microbiologia , Citosol/microbiologia , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/química , Mutação Puntual/genética , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/química , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade
12.
Fertil Steril ; 111(3): 510-518, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30827523

RESUMO

OBJECTIVE: To investigate the genetic cause of fertilization failure or poor fertilization. DESIGN: Genetic analysis. SETTING: University-affiliated center. PATIENT(S): Twenty-four Chinese women who underwent assisted reproductive technology (ART) and had repeated fertilization failure or poor fertilization. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Twenty-four affected patients were subjected to whole-exome sequencing and candidate mutations were validated by Sanger sequencing. Single-cell reverse transcription was used to analyze the functional characterization of the splice-site mutation in vivo. Evolutionary conservation and molecular modeling analyses were used to predict the impact of missense mutations on secondary protein structure. Immunofluorescence was used to analyze the protein levels of WEE2 and phosphorylated CDC2. RESULT(S): Biallelic mutations in WEE2 were identified in 5 of 24 (20.8%) Chinese patients with fertilization failure or poor fertilization. Among these individuals we found a novel splice-site mutation, two novel missense mutations, and a previously reported frame-shift mutation. Splicing mutation c.1136-2A>G of WEE2 caused an alteration of the reading frame and introduced a premature stop codon (p.Gly379Glufs*6/p.Asp380Leufs*39). The missense mutations c.585G>C (p.Lys195Asn) and c.1228C>T (p.Arg410Trp) produced obvious changes in secondary protein structures. Immunostaining indicated that mutated WEE2 resulted in the loss of phosphorylated CDC2. The phenotypes of women carrying WEE2 mutations exhibited slight variability, from total fertilization failure to poor fertilization. CONCLUSION(S): Novel mutations in the known causative gene WEE2 were identified in 5 of 24 women with fertilization failure or poor fertilization, indicating a high prevalence of WEE2 mutations in Chinese women experiencing fertilization failure or poor fertilization.


Assuntos
Proteínas de Ciclo Celular/genética , Fertilidade/genética , Infertilidade Feminina/genética , Infertilidade Feminina/terapia , Mutação , Proteínas Tirosina Quinases/genética , Técnicas de Reprodução Assistida/efeitos adversos , Adulto , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , China , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Humanos , Infertilidade Feminina/enzimologia , Infertilidade Feminina/fisiopatologia , Modelos Moleculares , Taxa de Mutação , Fenótipo , Fosforilação , Gravidez , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Relação Estrutura-Atividade , Falha de Tratamento , Sequenciamento Completo do Exoma
13.
Fertil Steril ; 111(3): 519-526, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30827524

RESUMO

OBJECTIVE: To determine whether variants in the WEE2 (WEE1 homolog 2, also known as WEE1B) gene, which has been known to function in the formation of pronuclei during fertilization, contribute to fertilization failure. DESIGN: Case-control genetic study. SETTING: University hospital. PATIENT(S): Ninety infertile women with repeated cycles of pronucleus formation failure undergoing in vitro fertilization and/or intracytoplasmic sperm injection treatment as well as 200 fertile control women. INTERVENTION(S): Genomic DNA was extracted from the peripheral blood. The whole exons of WEE2 were amplified by means of polymerase chain reaction and then Sanger sequencing was performed. MAIN OUTCOME MEASURE(S): Variants analysis of WEE2 gene. RESULT(S): We identified five subjects that were subjected to homozygous or compound-heterozygous variants of WEE2: case 1 (from a consanguineous family) with homozygous frameshift variant: c.293_294insCTGAGACACCAGCCCAACC (p.Pro98Pro fsX2); case 2 with homozygous missense variant: c.1576T>G (p.Tyr526Asp); and three cases with compound-heterozygous variants: case 3: c.991C>A (p.His331Asn) and c.1304_1307delCCAA (p.Thr435Met fsX31); case 4: c.341_342 del AA (p.Lys114Asn fsX20) and c.864G>C (p.Gln288His); and case 5: c.1A>G (p.0?) and c.1261G>A (p.Gly421Arg). Besides c.1576T>G (from case 2) and c.864G>C (from case 4), which have been previously reported as rare single nucleotide polymorphisms (SNPs), the other six variants were novel and predicted by software to be deleterious. The parental genotypes of case 1 and case 2 indicated that the detected homozygous variants were inherited in an autosomal recessive mode. All of the detected variants were absent from the control cohort. CONCLUSION(S): Novel variants found in WEE2, which is autosomal-recessive inherited, may be related to recurrent pronucleus formation failure and female infertility.


Assuntos
Proteínas de Ciclo Celular/genética , Fertilidade/genética , Fertilização In Vitro/efeitos adversos , Infertilidade Feminina/genética , Infertilidade Feminina/terapia , Mutação , Proteínas Tirosina Quinases/genética , Adulto , Estudos de Casos e Controles , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Hereditariedade , Humanos , Infertilidade Feminina/enzimologia , Infertilidade Feminina/fisiopatologia , Taxa de Mutação , Linhagem , Fenótipo , Gravidez , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Fatores de Risco , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Falha de Tratamento , Sequenciamento Completo do Exoma , Adulto Jovem
14.
Eur J Med Chem ; 166: 304-317, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30731399

RESUMO

Cdc2-like kinase 1 (CLK1) and dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) are involved in the regulation of alternative pre-mRNA splicing. Dysregulation of this process has been linked to cancer progression and neurodegenerative diseases, making CLK1 and DYRK1A important therapeutic targets. Here we describe the synthesis of new pyrido[3,4-g]quinazoline derivatives and the evaluation of the inhibitory potencies of these compounds toward CDK5, CK1, GSK3, CLK1 and DYRK1A. Introduction of aminoalkylamino groups at the 2-position resulted in several compounds with low nanomolar affinity and selective inhibition of CLK1 and/or DYRK1A. Their evaluation on several immortalized or cancerous cell lines showed varying degree of cell viability reduction. Co-crystal structures of CLK1 with two of the most potent compounds revealed two alternative binding modes of the pyrido[3,4-g]quinazoline scaffold that can be exploited for future inhibitor design.


Assuntos
Desenho de Drogas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Quinazolinas/síntese química , Quinazolinas/farmacologia , Linhagem Celular Tumoral , Técnicas de Química Sintética , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Quinazolinas/química , Quinazolinas/metabolismo , Relação Estrutura-Atividade
15.
Pharm Res ; 36(4): 56, 2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30796596

RESUMO

PURPOSE: Lipid suspensions have been shown to be a suitable bio-enabling formulation approach for highly lipophilic or 'grease ball' drug molecules, but studies on 'brick dust' drugs are lacking. This study explored the utility of lipid suspensions for enhancing oral bioavailability of the rather hydrophobic drug nilotinib in vivo in rats. METHODS: Four lipid suspensions were developed containing long chain triglycerides, medium chain triglyceride, long chain monoglycerides and medium chain monoglycerides and in vivo bioavailability was compared to an aqueous suspension. Additionally, in vitro lipolysis and wettability tests were conducted. RESULTS: Nilotinib lipid suspensions did not show a bioavailability increase compared to an aqueous suspension. The bioavailability was lower for triglyceride suspensions, relative to both monoglyceride and an aqueous suspension. The long chain monoglyceride displayed a significantly higher bioavailability relative to triglycerides. In vitro lipolysis results suggested entrapment of nilotinib crystals within poorly dispersible triglycerides, leading to slower nilotinib release and absorption. This was further supported by higher wettability of nilotinib by lipids. CONCLUSION: Monoglycerides improved oral bioavailability of nilotinib in rats, relative to triglycerides. For 'brick dust' drugs formulated as lipid suspensions, poorly dispersible formulations may delay the release of drug crystals from the formulation leading to reduced absorption. Graphical Abstract An aqueous and four lipid suspensions have been evaluated in in vitro and in vivo to gain insights into the potential benefits and limitations of lipid suspensions.


Assuntos
Antineoplásicos/farmacocinética , Excipientes/química , Monoglicerídeos/química , Proteínas Tirosina Quinases/farmacocinética , Pirimidinas/farmacocinética , Triglicerídeos/química , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Disponibilidade Biológica , Química Farmacêutica/métodos , Composição de Medicamentos , Absorção Gastrointestinal , Interações Hidrofóbicas e Hidrofílicas , Lipólise , Masculino , Soluções Farmacêuticas , Proteínas Tirosina Quinases/administração & dosagem , Proteínas Tirosina Quinases/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Ratos Sprague-Dawley , Suspensões , Molhabilidade
16.
J Mol Model ; 25(2): 41, 2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30673861

RESUMO

DYRK1B protein kinase is an emerging anticancer target due to its overexpression in a variety of cancers and its role in cancer chemoresistance through maintaining cancer cells in the G0 (quiescent) state. Consequently, there is a growing interest in the development of potent and selective DYRK1B inhibitors for anticancer therapy. One of the major off-targets is another protein kinase, GSK3ß, which phosphorylates an important regulator of cell cycle progression on the same residue as DYRK1B and is involved in multiple signaling pathways. In the current work, we performed a detailed comparative structural analysis of DYRK1B and GSK3ß ATP-binding sites and identified key regions responsible for selectivity. As the crystal structure of DYRK1B has never been reported, we built and optimized a homology model by comparative modeling and metadynamics simulations. Calculation of interaction energies between docked ligands in the ATP-binding sites of both kinases allowed us to pinpoint key residues responsible for potency and selectivity. Specifically, the role of the gatekeeper residues in DYRK1B and GSK3ß is discussed in detail, and two other residues are identified as key to selectivity of DYRK1B inhibition versus GSK3ß. The analysis presented in this work was used to support the design of potent and selective azaindole-quinoline-based DYRK1B inhibitors and can facilitate development of more selective inhibitors for DYRK kinases.


Assuntos
Desenho de Drogas , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Glicogênio Sintase Quinase 3 beta/química , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Domínios Proteicos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Homologia de Sequência de Aminoácidos
17.
J Biol Chem ; 294(12): 4608-4620, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30659095

RESUMO

Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains has been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr89 and Tyr134 in ABL1 or the homologous residues Tyr116 and Tyr161 in ABL2 induces only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine phosphorylation-induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed not only that the residues phosphorylated in our model systems are well-conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome.


Assuntos
Tirosina/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Humanos , Ligantes , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , Espalhamento a Baixo Ângulo
18.
J Biomol Struct Dyn ; 37(3): 611-622, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29380674

RESUMO

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of ß-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the ß-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.


Assuntos
Trifosfato de Adenosina/metabolismo , Aminoácidos/química , Compostos de Anilina/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Compostos Heterocíclicos com 2 Anéis/química , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Especificidade por Substrato , Termodinâmica
19.
Curr Med Chem ; 26(31): 5811-5824, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30009703

RESUMO

Tyrosine kinases are a subgroup of a large class of protein kinases that transfer phosphate groups from ATP to various amino acid residues. By phosphorylating the tyrosine residues, the tyrosine kinases are responsible for the activation of various proteins through signal transduction cascades, which serves as a ubiquitous mechanism of cell signaling. The frequent success of many tyrosine kinase inhibitors (TKIs) in clinical success and diseasecausing mutations in protein kinases suggests that a large number of kinases may represent therapeutically relevant targets. To date, most of the clinical and preclinical TKIs are ATPcompetitive non-covalent inhibitors, which achieve their selectivity by recognizing the unique features of specific protein kinases. Of growing interest now in the scientific community is the development of irreversible inhibitors that form covalent bonds with cysteines or other nucleophilic residues in the ATP binding pocket. Irreversible TKIs have many potential advantages including prolonged pharmacodynamics, reasonable compound design suitability, high potency, and the ability to validate pharmacological specificity by mutations in reactive cysteine residues. Here, we review recent efforts to develop cysteine-targeting irreversible TKIs and to discuss their patterns of configuration that identify adenosine triphosphate binding pockets and their biological activities.


Assuntos
Cisteína/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Cisteína/metabolismo , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo
20.
Pharmacol Ther ; 194: 199-221, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30268771

RESUMO

The dosage of the serine threonine kinase DYRK1A is critical in the central nervous system (CNS) during development and aging. This review analyzes the functions of this kinase by considering its interacting partners and pathways. The role of DYRK1A in controlling the differentiation of prenatal newly formed neurons is presented separately from its role at the pre- and post-synaptic levels in the adult CNS; its effects on synaptic plasticity are also discussed. Because this kinase is positioned at the crossroads of many important processes, genetic dosage errors in this protein produce devastating effects arising from DYRK1A deficiency, such as in MRD7, an autism spectrum disorder, or from DYRK1A excess, such as in Down syndrome. Effects of these errors have been shown in various animal models including Drosophila, zebrafish, and mice. Dysregulation of DYRK1A levels also occurs in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Finally, this review describes inhibitors that have been assessed in vivo. Accurate targeting of DYRK1A levels in the brain, with either inhibitors or activators, is a future research challenge.


Assuntos
Cognição , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Animais , Transtorno do Espectro Autista/metabolismo , Síndrome de Down/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Neurogênese , Neurônios/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Transmissão Sináptica
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