RESUMO
Long-chain scorpion toxin AaH-II isolated from Androctonus australis Hector can selectively inhibit mammalian voltage-gated sodium ion channel Nav 1.7 responsible for pain sensation. Efficient chemical synthesis of AaH-II and its derivatives is beneficial to the study of the function and mechanism of Nav 1.7 and the development of potential peptide inhibitors. Herein, we compared three different strategies, namely, direct solid-phase peptide synthesis, hydrazide-based two-segment native chemical ligation, and hydrazide-based three-segment native chemical ligation for the synthesis of AaH-II. The hydrazide-based two-segment native chemical ligation affords the target toxin with the optimal efficiency, which provides a practically robust procedure for the preparation of tool molecules derived from AaH-II to study the biological functions and modulation of Nav 1.7. Our work highlights the importance of selecting suitable segment condensation approach in the chemical synthesis of protein toxins.
Assuntos
Venenos de Escorpião , Animais , Peptídeos , Escorpiões , SódioRESUMO
Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell-penetrating peptides (CPPs) and pore-forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)- with DNA-based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP-FIT PNA conjugates were affected. Live-cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob-like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin-O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA-based FIT probes were brighter and more responsive than PNA-based FIT probes. Optimized conditions enabled live-cell multicolor imaging of three different mRNA target sequences.
Assuntos
DNA/química , Microscopia de Fluorescência , Ácidos Nucleicos Peptídicos/química , RNA Mensageiro/metabolismo , Peptídeos Penetradores de Células/química , DNA/metabolismo , Sondas de DNA/química , Sondas de DNA/metabolismo , Corantes Fluorescentes/química , Células HEK293 , Humanos , Substâncias Intercalantes/química , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/metabolismo , RNA Mensageiro/químicaRESUMO
A new robust strategy was reported for the epimerization-free synthesis of C-terminal Cys-containing peptide acids through mercaptoethanol-mediated hydrolysis of peptide thioesters prepared in situ from peptide hydrazides. This simple-to-operate and highly efficient method avoids the use of derivatization reagents for resin modification, thus providing a practical avenue for the preparation of C-terminal Cys-containing peptide acids.
Assuntos
Ácidos/síntese química , Cisteína/química , Peptídeos/síntese química , Sequência de Aminoácidos , Peptídeos/química , Conformação ProteicaRESUMO
A series of cyclic Arg-rich mitochondria-penetrating peptides were prepared with variation in the macrocycle size and the chirality of Arg residues. A cyclic heptapeptide was demonstrated to be an efficient mitochondria-specific delivery vector for delivering membrane impermeable peptides.
Assuntos
Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Mitocôndrias/metabolismo , Membrana Celular/química , Sobrevivência Celular , Peptídeos Penetradores de Células/química , Ciclização , Células HeLa , Humanos , Mitocôndrias/química , Conformação MolecularRESUMO
Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached through covalent bonding to an oligonucleotide probe. However, the additional conjugation step increases costs. Here we introduce a method that avoids the requirement for the preparation of fluorescence-labelled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine-containing peptide nucleic acid (PNA) probes to form a bipartite tetracysteine motif that binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases in fluorescence emission (with response factors of up to 80-fold and high brightness up to 50â mL mol-1 cm-1 ). The detection system provides sub-nanomolar limits of detection and allows discrimination of single nucleotide variations through more than 20-fold changes in fluorescence intensity. To demonstrate its usefulness, the FIAsH-based readout of the bivalent CysCys-PNA display was interfaced with a rolling-circle amplification (RCA) assay used to detect disease-associated microRNA let-7a.
Assuntos
DNA/análise , Corantes Fluorescentes/química , Hibridização in Situ Fluorescente , Ácidos Nucleicos Peptídicos/química , RNA/análise , Sequência de Bases , Cisteína/química , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/metabolismo , Espectrometria de FluorescênciaRESUMO
Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO2 in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicinâ Q.
Assuntos
Anilidas/química , Histonas/síntese química , Peptídeos/química , Aminação , Sequência de Aminoácidos , Anilidas/síntese química , Ésteres , Histonas/química , Modelos Moleculares , Dados de Sequência Molecular , Nitrogênio/química , Peptídeos/síntese química , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/químicaRESUMO
The ligation of peptide hydrazides at a Gly site carrying a removal auxiliary was found to be an efficient process. This technology was successfully used for the synthesis of ubiquitin C-terminal conjugates. Recombinant Ub(1-75)-NHNH2 was prepared through the hydrozinolysis of the Ub(1-75)-intein fusion protein. It was ligated with a glycine derivative modified with an acid-sensitive thiol auxiliary. The final acid treatment produced the desired bioactive ubiquitin conjugates in practical quantities. Thus, the method described here extends the protocols of expressed protein ligation.
Assuntos
Técnicas de Química Sintética/métodos , Cumarínicos/química , Ubiquitina C/síntese química , Eletroforese em Gel de Poliacrilamida , Estrutura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ubiquitina C/químicaRESUMO
With the increasing attention paid to macrocyclic scaffolds in peptide drug development, genetically encoded peptide macrocycle libraries have become invaluable sources for the discovery of high-affinity peptide ligands targeting disease-associated proteins. The traditional phage display technique of constructing disulfide-tethered macrocycles by cysteine oxidation has the inherent drawback of reduction instability of the disulfide bond. Chemical macrocyclization solves the problem of disulfide bond instability, but the involved highly electrophilic reagents are usually toxic to phages and may bring undesirable side reactions. Here, we report a unique Sortase-mediated Peptide Ligation and One-pot Cyclization strategy (SPLOC) to generate peptide macrocycle libraries, avoiding the undesired reactions of electrophiles with phages. The key to this platform is to mine the unnatural promiscuity of sortase on the X residue of the pentapeptide recognition sequence (LPXTG). Low reactive electrophiles are incorporated into the X-residue side chain, enabling intramolecular cyclization with the cysteine residue of the phage-displayed peptide library. Utilizing the genetically encoded peptide macrocycle library constructed by the SPLOC platform, we found a high-affinity bicyclic peptide binding TEAD4 with a nanomolar KD value (63.9 nM). Importantly, the binding affinity of the bicyclic peptide ligand is 102-fold lower than that of the acyclic analogue. To our knowledge, this is the first time to mine the unnatural promiscuity of ligases to generate peptide macrocycles, providing a new avenue for the construction of genetically encoded cyclic peptide libraries.
RESUMO
We describe a simple and robust oxidation strategy for preparing N-terminal thiazolidine-containing peptide thioesters from peptide hydrazides. We find for the first time that l-thioproline can be used as a protective agent to prevent the nitrosation of N-terminal thiazolidine during peptide hydrazide oxidation. The thioproline-based oxidation strategy has been successfully applied to the chemical synthesis of CC chemokine ligand-2 (69aa) and omniligase-C (113aa), thereby demonstrating its utility in hydrazide-based native chemical ligation.
Assuntos
Oxirredução , Peptídeos , Tiazolidinas , Tiazolidinas/química , Tiazolidinas/síntese química , Estrutura Molecular , Peptídeos/química , Peptídeos/síntese química , Hidrazinas/química , Prolina/química , Ésteres/química , Compostos de Sulfidrila/químicaRESUMO
We report here an enzymatic strategy for asparaginyl endopeptidase-mediated peptide cyclization. Incorporation of chloroacetyl groups into the recognition sequence of OaAEP1 enabled intramolecular cyclization with Cys residues. Combining this strategy and phage display, we identified nanomolar macrocyclic peptide ligands targeting TEAD4. One of the bicyclic peptides binds to TEAD4 with a KD value of 139 nM, 16 times lower than its linear analogue, demonstrating the utility of this platform in discovering high-affinity macrocyclic peptide ligands.
Assuntos
Bacteriófagos , Peptídeos , Ciclização , Peptídeos/química , Cisteína Endopeptidases , Ligantes , Bacteriófagos/metabolismo , Biblioteca de Peptídeos , Peptídeos Cíclicos/químicaRESUMO
We report here a robust and practical strategy for chemical protein synthesis using an o-nitrobenzyl group as a temporary protective group for an N-terminal cysteine residue of intermediate hydrazide fragments. By reinvestigating the photoremoval of an o-nitrobenzyl group, we establish a robust and reliable strategy for its quantitative photodeprotection. The o-nitrobenzyl group is completely stable to oxidative NaNO2 treatment and has been applied to the convergent chemical synthesis of programmed death ligand 1 fragment, providing a practical avenue for hydrazide-based native chemical ligation.
Assuntos
Cisteína , Proteínas , Cisteína/química , Ligadura , Nitrito de Sódio/químicaRESUMO
Native chemical ligation is a widely used technique for peptide fragment condensation in aqueous solutions, which has broken through the length limitation of traditional solid-phase peptide synthesis. It can achieve high-efficient chemical synthesis of proteins containing more than 300 amino acid residues. Peptide hydrazide, as a valuable reagent equivalent to a thioester peptide, can be easily and efficiently prepared by the Fmoc-based SPPS method and has been widely used in native chemical ligation. Here we take the chemical synthesis of a SARS-CoV-2 miniprotein inhibitor LCB1 as an example to describe the detailed procedure of hydrazide-based native chemical ligation.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Hidrazinas , Peptídeos/química , Técnicas de Síntese em Fase SólidaRESUMO
We report a new pattern of a bicyclic helical peptide constructed through head-to-tail cross-linking. The described bicyclic helical peptide has a head-to-tail cross-linking arm and a C-terminal i, i + 4 cross-linking arm. This scaffold will provide a promising scaffold for designing a proteolytically resistant helix-constrained peptide.
Assuntos
PeptídeosRESUMO
We present the finding of a dimeric ACE2 peptide mimetic designed through side chain cross-linking and covalent dimerization. It has a binding affinity of 16 nM for the SARS-CoV-2 spike RBD, and effectively inhibits the SARS-CoV-2 pseudovirus in Huh7-hACE2 cells with an IC50 of 190 nM and neutralizes the authentic SARS-CoV-2 in Caco2 cells with an IC50 of 2.4 µM. Our study should provide a new insight for the optimization of peptide-based anti-SARS-CoV-2 inhibitors.
Assuntos
Antivirais/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptidomiméticos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Antivirais/síntese química , Antivirais/metabolismo , Linhagem Celular Tumoral , Humanos , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Ligação Proteica , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Facile head-to-side chain cross-linking strategies are developed to generate helix-constrained peptides. In our strategies, a covalent cross-linker is incorporated at N, i+7 or N, i+1 positions to lock the peptide into a helical conformation. The described patterns of head-to-side chain cross-linking will provide new frameworks for constrained helical peptide.
Assuntos
Peptídeos/síntese química , Sequência de Aminoácidos , Dicroísmo Circular , Reagentes de Ligações Cruzadas , Estrutura Molecular , Peptídeos/química , Estrutura Secundária de ProteínaRESUMO
Previous studies have led to opposing hypotheses about the requirement of intermolecular disulfide exchange in the binding of the iron regulatory peptide hepcidin to its receptor ferroportin. To clarify this issue, we used the diaminodiacid approach to replace the disulfide bonds in hepcidin with non-reducible thioether bonds. Our results implied that disulfide exchange is not required for the interaction between hepcidin and ferroportin. This theory is further supported by our development of biologically active minihepcidins that do not show activity dependence on cysteine.
RESUMO
Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, in vitro and in cellulo. A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C â U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization. Herein we introduce a binary probe system based on fluorescence resonance energy transfer (FRET) that distinguishes three possible states i.e. (i) absence of target, (ii) presence of edited (matched) and (iii) unedited (single base mismatched) target. To address the shortcomings of read-out via FRET, we designed donor probes that avoid bleed through into the acceptor channel and nevertheless provide a high intensity of FRET signaling. We show the combined use of thiazole orange (TO) and an oxazolopyridine analogue (JO), linked as base surrogates in modified PNA FIT-probes that serve as FRET donor for a second, near-infrared (NIR)-labeled strand. In absence of target, donor emission is low and FRET cannot occur in lieu of the lacking co-alignment of probes. Hybridization of the TO/JO-PNA FIT-probe with the (unedited RNA) target leads to high brightness of emission at 540 nm. Co-alignment of the NIR-acceptor strand ensues from recognition of edited RNA inducing emission at 690 nm. We show imaging of mRNA in fixed and live cells and discuss the homogeneous detection and intracellular imaging of a single nucleotide mRNA edit used by nature to post-transcriptionally modify the function of the Glycine Receptor (GlyR).
RESUMO
A new strategy was developed for the synthesis of peptide disulfide-bond mimics using fully orthogonally protected diaminodiacids. This method overcomes the previous problems of heavy-metal contamination and poor compatibility with Fmoc chemistry and provides a practical avenue for the efficient preparation of peptide disulfide-bond mimics.
RESUMO
"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low-cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc.