RESUMO
Reactive oxygen species (ROS) including the superoxide anion (O2â¢-) are typically studied in cell cultures using fluorescent dyes, which provide only discrete single-point measurements. These methods lack the capabilities for assessing O2â¢- kinetics and release in a quantitative manner over long monitoring times. Herein, we present the fabrication and application of an electrochemical biosensor that enables real-time continuous monitoring of O2â¢- release in cell cultures for extended periods (> 8 h) using an O2â¢- specific microelectrode. To achieve the sensitivity and selectivity requirements for cellular sensing, we developed a biohybrid system consisting of superoxide dismutase (SOD) and Ti3C2Tx MXenes, deposited on a gold microwire electrode (AuME) as O2â¢- specific materials with catalytic amplification through the synergistic action of the enzyme and the biomimetic MXenes-based structure. The biosensor demonstrated a sensitivity of 18.35 nA/µM with a linear range from 147 to 930 nM in a cell culture medium. To demonstrate its robustness and practicality, we applied the biosensor to monitor O2â¢- levels in human leukemia monocytic THP-1 cells upon stimulation with lipopolysaccharide (LPS). Using this strategy, we successfully monitored LPS-induced O2â¢- in THP-1 cells, as well as the quenching effect induced by the ROS scavenger N-acetyl-L-cysteine (NAC). The biosensor is generally useful for exploring the role of oxidative stress and longitudinally monitoring O2â¢- release in cell cultures, enabling studies of biochemical processes and associated oxidative stress mechanisms in cellular and other biological environments.
Assuntos
Técnicas Biossensoriais , Superóxido Dismutase , Superóxidos , Humanos , Superóxidos/metabolismo , Superóxidos/análise , Técnicas Biossensoriais/métodos , Superóxido Dismutase/metabolismo , Células THP-1 , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Lipopolissacarídeos/farmacologia , Limite de DetecçãoRESUMO
Proteins with a functionalized C-terminus such as a C-terminal thioester are key to the synthesis of larger proteins via expressed protein ligation. They are usually made by recombinant fusion to intein. Although powerful, the intein fusion approach suffers from premature hydrolysis and low compatibility with denatured conditions. To totally bypass the involvement of an enzyme for expressed protein ligation, here we showed that a cysteine in a recombinant protein was chemically activated by a small molecule cyanylating reagent at its N-side amide for undergoing nucleophilic acyl substitution with amines including a number of l- and d-amino acids and hydrazine. The afforded protein hydrazides could be used further for expressed protein ligation. We demonstrated the versatility of this activated cysteine-directed protein ligation (ACPL) approach with the successful synthesis of ubiquitin conjugates, ubiquitin-like protein conjugates, histone H2A with a C-terminal posttranslational modification, RNase H that actively hydrolyzed RNA, and exenatide that is a commercial therapeutic peptide. The technique, which is exceedingly simple but highly useful, expands to a great extent the synthetic capacity of protein chemistry and will therefore make a large avenue of new research possible.
Assuntos
Inteínas/genética , Proteínas Recombinantes/química , HumanosRESUMO
With the current trajectory of the 2019-nCoV outbreak unknown, public health and medicinal measures will both be needed to contain spreading of the virus and to optimize patient outcomes. Although little is known about the virus, an examination of the genome sequence shows strong homology with its better-studied cousin, SARS-CoV. The spike protein used for host cell infection shows key nonsynonymous mutations that might hamper the efficacy of previously developed therapeutics but remains a viable target for the development of biologics and macrocyclic peptides. Other key drug targets, including RNA-dependent RNA polymerase and coronavirus main proteinase (3CLpro), share a strikingly high (>95 %) homology to SARS-CoV. Herein, we suggest four potential drug candidates (an ACE2-based peptide, remdesivir, 3CLpro-1 and a novel vinylsulfone protease inhibitor) that could be used to treat patients suffering with the 2019-nCoV. We also summarize previous efforts into drugging these targets and hope to help in the development of broad-spectrum anti-coronaviral agents for future epidemics.
Assuntos
Antivirais/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/prevenção & controle , Pneumonia Viral/prevenção & controle , Antivirais/química , Betacoronavirus/enzimologia , Betacoronavirus/genética , COVID-19 , Proteases 3C de Coronavírus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/transmissão , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Desenho de Fármacos , Humanos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/transmissão , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2 , Tratamento Farmacológico da COVID-19RESUMO
An amido cuprate formed from CuCN and LDA allows a general deconjugative α-alkylation of cyclic alkenenitriles. Deprotonating cyclic alkenenitriles with LDA-CuCN avoids polymerization that otherwise plagues these alkylations and generates a reactive metalated nitrile for alkylations with a range of carbon and heteroatom electrophiles. The strategy provides an effective synthesis of quaternary 5-, 6-, and 7-membered cycloalk-1-enecarbonitriles substituted on the nitrile-bearing carbon.
RESUMO
Chloride plays a crucial role in various cellular functions, and its level is regulated by a variety of chloride transporters and channels. However, to date, we still lack the capability to image instantaneous ion flux through chloride channels at single-cell level. Here, we developed a series of cell-permeable, pH-independent, chloride-sensitive fluorophores for real-time cytosolic chloride imaging, which we call CytoCl dyes. We demonstrated the ability of CytoCl dyes to monitor cytosolic chloride and used it to uncover the rapid changes and transient events of halide flux, which cannot be captured by steady-state imaging. Finally, we successfully imaged the proton-activated chloride channel-mediated ion flux at single-cell level, which is, to our knowledge, the first real-time imaging of ion flux through a chloride channel in unmodified cells. By enabling the imaging of single-cell level ion influx through chloride channels and transporters, CytoCl dyes can expand our understanding of ion flux dynamics, which is critical for characterization and modulator screening of these membrane proteins. A conjugable version of CytoCl dyes was also developed for its customization across different applications.
RESUMO
The cGAS-STING signaling pathway has emerged as a key mediator of inflammation. However, the roles of chloride homeostasis on this pathway are unclear. Here, we uncovered a correlation between chloride homeostasis and cGAS-STING signaling. We found that dysregulation of chloride homeostasis attenuates cGAS-STING signaling in a lysosome-independent manner. Treating immune cells with chloride channel inhibitors attenuated 2'3'-cGAMP production by cGAS and also suppressed STING polymerization, leading to reduced cytokine production. We also demonstrate that non-selective chloride channel blockers can suppress the NPC1 deficiency-induced, hyper-activated STING signaling in skin fibroblasts derived from Niemann Pick disease type C (NPC) patients. Our findings reveal that chloride homeostasis majorly affects cGAS-STING pathway and suggest a provocative strategy to dampen STING-mediated inflammation via targeting chloride channels.
RESUMO
Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+ -dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to coordinate the catalytic zinc ion in HDACs. Using a previously evolved Nð -acetyl-lysyl-tRNA synthetase in combination with tRNAPyl , we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that matched existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+ -chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent compound GH8HA01, which has an HDAC8-inhibition Ki value of 0.67 nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA01Δ1Δ7 that has an HDAC8-inhibition Ki value of 0.31 nM are two of the most potent HDAC8 inhibitors that have been developed. Furthermore, both are highly selective against HDAC8 compared with other HDACs tested, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets with conserved active sites among homologues.
Assuntos
Bacteriófagos , Inibidores de Histona Desacetilases , Aminoácidos/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Domínio Catalítico , Códon de Terminação , Escherichia coli/genética , Escherichia coli/metabolismo , Histona Desacetilases/química , Cetonas , Ligantes , Peptídeos/químicaRESUMO
With the current trajectory of the 2019-nCoV outbreak unknown, public health and medicinal measures will both be needed to contain spreading of the virus and to optimize patient outcomes. While little is known about the virus, an examination of the genome sequence shows strong homology with its more well-studied cousin, SARS-CoV. The spike protein used for host cell infection shows key nonsynonymous mutations which may hamper efficacy of previously developed therapeutics but remains a viable target for the development of biologics and macrocyclic peptides. Other key drug targets, including RdRp and 3CLpro, share a strikingly high (>95%) homology to SARS-CoV. Herein, we suggest 4 potential drug candidates (an ACE2-based peptide, remdesivir, 3CLpro-1 and a novel vinylsulfone protease inhibitor) that can be used to treat patients suffering with the 2019-nCoV. We also summarize previous efforts into drugging these targets and hope to help in the development of broad spectrum anti-coronaviral agents for future epidemics.
RESUMO
Although noncanonical amino acids (ncAAs) were first incorporated into phage libraries through amber suppression nearly two decades ago, their application for use in drug discovery has been limited due to inherent library bias towards sense-containing phages. Here, we report a technique based on superinfection immunity of phages to enrich amber-containing clones, thus avoiding the observed bias that has hindered incorporation of ncAAs into phage libraries. We then take advantage of this technique for development of active site-directed ligand evolution of peptides, where the ncAA serves as an anchor to direct the binding of its peptides to the target's active site. To demonstrate this, phage-displayed peptide libraries are developed that contain a genetically encoded butyryl lysine and are subsequently used to select for ligands that bind SIRT2. These ligands are then modified to develop low nanomolar inhibitors of SIRT2.