RESUMO
The spider venom protein, double-knot toxin (DkTx), partitions into the cellular membrane and binds bivalently to the pain-sensing ion channel, TRPV1, triggering long-lasting channel activation. In contrast, its monovalent single knots membrane partition poorly and invoke rapidly reversible TRPV1 activation. To discern the contributions of the bivalency and membrane affinity of DkTx to its sustained mode of action, here, we developed diverse toxin variants including those containing truncated linkers between individual knots, precluding bivalent binding. Additionally, by appending the single-knot domains to the Kv2.1 channel-targeting toxin, SGTx, we created monovalent double-knot proteins that demonstrated higher membrane affinity and more sustained TRPV1 activation than the single-knots. We also produced hyper-membrane affinity-possessing tetra-knot proteins, (DkTx)2 and DkTx-(SGTx)2, that demonstrated longer-lasting TRPV1 activation than DkTx, establishing the central role of the membrane affinity of DkTx in endowing it with its sustained TRPV1 activation properties. These results suggest that high membrane affinity-possessing TRPV1 agonists can potentially serve as long-acting analgesics.
Assuntos
Membrana Celular , Venenos de Aranha , Canais de Cátion TRPV , Membrana Celular/metabolismo , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Animais , Canais de Cátion TRPV/metabolismo , Dor/metabolismo , Ligação Proteica , Analgésicos , Transporte de ÍonsRESUMO
Sulfinyls are valuable structural moieties used for developing synthetically new pharmaceuticals and agrochemicals. Herein, we disclose a straightforward synthesis of sulfinylated spiro[5.5]trienones proceeding via an unprecedented BF3·Et2O-promoted spirocyclization of biaryl ynones. The availability of relatively inexpensive BF3·Et2O to carry out transformations on a bulk scale along with its further application towards the synthesis of dibenzocyclohepten-5-ones delivers a unique opportunity to deploy it in various synthetic directions.
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Bromine induced spiro cyclization of biaryl ynones facilitated the synthesis of spiro[5,5]trienones suitable for extended functionality at the C(3') position. Herein, a step-economic photo-oxidative brominative carbannulation of biaryl ynones employing ammonium bromide and riboflavin tetraacetate (RFTA) has been developed. The reactivity between distal phenyl C-H activated ortho-annulation and dearomative ipso-annulation is well exemplified. The eminent features of the methodology include metal-free, external additive free, low-loading photocatalyst (0.1 mol %), and use of a simple precursor.
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The dearomative spiro-etherification of naphthols is achieved using catalytic amounts of riboflavin tetracetate (RFTA) as a photosensitizer and molecular oxygen as a terminal oxidizing agent under blue light (440 nm) irradiation in the presence of acid. The presence of acid increases the photooxidation power of RFTA and facilitates the dearomatization reaction.
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Fever is a conserved and prominent response to infection. Yet, the issue of how CD4 T cell responses are modulated if they occur at fever temperatures remains poorly addressed. We have examined the priming of naive CD4 T cells in vitro at fever temperatures, and we report notable fever-mediated modulation of their cytokine commitment. When naive CD4 T cells were primed by plate-bound anti-CD3 and anti-CD28 monoclonal antibodies at moderate fever temperature (39 °C), they enhanced commitment to IL4/5/13 (Th2) and away from IFNg (Th1). This was accompanied by up-regulation of the Th2-relevant transcription factor GATA3 and reduction in the Th1-relevant transcription factor Tbet. Fever sensing by CD4 T cells involved transient receptor potential vanilloid cation channels (TRPVs) since TRPV1/TRPV4 antagonism blocked the febrile Th2 switch, while TRPV1 agonists mediated a Th2 switch at 37 °C. The febrile Th2 switch was IL4 independent, but a γ-secretase inhibitor abrogated it, and it was not found in Notch1-null CD4 T cells, identifying the Notch pathway as a major mediator. However, when naive CD4 T cells were primed via antigen and dendritic cells (DCs) at fever temperatures, the Th2 switch was abrogated via increased production of IL12 from DCs at fever temperatures. Thus, immune cells directly sense fever temperatures with likely complex physiological consequences.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Diferenciação Celular/fisiologia , Febre/fisiopatologia , Receptores Notch/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Temperatura Corporal/fisiologia , Linfócitos T CD4-Positivos/citologia , Células Cultivadas , Temperatura Alta , Camundongos , Modelos BiológicosRESUMO
Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these toxins for their target ion channels enables them to serve as powerful tools for ion channel biology. Toxins labeled with fluorescent dyes are employed for the cellular imaging of channels and also for studying toxin-channel and toxin-membrane interactions. Several of these toxins are cysteine-rich, rendering the production of properly folded fluorescently labeled toxins technically challenging. Herein, we evaluate a variety of site-specific protein bioconjugation approaches for producing fluorescently labeled double-knot toxin (DkTx), a potent TRPV1 ion channel agonist that contains an uncommonly large number of cysteines (12 out of a total of 75 amino acids present in the protein). We find that popular cysteine-mediated bioconjugation approaches are unsuccessful as the introduction of a non-native cysteine residue for thiol modification leads to the formation of misfolded toxin species. Moreover, N-terminal aldehyde-mediated bioconjugation approaches are also not suitable as the resultant labeled toxin lacks activity. In contrast to these approaches, C-terminal bioconjugation of DkTx via the sortase bioconjugation technology yields functionally active fluorescently labeled DkTx. We employ this labeled toxin for imaging rat TRPV1 heterologously expressed in Xenopus laevis oocytes, as well as for performing membrane binding studies on giant unilamellar vesicles composed of different lipid compositions. Our studies set the stage for using fluorescent DkTx as a tool for TRPV1 biology and provide an informative blueprint for labeling cysteine-rich proteins.
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Cisteína , Toxinas Biológicas , Aldeídos , Animais , Cisteína/química , Corantes Fluorescentes , Lipídeos , Mamíferos/metabolismo , Peptídeos/química , Ratos , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Lipossomas UnilamelaresRESUMO
A temperature-controlled facile synthesis of multisubstituted 4-alkynyl/trans 4-alkenyl coumarins with a metal salt cascade approach is reported. H2O serves both as a nucleophile and hydrogen source. The presence of metal salt facilitates the reduction of alkyne. The present protocol bypasses the structural shortcomings of the existing Sonogashira and Heck coupling reactions. In addition, the obtained 2,3-disubstituted coumarins are readily transformed into 2,3-disubstituted dihydrocoumarins, which serve as important building blocks in organic transformations.
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Alcinos , Cumarínicos , Cumarínicos/química , Temperatura , Alcinos/química , HidrogênioRESUMO
A new mechanistic pathway of propargylic alcohol activation by gold(I) catalysis has been proposed toward the efficient synthesis of N-protected pyrroles, 5,6-dihydropyridin-3(4H)-ones from N-protected 5-aminopent-2-yn-1-ol, and 5-aminopent-2-yn-1-ol. Control experiments support that the reaction proceeded via the neighboring group participation of the oxygen atom of propargylic alcohol to form an allene oxide intermediate where the nucleophilic heteroatom attacks intramolecularly. Further, this methodology is successfully extrapolated toward the atom-economic synthesis of hydroxyalkyl indoles and benzofurans. The short reaction time of 30 s, low catalyst loading of 0.5 mol %, high yield, variation in the substrate scope, and procedurally simple open-flask reaction conditions make this methodology highly applied.
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Alcadienos , Ouro , Catálise , ÓxidosRESUMO
Synthesis of ß-hydroxyenones and its application toward development of tetrahydro-4H-pyran-4-one in an atom-economic fashion is limited. This manuscript describes a ruthenium-catalyzed atom-economic coupling of pent-2-yne-1,5-diols and Michael acceptors as an efficient route for the synthesis of ß-hydroxyenones with excellent yields and high regioselectivity. The ß-hydroxyenones further undergo a 6-endo trig cyclization under acid-catalyzed conditions to deliver the tetrahydro-4H-pyran-4-ones with high diastereoselectivity. An intramolecular aldol condensation under mild basic conditions and palladium-catalyzed oxidative aromatization was developed for the synthesis of hexahydro-6H-isochromen-6-ones and isochromanols, respectively, from highly substituted tetrahydro-4H-pyran-4-ones with excellent yield and diastereoselectivity. Overall, this work demonstrates the synthetic potential toward the synthesis of oxacycles like tetrahydro-4H-pyran-4-ones, hexahydro-6H-isochromen-6-ones, and isochromanols via an atom-economic catalysis.
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Rutênio , Álcoois , Catálise , Ciclização , PiranosRESUMO
An efficient two-component copper-catalyzed cyclization cascade approach toward highly functionalized indolizinone heterocycles has been developed from reactions of pyridine-, isoquinoline-, and quinoline ynones, via 5-exo-dig cyclization. The catalysis involves the activation by diorgano diselenide and diorgano disulfide and also their incorporation into the indolizinone core. In addition, the obtained substituted indolizinones were readily transformed into 1-(organochalcogenyl)indolizin-2-ols, which are important building blocks in organic synthesis.
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An efficient copper-catalyzed cyclization cascade approach towards highly functionalized methylene 4-chromanol and aurone derivatives has been developed from reactions of ynols via 6-exo-dig and 5-exo-dig cyclization respectively. The catalysis involves alkyne activation via diorgano-diselenides and also their regioselective incorporation into the methylene 4-chromanol and aurone derivative core and is an open-air transformation.
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Understanding the topology adopted by individual G-quadruplex (GQ)-forming sequences in vivo and targeting a specific GQ motif among others in the genome will have a profound impact on GQ-directed therapeutic strategies. However, this remains a major challenge as most of the tools poorly distinguish different GQ conformations and are not suitable for both cell-free and in-cell analysis. Here, we describe an innovative probe design to investigate GQ conformations and recognition in both cell-free and native cellular environments by using a conformation-sensitive dual-app nucleoside analogue probe. The nucleoside probe, derived by conjugating fluorobenzofuran at the 5-position of 2'-deoxyuridine, is composed of a microenvironment-sensitive fluorophore and an in-cell NMR compatible 19F label. This noninvasive nucleoside, incorporated into the human telomeric DNA oligonucleotide repeat, serves as a common probe to distinguish different GQ topologies and quantify topology-specific binding of ligands by fluorescence and NMR techniques. Importantly, unique signatures displayed by the 19F-labeled nucleoside for different GQs enabled a systematic study in Xenopus laevis oocytes to provide new structural insights into the GQ topologies adopted by human telomeric overhang in cells, which so far has remained unclear. Studies using synthetic cell models, immunostaining on fixed cells, and crystallization conditions suggest that parallel GQ is the preferred conformation of telomeric DNA repeat. However, our findings using the dual-app probe clearly indicate that multiple structures including hybrid-type parallel-antiparallel and parallel GQs are formed in the cellular environment. Taken together, our findings open new experimental strategies to investigate topology, recognition, and therapeutic potential of individual GQ-forming motifs in a biologically relevant context.
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DNA/química , Corantes Fluorescentes/química , Sondas Moleculares/química , Nucleosídeos/química , Telômero/química , Animais , Benzofuranos/química , Sistema Livre de Células , Desoxiuridina/química , Quadruplex G , Humanos , Espectrofotometria Ultravioleta , Xenopus laevisRESUMO
A de novo palladium carbon-catalyzed synthesis of trisubstituted nicotinonitriles from easily synthesized homopropagylic or homoallylic aromatic alcohols in the presence of nitriles has been explored. The mechanism proceeds with an interesting generation of a Pd(II)-C palladacycle followed by an oxidative aromatization to generate the pyridine core. The pyridine core is generated with a noteworthy C-C bond cleavage in the case of the substituted nitriles. The moderate yields and easy separation of the products lend a unique importance to this one-pot methodology.
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This paper entails the first recognition of Phenyl Trimethyl Ammonium Tribromide (PTAB) as an effective reagent for spiro-cyclizations proceeding via oxidative dearomatization. The experiment exhibits economical, metal and ligand free one-pot accomplishment of these significant transformations. The described protocol presents the first generalised methodology of spiro-oxacycle synthesis which can be applied towards various directions. A stereoselective synthesis of oxa-spirocyclooxadieneones has been accomplished.
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Synthetically challenging spirolactams were developed in good yields and regio-selectivity through a step-up oxidative dearomatization of easily accessible arenols tethered to esters in the presence of quaternary ammonium tribromide as an economic oxidant and amines. The reaction mechanism associated with this unprecedented dearomative lactamisation has been forecasted with a series of controlled experiments and DFT studies.
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Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.
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Canais Iônicos Sensíveis a Ácido , Lisina , Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/genética , Lisina/química , Lisina/metabolismo , Humanos , Animais , Modelos Moleculares , Processamento de ProteínaRESUMO
An efficient photocatalytic para- and ortho-selective amination and aminative dearomatization of phenols, naphthols, and anilines with azodicarboxylates was developed using riboflavin tetraacetate (RFTA) as an organic photocatalyst. The site selectivity was controlled using tetrabutylammonium bromide (TBAB), which also acts as a phase transfer catalyst. The reaction conditions are simple and mild, giving high regioselectivity with good to excellent yields. A broad substrate scope and nice functional group tolerance with scalability and post-functionalization make this protocol both useful and regioselective.
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Severe Acute Respiratory Syndrome (SARS) aka SARS-CoV spread over southern China for the first time in 2002-2003 and history repeated again since last year and took away lives of more than two million people so far. On March 11, 2020 COVID-19 outbreak was officially declared as pandemic by World Health Organization (WHO). The entire world united to fight back against this ultimate destruction. Around 90 vaccines are featured against SARS-CoV-2 and more than 300 active clinical trials are underway by several groups and individuals. So far, no drugs have been currently approved that can completely eliminate the deadly coronavirus. The promising SARS-CoV-2 antiviral drugs are favipiravir, remdesivir, lopinavir, ribavirin and avifavir. In this review, we have discussed the synthetic approaches elaborately made so far by different groups and chemical companies all around the world towards top three convincing anti-viral drugs against SARS-CoV-2, which are favipiravir, remdesivir and lopinavir.
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Antivirais/síntese química , Antivirais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/síntese química , Alanina/análogos & derivados , Alanina/síntese química , Amidas/síntese química , Antivirais/uso terapêutico , COVID-19/virologia , Humanos , Lopinavir/síntese química , Pirazinas/síntese química , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND AND PURPOSE: P2X receptors are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains. EXPERIMENTAL APPROACH: Here, we examine the impact of P2X2 receptor inter-subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. KEY RESULTS: We demonstrate that predicted extracellular domain inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity. CONCLUSION AND IMPLICATIONS: We provide the first structural mapping of the mutational distribution across the human population in a ligand-gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand-gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.
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Ativação do Canal Iônico , Mutação de Sentido Incorreto , Receptores Purinérgicos P2X2 , Trifosfato de Adenosina/metabolismo , Humanos , Mutação , Receptores Purinérgicos P2X2/genéticaRESUMO
Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion channels. Recently, this has increasingly been complemented by semi-synthetic strategies, in which part of the protein is generated synthetically. This means a vast range of chemical modifications, including non-canonical amino acids (ncAA), backbone modifications, chemical handles, fluorescent or spectroscopic labels and any combination of these can be incorporated. Among these approaches, protein trans-splicing (PTS) is particularly promising for protein reconstitution in live cells. It relies on one or more split inteins, which can spontaneously and covalently link flanking peptide or protein sequences. Here, we describe the use of PTS and its variant tandem PTS (tPTS) in semi-synthesis of ion channels in Xenopus laevis oocytes to incorporate ncAAs, post-translational modifications or metabolically stable mimics thereof. This strategy has the potential to expand the type and number of modifications in ion channel research.