RESUMO
Activated esters are widely used to label proteins at lysine side chains and Nâ termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins.
Assuntos
Lisina/metabolismo , Proteínas/metabolismo , Cristalografia por Raios X , Teoria da Densidade Funcional , Humanos , Conformação Proteica , Proteínas/química , TermodinâmicaRESUMO
Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.
Assuntos
Lisina/química , Proteínas/química , Pirróis/química , Meios de Cultura , Escherichia coli/genética , Ressonância Magnética Nuclear BiomolecularRESUMO
D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.
Assuntos
Lisina/análogos & derivados , Ornitina/farmacologia , Sequência de Aminoácidos , Escherichia coli , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Lisina/biossíntese , Lisina/química , Methanosarcina/genética , Methanosarcina/metabolismo , Estrutura Molecular , Ornitina/química , Ornitina/metabolismo , Plasmídeos , Regiões Promotoras GenéticasRESUMO
Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.
Assuntos
Lisina/análogos & derivados , Proteínas/química , Proteínas/isolamento & purificação , Benzaldeídos/química , Sítios de Ligação , Lisina/química , Lisina/metabolismo , Estrutura MolecularRESUMO
A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.
Assuntos
Aminoácidos/química , Marcação por Isótopo/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Proteínas/química , Isótopos de Carbono/química , Códon de Terminação , Flúor/química , Isótopos de Nitrogênio/química , Células Procarióticas/química , Marcadores de SpinRESUMO
A novel series of benzoazepin-2-ones were designed and synthesized targeting the PIF pocket of AGC protein kinases, among which a series of thioether-linked benzoazepin-2-ones were discovered to bind to the PIF pocket of 3-phosphoinositide-dependent kinase-1 (PDK1), and to displace the PIF peptide with an EC(50) values in the lower micromolar range. The structure-activity relationships (SARs) of the linker region, tail region, and distal region were explored to further optimize these novel binders which target the PIF pocket of PDK1. When tested in an in vitro PDK1 enzymatic assay using a peptide substrate, the benzodiazepin-2-ones increased the activity of the enzyme in a concentration-dependent fashion, indicating these compounds act as PDK1 allosteric activators. These new compounds may be further developed as therapeutic agents for the treatment of diseases where the PDK1-mediated AGC protein kinases are dysregulated.
Assuntos
Azepinas/síntese química , Azepinas/farmacologia , Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Azepinas/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Loss of ß-cell mass and function can lead to insufficient insulin levels and ultimately to hyperglycemia and diabetes mellitus. The mainstream treatment approach involves regulation of insulin levels; however, approaches intended to increase ß-cell mass are less developed. Promoting ß-cell proliferation with low-molecular-weight inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) offers the potential to treat diabetes with oral therapies by restoring ß-cell mass, insulin content and glycemic control. GNF4877, a potent dual inhibitor of DYRK1A and glycogen synthase kinase 3ß (GSK3ß) was previously reported to induce primary human ß-cell proliferation inâ vitro and inâ vivo. Herein, we describe the lead optimization that lead to the identification of GNF4877 from an aminopyrazine hit identified in a phenotypic high-throughput screening campaign measuring ß-cell proliferation.
Assuntos
Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Células Secretoras de Insulina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Relação Estrutura-Atividade , Quinases DyrkRESUMO
Autoimmune deficiency and destruction in either ß-cell mass or function can cause insufficient insulin levels and, as a result, hyperglycemia and diabetes. Thus, promoting ß-cell proliferation could be one approach toward diabetes intervention. In this report we describe the discovery of a potent and selective DYRK1A inhibitor GNF2133, which was identified through optimization of a 6-azaindole screening hit. In vitro, GNF2133 is able to proliferate both rodent and human ß-cells. In vivo, GNF2133 demonstrated significant dose-dependent glucose disposal capacity and insulin secretion in response to glucose-potentiated arginine-induced insulin secretion (GPAIS) challenge in rat insulin promoter and diphtheria toxin A (RIP-DTA) mice. The work described here provides new avenues to disease altering therapeutic interventions in the treatment of type 1 diabetes (T1D).
Assuntos
Compostos Aza/química , Compostos Aza/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Indóis/química , Indóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Compostos Aza/farmacocinética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Hipoglicemiantes/farmacocinética , Indóis/farmacocinética , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Quinases DyrkRESUMO
In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Fenilalanina/análogos & derivados , Fenilpropionatos/química , Proteínas/análise , Tirosina/análogos & derivados , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Isótopos de Carbono , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/química , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Marcação por Isótopo , Isótopos de Nitrogênio , Fenilpropionatos/metabolismo , Plasmídeos/genética , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismoRESUMO
[Structure: see text] Pd-catalyzed selective oxidation of Boc-protected N-methylamines with IOAc as the oxidant is described. Evidence for the involvement of a Boc-directed C-H activation process is provided.
RESUMO
Our research groups recently described a series of small-molecule inducers of ß-cell proliferation that could be used to increase ß-cell mass. To mitigate the risk of nonspecific proliferation of other cell types, we devised a delivery strategy built on the tissue specificity observed in the experimental ß-cell imaging agent (+)-dihydrotetrabenazine (DTBZ). The ß-cell proliferator agent aminopyrazine (AP) was covalently linked with (+)-DTBZ to afford conjugates that retain both the proliferation activity and binding affinity for vesicular monoamine transporter-2 (VMAT2). Inâ vivo mouse tissue distribution studies of a prototypical AP-DTBZ conjugate showed 15-fold pancreas exposure over plasma. Tissue-to-plasma ratios in liver and kidneys were two- and five-fold, respectively. This work is the first demonstration of enhanced delivery of ß-cell-proliferating molecules to the pancreas by leveraging the intrinsic tissue specificity of a ß-cell imaging agent.
Assuntos
Pâncreas/metabolismo , Tetrabenazina/análogos & derivados , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Portadores de Fármacos/química , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Piridazinas/química , Coelhos , Espectrometria de Massas em Tandem , Tetrabenazina/sangue , Tetrabenazina/química , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Distribuição Tecidual , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Insufficient pancreatic ß-cell mass or function results in diabetes mellitus. While significant progress has been made in regulating insulin secretion from ß-cells in diabetic patients, no pharmacological agents have been described that increase ß-cell replication in humans. Here we report aminopyrazine compounds that stimulate robust ß-cell proliferation in adult primary islets, most likely as a result of combined inhibition of DYRK1A and GSK3B. Aminopyrazine-treated human islets retain functionality in vitro and after transplantation into diabetic mice. Oral dosing of these compounds in diabetic mice induces ß-cell proliferation, increases ß-cell mass and insulin content, and improves glycaemic control. Biochemical, genetic and cell biology data point to Dyrk1a as the key molecular target. This study supports the feasibility of treating diabetes with an oral therapy to restore ß-cell mass, and highlights a tractable pathway for future drug discovery efforts.
Assuntos
Proliferação de Células , Quinase 3 da Glicogênio Sintase/genética , Células Secretoras de Insulina/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Animais , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Regulação para Baixo/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Piridazinas/farmacologia , Quinases DyrkRESUMO
[reaction: see text] It is observed that the iodohydroxylation of 1,2-allenyl sulfides with I(2) and water in aqueous acetone showed Z-selectivity, which is opposite to that of the iodohydroxylation of 1,2-allenyl sulfoxides.
RESUMO
Z-selectivity was observed for iodohydroxylation of Se-substituted allenes with I2 and H2O, which is opposite to that of 1,2-allenyl sulfoxides. With n-hexane as the co-solvent Z-iodoamination leading to N-(3-organoseleno-2-iodo-2(Z)-propenyl)acetamide was observed. A brief rational for the stereoselectivity of this reaction is provided.
RESUMO
The combination of directed C-H activation, batch-wise addition of tetraalkyltin reagents, and rate enhancement by benzoquinone and microwave irradiation provides a promising strategy for the direct coupling of C-H bonds with organometallic reagents. A variety of tetraalkyltins were coupled to C-H bonds to give the alkylated products in good yields by using 5 mol % Pd(OAc)2 as the catalyst. Benzoquinone was shown to be essential for the C-H activation when substrates containing non-pi-conjugated chelating groups are used. Monitoring the formation and reductive elimination of the Pd(Ar)(Me)L2 complex also revealed that benzoquinone promotes the reductive elimination step. Microwave irradiation enhances the reaction rate drastically. The versatility of this protocol was demonstrated by using substrates containing either oxazoline or pyridine as directing groups.
RESUMO
Cu(II)-catalyzed acetoxylation and halogenation of aryl C-H bonds are developed. ortho-Selectivity was observed with a wide range of 2-arylpyridine substrates. Both mono- and difunctionalizations are achieved by tuning the reaction conditions. Excellent functional group tolerance and use of O2 as a stoichiometric oxidant are significant advantages over our recently developed Pd-catalyzed C-H functionalization reactions. These newly discovered reaction conditions are also applicable for cyanation, amination, etherification, and thioetherification of aryl C-H bonds. Mechanistic investigations are carried out to gain insights into the Cu(II)-catalyzed C-H functionalization reactions.
RESUMO
It was observed that the halohydroxylation of 1,2-allenyl sulfides or selenides with Br2 (CuBr2 or NBS) or I2 and water demonstrated a fairly good regioselectivity (i.e., the C=C bond that is remote from the S or Se atom was halohydroxylated with the halogen atom connecting to the middle carbon atom and the hydroxyl group connecting to the non-S terminal carbon or Se-substituted terminal carbon atom of the allene moiety), leading to the synthesis of synthetically important 3-organosulfur or seleno-2-haloallylic alcohols. The stereoselectivity depends on the nature of X+ and S or Se, showing a Z-selectivity with the matched Lewis acid-base pair.