RESUMO
We have developed radical C-glycosylation using photoexcitable unprotected glycosyl borate. The direct excitation of glycosyl borate under visible light irradiation enabled the generation of anomeric radical without any photoredox catalysts. The inâ situ generated anomeric radical was applicable to the radical addition such as Giese-type addition and Minisci-type reaction to introduce alkyl and heteroaryl groups at the anomeric position. In addition, the radical-radical coupling between the glycosyl borate and acyl imidazolide provided unprotected acyl C-glycosides.
RESUMO
Xenobiotic metabolic reactions in the hepatocyte endoplasmic reticulum (ER) including UDP-glucuronosyltransferase and carboxylesterase play central roles in the detoxification of medical agents with small- and medium-sized molecules. Although the catalytic sites of these enzymes exist inside of ER, the molecular mechanism for membrane permeation in the ER remains enigmatic. Here, we investigated that organic anion transporter 2 (OAT2) regulates the detoxification reactions of xenobiotic agents including anti-cancer capecitabine and antiviral zidovudine, via the permeation process across the ER membrane in the liver. Pharmacokinetic studies in patients with colorectal cancer revealed that the half-lives of capecitabine in rs2270860 (1324C > T) variants was 1.4 times higher than that in the C/C variants. Moreover, the hydrolysis of capecitabine to 5'-deoxy-5-fluorocytidine in primary cultured human hepatocytes was reduced by OAT2 inhibitor ketoprofen, whereas capecitabine hydrolysis directly assessed in human liver microsomes were not affected. The immunostaining of OAT2 was merged with ER marker calnexin in human liver periportal zone. These results suggested that OAT2 is involved in distribution of capecitabine into ER. Furthermore, we clarified that OAT2 plays an essential role in drug-drug interactions between zidovudine and valproic acid, leading to the alteration in zidovudine exposure to the body. Our findings contribute to mechanistically understanding medical agent detoxification, shedding light on the ER membrane permeation process as xenobiotic metabolic machinery to improve chemical changes in hydrophilic compounds.
Assuntos
Retículo Endoplasmático , Humanos , Retículo Endoplasmático/metabolismo , Interações Medicamentosas/fisiologia , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Masculino , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Zidovudina/metabolismo , Zidovudina/farmacocinética , Feminino , Microssomos Hepáticos/metabolismoRESUMO
Photo-caged methodologies have been indispensable for elucidating the functional mechanisms of pharmacologically active molecules at the cellular level. A photo-triggered removable unit enables control of the photo-induced expression of pharmacologically active molecular function, resulting in a rapid increase in the concentration of the bioactive compound near the target cell. However, caging the target bioactive compound generally requires specific heteroatom-based functional groups, limiting the types of molecular structures that can be caged. We have developed an unprecedented methodology for caging/uncaging on carbon atoms using a unit with a photo-cleavable carbon-boron bond. The caging/uncaging process requires installation of the CH2-B group on the nitrogen atom that formally assembles an N-methyl group protected with a photoremovable unit. N-Methylation proceeds by photoirradiation via carbon-centered radical generation. Using this radical caging strategy to cage previously uncageable bioactive molecules, we have photocaged molecules with no general labeling sites, including acetylcholine, an endogenous neurotransmitter. Caged acetylcholine provides an unconventional tool for optopharmacology to clarify neuronal mechanisms on the basis of photo-regulating acetylcholine localization. We demonstrated the utility of this probe by monitoring uncaging in HEK cells expressing a biosensor to detect ACh on the cell surface, as well as Ca2+ imaging in Drosophila brain cells (ex vivo).
Assuntos
Acetilcolina , Neurotransmissores , Neurotransmissores/química , Neurônios , Estrutura Molecular , ColinérgicosRESUMO
Native Oplophorus luciferase (OpLase) and its catalytic 19 kDa protein (wild KAZ) show highest luminescence activity with coelenterazine (CTZ) among CTZ analogs. Mutated wild KAZ with 16 amino acid substitutions (nanoKAZ/nanoLuc) utilizes bis-coelenterazine (bis-CTZ) as the preferred substrate and exhibits over 10-fold higher maximum intensity than CTZ. To understand the substrate selectivity of nanoKAZ between CTZ and bis-CTZ, we prepared the reverse mutants of nanoKAZ by amino acid replacements with the original amino acid residue of wild KAZ. The reverse mutant with L18Q and V27L substitutions (QL-nanoKAZ) exhibited 2.6-fold higher maximum intensity with CTZ than that of nanoKAZ with bis-CTZ. The catalytic properties of QL-nanoKAZ including substrate specificity, luminescence spectrum, luminescence kinetics, luminescence products of CTZ, and luminescence inhibition by deaza-CTZ analogs were characterized and were compared with other CTZ-utilizing luciferases such as Gaussia and Renilla luciferases. Thus, QL-nanoKAZ with CTZ could be used as a potential reporter protein for various luminescence assay systems. Furthermore, the crystal structure of QL-nanoKAZ was determined at 1.70 Å resolution. The reverse mutation at the L18Q and V27L positions of α2-helix in nanoKAZ led to changes in the local structures of the α4-helix and the ß6- and ß7-sheets, and might enhance its binding affinity and oxidation efficiency with CTZ to emit light.
Assuntos
Decápodes , Aminoácidos , Animais , Decápodes/metabolismo , Imidazóis , Luciferases/metabolismo , Luciferases de Renilla/genética , Medições Luminescentes , Proteínas Mutantes/metabolismo , PirazinasRESUMO
DYRK1A phosphorylates proteins involved in neurological disorders in an intermolecular manner. Meanwhile, during the protein folding process of DYRK1A, a transitional folding intermediate catalyzes the intramolecular autophosphorylation required for the "one-off" inceptive activation and stabilization. In our previous study, a small molecule termed FINDY (1) was identified, which inhibits the folding intermediate-catalyzed intramolecular autophosphorylation of DYRK1A but not the folded state-catalyzed intermolecular phosphorylation. However, the structural features of FINDY (1) responsible for this intermediate-selective inhibition remain elusive. In this study, structural derivatives of FINDY (1) were designed and synthesized according to its predicted binding mode in the ATP pocket of DYRK1A. Quantitative structure-activity relationship (QSAR) of the derivatives revealed that the selectivity against the folding intermediate is determined by steric hindrance between the bulky hydrophobic moiety of the derivatives and the entrance to the pocket. In addition, a potent derivative 3 was identified, which inhibited the folding intermediate more strongly than FINDY (1); it was designated as dp-FINDY. Although dp-FINDY (3) did not inhibit the folded state, as well as FINDY (1), it inhibited the intramolecular autophosphorylation of DYRK1A in an in vitro cell-free protein synthesis assay. Furthermore, dp-FINDY (3) destabilized endogenous DYRK1A in HEK293 cells. This study provides structural insights into the folding intermediate-selective inhibition of DYRK1A and expands the chemical options for the design of a kinase inhibitor.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Tiazóis/química , Quinases DyrkRESUMO
A new type of alkylborate was developed for the purpose of generating radicals via direct photoexcitation. These borates were prepared using 2,2'-(pyridine-2,6-diyl)diphenol as a tridentate ligand together with organoboronic acids or potassium trifluoroborates. The ready availability of organoboron compounds is a significant advantage of this direct photoexcitation protocol. The excited states of these borates can also serve as strong reductants, enabling various transformations.
RESUMO
The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.
Assuntos
Equorina/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Cálcio/metabolismo , Equorina/química , Sítios de Ligação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMO
The optical property of fluorescent unit-conjugated aliphatic oxaboroles has been investigated. The oxaboroles provide good fluorescence quantum yields and selective recognition toward D-ribose and D-ribose containing molecules. The molecular recognition induced significant fluorescence quenching. The property of the boroles showed the possibility of the boron-based nicotinamide adenine dinucleotide (NAD) sensor probe.
Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , NAD/química , Açúcares/análise , Compostos de Boro/síntese química , Corantes Fluorescentes/síntese química , Estrutura Molecular , Fenômenos ÓpticosRESUMO
Visible-light-mediated chemical processes have been vigorously studied and have led to state-of-the-art synthetic chemistry since they enable the control of radical generation and excited-state-based transformations. The essential process is the generation of a radical species via single electron transfer (SET) between the substrate and catalyst. While photoredox chemistry is an important methodology, these systems essentially require photocatalysts and involve redox processes of the catalyst in the catalytic cycle, which often complicates the reaction. Hence, a seminal contribution in the area of photoredox chemistry is the development of a system free of a photoredox catalyst. In this tutorial review, we summarise the chronology of C-centred radicals, including photoredox chemistry, and shed light on the direct excitation strategy that enables the generation of radical species without exogenous photocatalysts. This strategy provides more straightforward methods, which are energetically efficient in principle, with the potential to open a new window into organic synthesis.
RESUMO
Boracene-based alkylborate enabled visible light-mediated metallaphotoredox catalysis. The directly excited borate was easily oxidatively quenched by an excited Ir photoredox catalyst. Ni/Ir hybrid catalysis afforded the products under significantly low irradiance.
RESUMO
An efficient transformation of dibenzoxaborins to dibenzofurans by deborylative ring contraction was achieved under mild conditions using a copper catalyst. The method showed a broad substrate scope enabling the preparation of various dibenzofurans, including those bearing a functional group. The ready availability of various dibenzoxaborins enhances the utility of this method, as demonstrated by the regiodivergent synthesis of dibenzofurans.
RESUMO
The generation of tertiary, secondary, and primary alkyl radicals has been achieved by the direct visible-light excitation of a boracene-based alkylborate. This system is based on the photophysical properties of the organoboron molecule. The protocol is applicable to decyanoalkylation, Giese addition, and nickel-catalyzed carbon-carbon bond formations such as alkyl-aryl cross-coupling or vicinal alkylarylation of alkenes, enabling the introduction of various C(sp3) fragments to organic molecules.
RESUMO
In this study, three reaction mechanisms of a benzyne-nickel (Ni) complex ([Ni(C6H4)(dcpe)]) with iodomethane during the methylation process were investigated, namely (a) SN2 reaction of the benzyne-Ni complex with iodomethane, (b) concerted σ-bond metathesis during the bond breaking/forming processes, and (c) oxidative addition of iodomethane to the Ni-center and the subsequent reductive elimination process. DFT calculations revealed that the reaction barrier of the SN2 reaction is slightly lower than those of the other mechanisms. The results of orbital analyses suggest that [Ni(C6H4)(dcpe)] forms a metallacycle structure between benzyne and the NiII (3d8) center instead of the η2-structure with the Ni0 (3d10) center. The metallacycle structures became inappropriate as the intermediates of oxidative addition in the formation of the NiII-Me bond, avoiding further oxidation to the high-valent NiIV. The high free energy along σ-bond metathesis was generated from the steric hindrance, thus invoking methylation and Ni-I bond formation concertedly.
RESUMO
Down syndrome (DS) caused by trisomy of chromosome 21 is the most common genetic cause of intellectual disability. Although the prenatal diagnosis of DS has become feasible, there are no therapies available for the rescue of DS-related neurocognitive impairment. A growth inducer newly identified in our screen of neural stem cells (NSCs) has potent inhibitory activity against dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) and was found to rescue proliferative deficits in Ts65Dn-derived neurospheres and human NSCs derived from individuals with DS. The oral administration of this compound, named ALGERNON (altered generation of neurons), restored NSC proliferation in murine models of DS and increased the number of newborn neurons. Moreover, administration of ALGERNON to pregnant dams rescued aberrant cortical formation in DS mouse embryos and prevented the development of abnormal behaviors in DS offspring. These data suggest that the neurogenic phenotype of DS can be prevented by ALGERNON prenatal therapy.
Assuntos
Síndrome de Down/tratamento farmacológico , Terapias Fetais , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/embriologia , Córtex Cerebral/patologia , Cognição/efeitos dos fármacos , Ciclina D1/metabolismo , Giro Denteado/efeitos dos fármacos , Giro Denteado/patologia , Síndrome de Down/patologia , Síndrome de Down/psicologia , Feminino , Células HEK293 , Humanos , Aprendizagem/efeitos dos fármacos , Masculino , Camundongos , Células-Tronco Neurais/patologia , Gravidez , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinases DyrkRESUMO
A facile method for preparing diverse aryne-nickel complexes from readily synthesized ortho-borylaryl triflates is described. Exploratory synthetic applications, including the synthesis of 1,2-difunctionalized arenes, based on the nucleophilic character of the aryne-nickel complexes are also demonstrated.
RESUMO
Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors.
Assuntos
Bioensaio , Dobramento de Proteína/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Sequência de Aminoácidos , Animais , Biomarcadores/metabolismo , Cantaridina/farmacologia , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Toxinas Marinhas , Dados de Sequência Molecular , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Fosforilação/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes , Alinhamento de Sequência , Tiazóis/química , Transfecção , Xenopus laevis/embriologia , Quinases DyrkRESUMO
The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer's disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases.
Assuntos
Proteínas de Ciclo Celular/genética , Chaperoninas/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Luciferases/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Benzofuranos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Luciferases/metabolismo , Chaperonas Moleculares/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Tiazolidinas/farmacologia , Triazóis/farmacologia , Quinases DyrkRESUMO
Dysregulation of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) has been demonstrated in several pathological conditions, including Alzheimer's disease and cancer progression. It has been recently reported that a gain of function-mutation in the human DYRK1B gene exacerbates metabolic syndrome by enhancing obesity. In the previous study, we developed an inhibitor of DYRK family kinases (INDY) and demonstrated that INDY suppresses the pathological phenotypes induced by overexpression of DYRK1A or DYRK1B in cellular and animal models. In this study, we designed and synthesized a novel inhibitor of DYRK family kinases based on the crystal structure of the DYRK1A/INDY complex by replacing the phenol group of INDY with dibenzofuran to produce a derivative, named BINDY. This compound exhibited potent and selective inhibitory activity toward DYRK family kinases in an in vitro assay. Furthermore, treatment of 3T3-L1 pre-adipocytes with BINDY hampered adipogenesis by suppressing gene expression of the critical transcription factors PPARγ and C/EBPα. This study indicates the possibility of BINDY as a potential drug for metabolic syndrome.
Assuntos
Benzofuranos/síntese química , Benzotiazóis/síntese química , Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , Benzofuranos/química , Benzofuranos/toxicidade , Benzotiazóis/química , Benzotiazóis/toxicidade , Sítios de Ligação , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , PPAR gama/genética , PPAR gama/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/toxicidade , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Quinases DyrkRESUMO
A novel synthetic method for v-coelenterazine (v-CTZ), which is a vinylene-bridged analog of native CTZ with a large red-shifted luminescence property, is described. The synthesis was achieved in a concise way through the use of three sequential cross-coupling reactions and ring-closing metathesis (RCM). A newly synthesized C2-modified trifluoromethyl analog cf3-v-CTZ showed slightly more red-shifted luminescence than v-CTZ when it was used as a substrate for Renilla luciferases.
Assuntos
Imidazóis/síntese química , Substâncias Luminescentes/síntese química , Pirazinas/síntese química , Imidazóis/química , Luminescência , Substâncias Luminescentes/química , Biologia Marinha , Estrutura Molecular , Pirazinas/químicaRESUMO
An alternative method for generating arynes from ortho-silylaryl triflates using cesium carbonate and 18-crown-6 is reported. The method was efficiently applied to a variety of reactions between several arynes and arynophiles. We also demonstrated that the efficiency of aryne generation is significantly affected by the alkali metal countercation of the carbonate.