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The interaction between the intrinsically disordered transcription factor HIF-1α and the coactivator proteins p300/CBP is essential in the fast response to low oxygenation. The negative feedback regulator, CITED2, switches off the hypoxic response through a very efficient irreversible mechanism. The negative cooperativity with HIF-1α relies on the formation of a ternary intermediate that leads to allosteric structural changes in p300/CBP, in which the cooperative folding/binding of the CITED2 sequence motifs plays a key role. Understanding the contribution of a binding motif to the structural changes in relation to competition efficiency provides invaluable insights into the molecular mechanism. Our strategy is to site-directedly perturb the p300-CITED2 complex's structure without significantly affecting binding thermodynamics. In this way, the contribution of a sequence motif to the negative cooperativity with HIF-1α would mainly depend on the induced structural changes, and to a lesser extent on binding affinity. Using biophysical assays and NMR measurements, we show here that the interplay between the N-terminal tail and the rest of the binding motifs of CITED2 is crucial for the unidirectional displacement of HIF-1α. We introduce an advantageous approach for evaluating the roles of the different sequence parts with the help of motif-by-motif backbone perturbations.
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SARS-CoV-2 and its variants are crossing the immunity barrier induced through vaccination. Recent Omicron sub-variants are highly transmissible and have a low mortality rate. Despite the low severity of Omicron variants, these new variants are known to cause acute post-infectious syndromes. Nowadays, novel strategies to develop new potential inhibitors for SARS-CoV-2 and other Omicron variants have gained prominence. For viral replication and survival the main protease of SARS-CoV-2 plays a vital role. Peptide-like inhibitors that mimic the substrate peptide have already proved to be effective in inhibiting the Mpro of SARS-CoV-2 variants. Our systematic canonical amino acid point mutation analysis on the native peptide has revealed various ways to improve the native peptide of the main protease. Multi mutation analysis has led us to identify and design potent peptide-analog inhibitors that act against the Mpro of the Omicron sub-variants. Our in-depth analysis of all-atom molecular dynamics studies has paved the way to characterize the atomistic behavior of Mpro in Omicron variants. Our goal is to develop potent peptide-analogs that could be therapeutically effective against Omicron and its sub-variants.
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Proteases 3C de Coronavírus , Simulação de Dinâmica Molecular , Peptídeos , SARS-CoV-2 , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Proteases 3C de Coronavírus/química , Humanos , Antivirais/química , Antivirais/farmacologia , Desenho de Fármacos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , COVID-19/virologiaRESUMO
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.
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Biomimética , Origem da Vida , Evolução Química , PeptídeosRESUMO
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.
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Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5' or 3' end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation.
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Cationic antimicrobial peptide PGLa gets into close contact with the anionic bacterial cell membrane, facilitating cross-membrane transport phenomena and membrane disruption depending on the concentration. The mechanisms of action are closely associated with the tilted insertion geometry of PGLa. Therefore, we aimed to understand the interaction between the transmembrane potential (TMP) and the orientation of the membrane-bound PGLa helix. Molecular dynamics simulations were performed with TMP, and we found that the PGLa tilt angle relative to the membrane is coupled with the TMP. Elevated TMP increases the population of the tilted state. We observed positive feedback between the tilt angle and the TMP, which occurs due to the electrostatic interaction between the peptidic helix and the Na+ cations at the membrane-water interface. These TMP coupled phenomena can contribute to understanding the direct antimicrobial and adjuvant effects of PGLa in combination with regular antibiotics.
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Anti-Infecciosos , Peptídeos Antimicrobianos , Potenciais da Membrana , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Antibacterianos/farmacologia , Água , Bicamadas Lipídicas/químicaRESUMO
S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein-protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally occurring S100 partners in the human proteome. Such information will be precious for future drug design to interfere with S100 related pathologies.
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Motivos EF Hand , Proteínas S100 , Proteínas de Ligação ao Cálcio/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Proteínas S100/metabolismoRESUMO
Cell delivery of therapeutic macromolecules and nanoparticles is a critical drug development challenge. Translocation through lipid raft-mediated endocytic mechanisms is being sought, as it can avoid rapid lysosomal degradation. Here, we present a set of short α/ß-peptide tags with high affinity to the lipid raft-associated ganglioside GM1. These sequences induce effective internalization of the attached immunoglobulin cargo. The structural requirements of the GM1-peptide interaction are presented, and the importance of the membrane components are shown. The results contribute to the development of a receptor-based cell delivery platform.
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The negative membrane potential of bacterial cells influences crucial cellular processes. Inspired by the molecular scaffold of the antimicrobial peptide PGLa, we have developed antimicrobial foldamers with a computer-guided design strategy. The novel PGLa analogues induce sustained membrane hyperpolarization. When co-administered as an adjuvant, the resulting compounds - PGLb1 and PGLb2 - have substantially reduced the level of antibiotic resistance of multi-drug resistant Escherichia coli, Klebsiella pneumoniae and Shigella flexneri clinical isolates. The observed antibiotic potentiation was mediated by hyperpolarization of the bacterial membrane caused by the alteration of cellular ion transport. Specifically, PGLb1 and PGLb2 are selective ionophores that enhance the Goldman-Hodgkin-Katz potential across the bacterial membrane. These findings indicate that manipulating bacterial membrane electrophysiology could be a valuable tool to overcome antimicrobial resistance.
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The incorporation of ß-amino acids into a peptide sequence has gained particular attention as ß- and α/ß-peptides have shown remarkable proteolytic stability, even after a single homologation at the scissile bond. Several peptidases have been shown to cleave such bonds with high specificity but at a much slower rate compared to α-peptide bonds. In this study, a series of analogs of dipeptidyl peptidase-4 (DPP-4) substrate inhibitors were synthesized in order to investigate whether ß-amino acid homologation at the scissile bond could be a valid approach to improving peptide stability towards DPP-4 degradation. DPP-4 cleaved the α/ß-peptide bond after the N-terminal penultimate Pro with a broad specificity and retained full activity regardless of the ß3 -amino acid side chain and peptide length. Significantly improved half-lives were observed for ß3 Ile-containing peptides. Replacing the penultimate Pro with a conformationally constrained Pro mimetic led to proteolytic resistance. DPP-4 cleavage of α/ß-peptide bonds with a broad promiscuity represents a new insight into the stability of peptide analogs containing ß-amino acids as such analogs were thought to be stable towards enzymatic degradation.
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Dipeptidil Peptidase 4/metabolismo , Peptídeos/metabolismo , Meia-Vida , Humanos , Peptídeos/síntese química , Peptídeos/química , Especificidade por SubstratoRESUMO
There is a pressing need to develop ways to deliver therapeutic macromolecules to their intracellular targets. Certain viral and bacterial proteins are readily internalized in functional form through lipid raft-mediated/caveolar endocytosis, but mimicking this process with protein cargoes at therapeutically relevant concentrations is a great challenge. Targeting ganglioside GM1 in the caveolar pits triggers endocytosis. A pentapeptide sequence WYKYW is presented, which specifically captures the glycan moiety of GM1 (K D = 24 nm). The WYKYW-tag facilitates the GM1-dependent endocytosis of proteins in which the cargo-loaded caveosomes do not fuse with lysosomes. A structurally intact immunoglobulin G complex (580 kDa) is successfully delivered into live HeLa cells at extracellular concentrations ranging from 20 to 160 nm, and escape of the cargo proteins to the cytosol is observed. The short peptidic WYKYW-tag is an advantageous endocytosis routing sequence for lipid raft-mediated/caveolar cell delivery of therapeutic macromolecules, especially for cancer cells that overexpress GM1.
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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.
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The absorption of drugs is limited by the epithelial barriers of the gastrointestinal tract. One of the strategies to improve drug delivery is the modulation of barrier function by the targeted opening of epithelial tight junctions. In our previous study the 18-mer amphiphilic PN159 peptide was found to be an effective tight junction modulator on intestinal epithelial and bloodâ»brain barrier models. PN159, also known as KLAL or MAP, was described to interact with biological membranes as a cell-penetrating peptide. In the present work we demonstrated that the PN159 peptide as a penetration enhancer has a dual action on intestinal epithelial cells. The peptide safely and reversibly enhanced the permeability of Caco-2 monolayers by opening the intercellular junctions. The penetration of dextran molecules with different size and four efflux pump substrate drugs was increased several folds. We identified claudin-4 and -7 junctional proteins by docking studies as potential binding partners and targets of PN159 in the opening of the paracellular pathway. In addition to the tight junction modulator action, the peptide showed cell membrane permeabilizing and antimicrobial effects. This dual action is not general for cell-penetrating peptides (CPPs), since the other three CPPs tested did not show barrier opening effects.
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Alzheimer's disease is one of the most common chronic neurodegenerative disorders. Despite several in vivo and clinical studies, the cause of the disease is poorly understood. Currently, amyloid ß (Aß) peptide and its tendency to assemble into soluble oligomers are known as a main pathogenic event leading to the interruption of synapses and brain degeneration. Targeting neurotoxic Aß oligomers can help recognize the disease at an early stage or it can be a potential therapeutic approach. Unnatural ß-peptidic foldamers are successfully used against many different protein targets due to their favorable structural and pharmacokinetic properties compared to small molecule or protein-like drug candidates. We have previously reported a tetravalent foldamer-dendrimer conjugate which can selectively bind Aß oligomers. Taking advantage of multivalency and foldamers, we synthesized different multivalent foldamer-based conjugates to optimize the geometry of the ligand. Isothermal titration calorimetry (ITC) was used to measure binding affinity to Aß, thereafter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based tissue viability assay and impedance-based viability assay on SH-SY5Y cells were applied to monitor Aß toxicity and protective effects of the compounds. Important factors for high binding affinity were determined and a good correlation was found between influencing the valence and the capability of the conjugates for Aß binding.
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Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Dendrímeros/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/química , Animais , Calorimetria , Dendrímeros/uso terapêutico , Humanos , Ligantes , Neurônios/química , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/uso terapêutico , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacosRESUMO
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.
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Antimicrobial peptides are promising alternative antimicrobial agents. However, little is known about whether resistance to small-molecule antibiotics leads to cross-resistance (decreased sensitivity) or collateral sensitivity (increased sensitivity) to antimicrobial peptides. We systematically addressed this question by studying the susceptibilities of a comprehensive set of 60 antibiotic-resistant Escherichia coli strains towards 24 antimicrobial peptides. Strikingly, antibiotic-resistant bacteria show a high frequency of collateral sensitivity to antimicrobial peptides, whereas cross-resistance is relatively rare. We identify clinically relevant multidrug-resistance mutations that increase bacterial sensitivity to antimicrobial peptides. Collateral sensitivity in multidrug-resistant bacteria arises partly through regulatory changes shaping the lipopolysaccharide composition of the bacterial outer membrane. These advances allow the identification of antimicrobial peptide-antibiotic combinations that enhance antibiotic activity against multidrug-resistant bacteria and slow down de novo evolution of resistance. In particular, when co-administered as an adjuvant, the antimicrobial peptide glycine-leucine-amide caused up to 30-fold decrease in the antibiotic resistance level of resistant bacteria. Our work provides guidelines for the development of efficient peptide-based therapies of antibiotic-resistant infections.
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Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas da Membrana Bacteriana Externa/genética , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Mutação , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
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.
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Mimicking the molecular recognition functionality of antibodies is a great challenge. Foldamers are attractive candidates because of their relatively small size and designable interaction surface. This paper describes a sandwich type enzyme-linked immunoassay with a tetravalent ß-peptide foldamer helix array as capture element and enzyme labeled tracer antibodies. The assay was found to be selective to ß-amyloid oligomeric species with surface features transiently present in ongoing aggregation. In optimized conditions, with special emphasis on the foldamer immobilization, a detection limit of 5 pM was achieved with a linear range of 10-500 pM. These results suggest that protein mimetic foldamers can be useful tools in biosensors and affinity assays.
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Peptídeos beta-Amiloides/química , Ensaio de Imunoadsorção Enzimática/métodos , Multimerização Proteica , Sequência de Aminoácidos , Modelos Moleculares , Agregados Proteicos , Conformação Proteica em alfa-Hélice , Estrutura Secundária de Proteína , Fatores de TempoRESUMO
The neurotransmitter γ-amino butyric acid (GABA) has a fundamental role in CNS function and ionotropic (GABAA) receptors that mediate many of the actions of GABA are important therapeutic targets. This study reports the mechanism of action of novel GABAA antagonists based on a tricyclic oxazolo-2,3-benzodiazepine scaffold. These compounds are orthosteric antagonists of GABA on heteropentameric GABAA receptors of αxß2γ2 configuration expressed in HEK293 cells. In silico modelling predicted that the test compounds docked in the GABA binding-pocket and would interact with amino-acid residues in the α- and ß-subunit interface that are known to be important for the binding of GABA. Intriguingly, optimal docking also required an interaction with the non-conserved amino-terminal segment of Loop-F of the α-subunit. Testing of a compound with altered regiochemistry of the oxazolone moiety supported the model with respect to the conserved GABA-interacting residues in vitro as well as in vivo. The prediction regarding loop-F was examined by replacing the amino-terminal variable segment of loop-F of the α5-subunit with the corresponding residues in the α1- and α2-subunits. When tested with the novel inhibitors, the receptors formed by the modified α5-subunits displayed the pharmacologic phenotype of the source of loop-F. In summary, these data show that the variable amino-terminal segment of loop-F of the α-subunit determines the pharmacologic selectivity of the novel tricyclic inhibitors of GABAA receptors.
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Benzodiazepinas/química , Benzodiazepinas/farmacologia , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacologia , Subunidades Proteicas/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Benzodiazepinas/metabolismo , Ligação Competitiva , Simulação por Computador , Antagonistas de Receptores de GABA-A/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Oxazóis/química , Conformação Proteica , Subunidades Proteicas/química , Relação Estrutura-Atividade , Ácido gama-Aminobutírico/metabolismoRESUMO
Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.