RESUMEN
Arsenic is highly toxic and a significant threat to human health, but certain bacteria have developed defense mechanisms initiated by AsIII binding to AsIII-sensing proteins of the ArsR family. The transcriptional regulator AfArsR responds to AsIII and SbIII by coordinating the metalloids with three cysteines, located in a short sequence of the same monomer chain. Here, we characterize the binding of AsIII and HgII to a model peptide encompassing this fragment of the protein via solution equilibrium and spectroscopic/spectrometric techniques (pH potentiometry, UV, CD, NMR, PAC, EXAFS, and ESI-MS) combined with DFT calculations and MD simulations. Coordination of AsIII changes the peptide structure from a random-coil to a well-defined structure of the complex. A trigonal pyramidal AsS3 binding site is formed with almost exactly the same structure as observed in the crystal structure of the native protein, implying that the peptide possesses all of the features required to mimic the AsIII recognition and response selectivity of AfArsR. Contrary to this, binding of HgII to the peptide does not lead to a well-defined structure of the peptide, and the atoms near the metal binding site are displaced and reoriented in the HgII model. Our model study suggests that structural organization of the metal site by the inducer ion is a key element in the mechanism of the metalloid-selective recognition of this protein.
Asunto(s)
Arsénico , Arsénico/química , Arsénico/metabolismo , Sitios de Unión , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Metaloides/química , Metaloides/metabolismo , Teoría Funcional de la Densidad , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
The concept of chemically evolvable replicators is central to abiogenesis. Chemical evolvability requires three essential components: energy-harvesting mechanisms for nonequilibrium dissipation, kinetically asymmetric replication and decomposition pathways, and structure-dependent selective templating in the autocatalytic cycles. We observed a UVA light-fueled chemical system displaying sequence-dependent replication and replicator decomposition. The system was constructed with primitive peptidic foldamer components. The photocatalytic formation-recombination cycle of thiyl radicals was coupled with the molecular recognition steps in the replication cycles. Thiyl radical-mediated chain reaction was responsible for the replicator death mechanism. The competing and kinetically asymmetric replication and decomposition processes led to light intensity-dependent selection far from equilibrium. Here, we show that this system can dynamically adapt to energy influx and seeding. The results highlight that mimicking chemical evolution is feasible with primitive building blocks and simple chemical reactions.
Asunto(s)
Biomimética , Origen de la Vida , Evolución Química , PéptidosRESUMEN
A ZnCl2-catalyzed diastereoselective Joullié-Ugi three-component reaction from 2 H-azirines, isocyanides, and carboxylic acids was established. The protocol allows the preparation of highly and diversely functionalized N-acylaziridine-2-carboxamide derivatives in up to 82% isolated yields. Moreover, the applicability of N-acylaziridines is demonstrated through a variety of transformations.
RESUMEN
Engineering water-soluble stand-alone ß-sandwich mimetics is a current challenge because of the difficulties associated with tailoring long-range interactions. In this work, single cis-(1R,2S)-2-aminocyclohexanecarboxylic acid mutations were introduced into the edge strands of the eight-stranded ß-sandwich mimetic structures from the betabellin family. Temperature-dependent NMR and CD measurements, together with thermodynamic analyses, demonstrated that the modified peripheral strands exhibited an irregular and partially disordered structure but were able to exert sufficient shielding on the hydrophobic core to retain the predominantly ß-sandwich structure. Although the frustrated interactions decreased the free energy of unfolding, the temperature of the maximum stabilities increased to or remained at physiologically relevant temperatures. We found that the irregular peripheral strands were able to prevent edge-to-edge association and fibril formation in the aggregation-prone model. These findings establish a ß-sandwich stabilization and aggregation inhibition approach, which does not interfere with the pillars of the peptide bond or change the net charge of the peptide.
RESUMEN
The mimicry of protein-sized ß-sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin-14 ß-sheet has been used as a template, and αâß residue mutations were carried out in the hydrophobic core (positions 12 and 19). ß-Residues with diverse structural properties were utilized: Homologous ß(3) -amino acids, (1R,2S)-2-aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex-3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3-carboxylic acid (ABHC). Six α/ß-peptidic chains were constructed in both monomeric and disulfide-linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced ß-sheet formation in the 64-residue foldameric systems. Core replacement with (1R,2S)-ACHC was found to be unique among the ß-amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen-bonding network and to fit sterically into the hydrophobic interior of the ß-sandwich. The novel ß-sandwich model containing 25 % unnatural building blocks afforded protein-like thermal denaturation behavior.
Asunto(s)
Pliegue de Proteína , Proteínas/química , Secuencia de Aminoácidos , Ácidos Ciclohexanocarboxílicos/química , Ciclohexilaminas/química , Cicloleucina/química , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Desnaturalización Proteica , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
The principles of ß-sheet folding and design for α-peptidic sequences are well established, while those for sheet mimetics containing homologated amino acid building blocks are still under investigation. To reveal the structure-function relations of ß-amino-acid-containing foldamers, we followed a top-down approach to study a series of α/ß-peptidic analogs of anginex, a ß-sheet-forming antiangiogenic peptide. Eight anginex analogs were developed by systematic α â ß(3) substitutions and analyzed by using NMR and CD spectroscopy. The foldamers retained the ß-sheet tendency, though with a decreased folding propensity. ß-Sheet formation could be induced by a micellar environment, similarly to that of the parent peptide. The destructuring effect was higher when the α â ß(3) exchange was located in the ß-sheet core. Analysis of the ß-sheet stability versus substitution pattern and the local conformational bias of the bulky ß(3)V and ß(3)I residues revealed that a mismatch between the H-bonding preferences of the α- and ß-residues played a minor role in the structure-breaking effect. Temperature-dependent CD and NMR measurements showed that the hydrophobic stabilization was scaled-down for the α/ß-peptides. Analysis of the biological activity of the foldamer peptides showed that four anginex derivatives dose-dependently inhibited the proliferation of a mouse endothelial cell line. The α â ß(3) substitution strategy applied in this work can be a useful approach to the construction of bioactive ß-sheet mimetics with a reduced aggregation tendency and improved pharmacokinetic properties.
Asunto(s)
Péptidos/química , Péptidos/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Ratones , Modelos Moleculares , Péptidos/síntesis química , Pliegue de Proteína , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The tea from the white mulberry (Morus alba L.) leaf is a worldwide known traditional medicine of type II diabetes. Here, we report the investigation of the dichloromethane-soluble fraction obtained in a 0.24% m/m yield from the hot water extract of mulberry leaves. A significant, dose-dependent activity was found by means of the 24-h glucose consumption of fully differentiated adipocytes both in the absence and presence of insulin. The fraction was characterized by HPLC-DAD, GC-MS and GC-FID. The main constituent (40.3% by means of GC-FID) was isolated and identified as loliolide by EIMS, HRESIMS and NMR spectroscopy. In the active fraction benzyl alcohol, ethyl benzoate, t-cinnamic acid, p-hydroxyacetophenone, t-coniferyl alcohol and synapil alcohol were also identified by GC-MS and quantified by GC-FID (0.7, 1.3, 1.5, 2.9, 7.5 and 2.6%, respectively).
Asunto(s)
Hipoglucemiantes/farmacología , Morus/química , Extractos Vegetales/farmacología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Animales , Benzofuranos/química , Benzofuranos/aislamiento & purificación , Hipoglucemiantes/química , Ratones , Extractos Vegetales/química , Hojas de la Planta/químicaRESUMEN
Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide-protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy-entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands.
RESUMEN
Ubiquitination of proliferating cell nuclear antigen (PCNA) triggers pathways of DNA damage tolerance, including mutagenic translesion DNA synthesis, and comprises a cascade of reactions involving the E1 ubiquitin-activating enzyme Uba1, the E2 ubiquitin-conjugating enzyme Rad6, and the E3 ubiquitin ligase Rad18. We report here the discovery of a series of xanthenes that inhibit PCNA ubiquitination, Rad6â¼ubiquitin thioester formation, and the Rad6-Rad18 interaction. Structure-activity relationship experiments across multiple assays reveal chemical and structural features important for different activities along the pathway to PCNA ubiquitination. The compounds that inhibit these processes are all a subset of the xanthen-3-ones we tested. These small molecules thus represent first-in-class probes of Rad6 function and the association of Rad6 and Rad18, the latter being a new inhibitory activity discovered for a small molecule, in the PCNA ubiquitination cascade and potential therapeutic agents to contain cancer progression.
RESUMEN
Saturation transfer difference (STD) is a valuable tool for studying the binding of small molecules to large biomolecules and for obtaining detailed information on the binding epitopes. Here, we demonstrate that the proposed (15)N/(13)C variants of group-selective, "GS-STD" experiments provide a powerful approach to mapping the binding epitope of a ligand even in the absence of efficient spin diffusion within the target protein. Therefore, these experimental variants broaden the scope of STD studies to smaller and/or more-dynamic targets. The STD spectra obtained in four different experimental setups (selective (1)H STD, (15)N GS-STD, (13)C(Ar) and (13)C(aliphatic) GS-STD approaches) revealed that the signal-intensity pattern of the difference spectra is affected by both the type and the spatial distribution of the excited "transmitter" atoms, as well as by the efficiency of the spin-diffusion-mediated magnetization transfer. The performance of the experiments is demonstrated on a system by using the lectin, galectin-1 and its carbohydrate ligand, lactose.
Asunto(s)
Ligandos , Resonancia Magnética Nuclear Biomolecular , Isótopos de Carbono/química , Dimerización , Galectina 1/química , Lactosa/química , Isótopos de Nitrógeno/química , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Galectin-1 (Gal-1), a ubiquitous beta-galactoside-binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal-1 depend on its affinity for beta-galactoside-containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr-Xxx-Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal-1-asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr-Xxx-Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr-Xxx-Tyr peptides studied do not bind to Gal-1, whereas their binding to ASF is clearly detected. (15)N,(1)H HSQC titrations with (15)N-labeled Gal-1 confirm the absence of any peptide-Gal-1 interaction. These data indicate that the Tyr-Xxx-Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.
Asunto(s)
Asialoglicoproteínas/metabolismo , Galectina 1/metabolismo , Glicoproteínas/metabolismo , Péptidos/farmacología , Tirosina/química , alfa-Fetoproteínas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Asialoglicoproteínas/antagonistas & inhibidores , Fetuínas , Galectina 1/antagonistas & inhibidores , Galectina 1/genética , Humanos , Células Jurkat , Espectroscopía de Resonancia Magnética , Péptidos/síntesis química , Péptidos/química , Unión Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa-Fetoproteínas/antagonistas & inhibidoresRESUMEN
The fragment-centric design promises a means to develop complex xenobiotic protein surface mimetics, but it is challenging to find locally biomimetic structures. To address this issue, foldameric local surface mimetic (LSM) libraries were constructed. Protein affinity patterns, ligand promiscuity and protein druggability were evaluated using pull-down data for targets with various interaction tendencies and levels of homology. LSM probes based on H14 helices exhibited sufficient binding affinities for the detection of both orthosteric and non-orthosteric spots, and overall binding tendencies correlated with the magnitude of the target interactome. Binding was driven by two proteinogenic side chains and LSM probes could distinguish structurally similar proteins with different functions, indicating limited promiscuity. Binding patterns displayed similar side chain enrichment values to those for native protein-protein interfaces implying locally biomimetic behavior. These analyses suggest that in a fragment-centric approach foldameric LSMs can serve as useful probes and building blocks for undruggable protein interfaces.
RESUMEN
Assembly language: The programmed sequences of stereochemical building blocks lead to novel biomimetic helices. The rational design approach offers new possibilities for creating periodic secondary structures.
Asunto(s)
Materiales Biomiméticos/química , Péptidos/química , Dicroismo Circular , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Estructura Secundaria de Proteína , EstereoisomerismoRESUMEN
We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structure-activity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.
Asunto(s)
Catequina/análogos & derivados , Té/química , Enzimas Activadoras de Ubiquitina/química , Ubiquitinación , Catequina/química , Catequina/farmacología , Flavonoides/química , Flavonoides/farmacología , Células HEK293 , Humanos , Antígeno Nuclear de Célula en Proliferación/química , Relación Estructura-Actividad , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Ubiquitinación/efectos de los fármacosRESUMEN
Aggregation of the amyloid-beta (Abeta) peptides has a pivotal role in Alzheimer's disease (AD). Small molecules and short peptides/peptidomimetics can exert their full protective effects against Abeta within a short time-frame, but the exact mechanism of action is unclear. Time-dependent NMR spectroscopic binding and replacement experiments were carried out for peptide LPFFD and thioflavine T (ThT) on neurotoxic fibrillar Abeta(1-42), which revealed transient binding behavior for both compounds, and complex time-dependent features in the replacement experiments. The results of particle size measurements through the use of diffuse light-scattering and transmission electron microscopy support the conclusions that the studied ligands induced interfibrillar association on a short timescale, which explains the NMR spectroscopic binding and replacement results. zeta-Potential measurements revealed a slightly increased electrostatic stability of the Abeta fibrils upon ligand binding; this suggests that the interfibrillar assembly is driven by specific noncovalent cross-linking interactions. A specific surface and mobility decrease due to the ligand-induced flocculation of the Abeta fibrils can explain the neuroprotective effects.
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Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Péptidos/química , Tiazoles/química , Benzotiazoles , Sitios de Unión , Floculación , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Microscopía Electrónica de Transmisión/métodos , Tamaño de la Partícula , Estándares de Referencia , Propiedades de Superficie , Factores de TiempoRESUMEN
Protein-protein interactions stabilized by multiple separate hot spots are highly challenging targets for synthetic scaffolds. Surface-mimetic foldamers bearing multiple recognition segments are promising candidate inhibitors. In this work, a modular bottom-up approach is implemented by identifying short foldameric recognition segments that interact with the independent hot spots, and connecting them through dynamic covalent library (DCL) optimization. The independent hot spots of a model target (calmodulin) are mapped with hexameric ß-peptide helices using a pull-down assay. Recognition segment hits are subjected to a target-templated DCL ligation through thiol-disulfide exchange. The most potent derivative displays low nanomolar affinity towards calmodulin and effectively inhibits the calmodulin-TRPV1 interaction. The DCL assembly of the folded segments offers an efficient approach towards the deâ novo development of a high-affinity inhibitor of protein-protein interactions.
RESUMEN
There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron-blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1-calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell-regulatory calmodulin-protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain-modulating behavior of these important pharmacons.
Asunto(s)
Calmodulina/metabolismo , Dolor/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Canales Catiónicos TRPV/metabolismo , Sitios de Unión , Calmodulina/química , Calmodulina/genética , Capsaicina/metabolismo , Capsaicina/farmacología , Diterpenos/metabolismo , Diterpenos/farmacología , Humanos , Dolor/tratamiento farmacológico , Unión Proteica , Conformación Proteica , Mapas de Interacción de Proteínas/genética , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/genéticaRESUMEN
The chloroform extract of Asclepias syriaca stem were investigated. Three triterpenes were isolated by TLC, VLC, and preparative chromatography, and their structures established by one and two-dimensional NMR spectroscopy. Lupenyl acetate has been isolated for the first time from A. syriaca; this is thefirst representative of a triterpene bearing a lupane skeleton in this species. In addition, alpha-amyrin acetate and alpha-amyrin butyrate were isolated.
Asunto(s)
Asclepias/química , Triterpenos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Triterpenos/químicaRESUMEN
Two histidine-rich branched peptides with one lysine as a branching unit have been designed and synthesized by solid-phase peptide synthesis. Their complex formation with Cu(II) and Zn(II) as well as their ability to attenuate the metal-ion induced amyloid aggregation has been characterized. Both peptides can keep Cu(II) and Zn(II) in complexed forms at pH 7.4 and can bind two equivalents of metal ions in solutions with excess metal. The stoichiometry, stability and structure of the complexes formed have been determined by pH potentiometry, UV-Vis spectrophotometry, circular dichroism, EPR and NMR spectroscopy and ESI-MS. Both mono- and bimetallic species have been detected over the whole pH range studied. The basic binding mode is either a tridentate {N(amino), N(amide), N(im)} or a histamine-type of coordination which is complemented by the binding of far imidazole or amino groups leading to macrochelate formation. The peptides were able to prevent Cu(II)-induced Aß(1-40) aggregation but could not effectively compete for Zn(II) in vitro. Our results suggest that branched peptides containing potential metal-binding sites may be suitable metal chelators for reducing the risk of amyloid plaque formation in Alzheimer's disease.