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1.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502335

RESUMO

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen responsible for the outbreak of a severe, rapidly developing pneumonia (Coronavirus disease 2019, COVID-19). The virus enzyme, called 3CLpro or main protease (Mpro), is essential for viral replication, making it a most promising target for antiviral drug development. Recently, we adopted the drug repurposing as appropriate strategy to give fast response to global COVID-19 epidemic, by demonstrating that the zonulin octapeptide inhibitor AT1001 (Larazotide acetate) binds Mpro catalytic domain. Thus, in the present study we tried to investigate the antiviral activity of AT1001, along with five derivatives, by cell-based assays. Our results provide with the identification of AT1001 peptide molecular framework for lead optimization step to develop new generations of antiviral agents of SARS-CoV-2 with an improved biological activity, expanding the chance for success in clinical trials.


Assuntos
Antivirais/farmacologia , Simulação de Acoplamento Molecular , Oligopeptídeos/química , Peptídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/virologia , Domínio Catalítico , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Reposicionamento de Medicamentos , Herpesvirus Humano 3/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
2.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360998

RESUMO

Candida albicans, an opportunistic fungus, causes dental caries and contributes to mucosal bacterial dysbiosis leading to a second infection. Furthermore, C.albicans forms biofilms that are resistant to medicinal treatment. To make matters worse, antifungal resistance has spread (albeit slowly) in this species. Thus, it has been imperative to develop novel, antifungal drug compounds. Herein, a peptide was engineered with the sequence of RRFSFWFSFRR-NH2; this was named P19. This novel peptide has been observed to exert disruptive effects on fungal cell membrane physiology. Our results showed that P19 displayed high binding affinity to lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the plasma membrane phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidylglycerol (PG), further indicating that the molecular mechanism of P19 was not associated with the receptor recognition, but rather related to competitive interaction with the plasma membrane. In addition, compared with fluconazole and amphotericin B, P19 has been shown to have a lower potential for resistance selection than established antifungal agents.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Oligopeptídeos/farmacologia , Antifúngicos/química , Candida albicans/fisiologia , Cardiolipinas/metabolismo , Membrana Celular/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Oligopeptídeos/química , Fosfatidilgliceróis/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Ácidos Teicoicos/metabolismo , Triptofano/química
3.
J Phys Chem Lett ; 12(34): 8416-8422, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34436909

RESUMO

Polyproline II (pPII) is a left-handed 31-helix conformation, which has been observed to be the most abundant secondary structure in unfolded peptides and proteins compared to α-helix and ß-sheet. Although pPII has been reported as the most stable conformation for several unfolded short chain peptides in aqueous solution, it is rarely observed in their solid state. Here, we show for the first time a glycine homopeptide (gly-gly-gly) adopting the pPII conformation in its crystalline dihydrate structure. The single crystal X-ray structure with molecular dynamic simulation suggests that a network of water and the charged carboxylate group is critical in stabilizing the pPII conformation in solid state, offering an insight into the structures of unfolded regions of proteins and the role of water in peptide crystallization.


Assuntos
Oligopeptídeos/química , Peptídeos/química , Água/química , Cristalização , Conformação Molecular
4.
Nat Commun ; 12(1): 5064, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417468

RESUMO

Ghrelin, also called "the hunger hormone", is a gastric peptide hormone that regulates food intake, body weight, as well as taste sensation, reward, cognition, learning and memory. One unique feature of ghrelin is its acylation, primarily with an octanoic acid, which is essential for its binding and activation of the ghrelin receptor, a G protein-coupled receptor. The multifaceted roles of ghrelin make ghrelin receptor a highly attractive drug target for growth retardation, obesity, and metabolic disorders. Here we present two cryo-electron microscopy structures of Gq-coupled ghrelin receptor bound to ghrelin and a synthetic agonist, GHRP-6. Analysis of these two structures reveals a unique binding pocket for the octanoyl group, which guides the correct positioning of the peptide to initiate the receptor activation. Together with mutational and functional data, our structures define the rules for recognition of the acylated peptide hormone and activation of ghrelin receptor, and provide structural templates to facilitate drug design targeting ghrelin receptor.


Assuntos
Oligopeptídeos/química , Receptores de Grelina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Microscopia Crioeletrônica , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Grelina/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Oligopeptídeos/metabolismo , Ligação Proteica , Receptores de Grelina/metabolismo , Receptores de Grelina/ultraestrutura
5.
Molecules ; 26(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34443366

RESUMO

The kappa opioid receptor (KOR) represents an attractive target for the development of drugs as potential antidepressants, anxiolytics and analgesics. A robust computational approach may guarantee a reduction in costs in the initial stages of drug discovery, novelty and accurate results. In this work, a virtual screening workflow of a library consisting of ~6 million molecules was set up, with the aim to find potential lead compounds that could manifest activity on the KOR. This in silico study provides a significant contribution in the identification of compounds capable of interacting with a specific molecular target. The main computational techniques adopted in this experimental work include: (i) virtual screening; (ii) drug design and leads optimization; (iii) molecular dynamics. The best hits are tripeptides prepared via solution phase peptide synthesis. These were tested in vivo, revealing a good antinociceptive effect after subcutaneous administration. However, further work is due to delineate their full pharmacological profile, in order to verify the features predicted by the in silico outcomes.


Assuntos
Simulação por Computador , Desenho de Fármacos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Receptores Opioides kappa/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Receptores Opioides kappa/química
6.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443374

RESUMO

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.


Assuntos
Neoplasias Encefálicas/patologia , Calcineurina/metabolismo , Glioma/patologia , Fatores de Transcrição NFATC/metabolismo , Oligopeptídeos/farmacologia , Transdução de Sinais , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Glioma/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fatores de Transcrição NFATC/química , Oligopeptídeos/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
7.
J Chem Phys ; 155(5): 055101, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34364337

RESUMO

Artificial amyloid-like nanofibers formed from short peptides are emerging as new supramolecular structures for catalysis and advanced materials. In this work, we analyze, by means of computational approaches, the preferred atomistic fibrillar architectures that result from the self-assembly of polar NY7, NF7, SY7, SF7, and GY7 peptides into steric zippers formed by two ß-sheets (describing an individual steric zipper) and by four ß-sheets. For all heptapeptides, except GY7, parallel ß-sheet organizations with polar residues packed at the steric zipper appear to be the preferred assemblies for the two ß-sheets system due to the formation of a strong network of hydrogen bonds. For GY7, however, an antiparallel organization with glycine at the steric zipper is the most stable one. The preferred architecture is mostly conserved when enlarging our model from two to four ß-sheets. The present work shows that the relative stability of different architectures results from a delicate balance between peptide composition, side chain hydrophobicity, and non-covalent interactions at the interface and provides the basis for a rational design of new improved artificial prion-inspired materials.


Assuntos
Proteínas Amiloidogênicas/química , Nanofibras/química , Oligopeptídeos/química , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Termodinâmica
8.
Phys Chem Chem Phys ; 23(33): 18001-18011, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34382985

RESUMO

A hydrophobic heptapeptide, with sequence AFILPTG, as part of a phage capsid protein binds effectively to silica particles carrying negative charge. Here, we explore the silica binding activity of the sequence as a short polypeptide with polar N and C terminals. To describe the structural changes that occur on binding, we fit experimental infrared, Raman and circular dichroism data for a number of structures simulated in the full configuration space of the hepta-peptide using replica exchange molecular dynamics. Quantum chemistry was used to compute normal modes of infrared and Raman spectra and establish a relationship to structures from MD data. To interpret the circular dichroism data, instead of empirical factoring of optical activity into helical/sheet/random components, we exploit natural transition orbital theory and specify the contributions of backbone amide units, side chain functional groups, water, sodium ions and silica to the observed transitions. Computed optical responses suggest a less folded backbone and importance of the N-terminal when close to silica. We further discuss the thermodynamics of the interplay of charged and hydrophobic moieties of the polypeptide on association with the silica surface. The outcomes of this study may assist in the engineering of novel artificial bio-silica heterostructures.


Assuntos
Oligopeptídeos/química , Dióxido de Silício/química , Teoria da Densidade Funcional , Interações Hidrofóbicas e Hidrofílicas , Nanopartículas/química , Desdobramento de Proteína , Dióxido de Silício/síntese química , Propriedades de Superfície
9.
Molecules ; 26(14)2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34299619

RESUMO

Radioiodine labeling of peptides and proteins is routinely performed by using various oxidizing agents such as Chloramine T, Iodobeads, and Iodogen reagent and radioactive iodide (I-), although some other oxidizing agents were also investigated. The main objective of the present study was to develop and test a novel reagent, inorganic monochloramine (NH2Cl), for radioiodine labeling of new chemical entities and biomolecules which is cost-effective, easy to make and handle, and is selective to label amino acids, peptides, and proteins. The data presented in this report demonstrate that the yields of the non-radioactive iodine labeling reactions using monochloramine are >70% for an amino acid (tyrosine) and a cyclic peptide (cyclo Arg-Gly-Asp-d-Tyr-Lys, cRGDyK). No evidence of the formation of N-chloro derivatives in cRGDyK was observed, suggesting that the reagent is selective in iodinating the tyrosine residue in the biomolecules. The method was successfully translated into radioiodine labeling of amino acid, a peptide, and a protein, Bovine Serum Albumin (BSA).


Assuntos
Radioisótopos do Iodo/química , Cloraminas/química , Marcação por Isótopo/métodos , Oligopeptídeos/química , Oxidantes/química , Peptídeos Cíclicos/química , Compostos Radiofarmacêuticos/química , Compostos de Tosil/química
10.
Biomolecules ; 11(7)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209793

RESUMO

The surface-enhanced Raman scattering (SERS) spectra of three amphiphilic oligopeptides derived from EAK16 (AEAEAKAK)2 were examined to study systematic amino acid substitution effects on the corresponding interaction with Ag colloidal nanoparticles. Such self-assembling molecular systems, known as "molecular Lego", are of particular interest for their uses in tissue engineering and as biomimetic coatings for medical devices because they can form insoluble macroscopic membranes under physiological conditions. Spectra were collected for both native and gamma-irradiated samples. Quantum mechanical data on two of the examined oligopeptides were also obtained to clarify the assignment of the prominent significative bands observed in the spectra. In general, the peptide-nanoparticles interaction occurs through the COO- groups, with the amide bond and the aliphatic chain close to the colloid surface. After gamma irradiation, mimicking a free oxidative radical attack, the SERS spectra of the biomaterials show that COO- groups still provide the main peptide-nanoparticle interactions. However, the spatial arrangement of the peptides is different, exhibiting a systematic decrease in the distance between aliphatic chains and colloid nanoparticles.


Assuntos
Materiais Biomiméticos/farmacologia , Oligopeptídeos/química , Análise Espectral Raman/métodos , Biomimética/métodos , Humanos , Nanopartículas/química , Oligopeptídeos/farmacologia , Peptídeos , Prata/química , Propriedades de Superfície
11.
Molecules ; 26(13)2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34279386

RESUMO

The selective inhibition of immunoproteasome is a valuable strategy to treat autoimmune, inflammatory diseases, and hematologic malignancies. Recently, a new series of amide derivatives as non-covalent inhibitors of the ß1i subunit with Ki values in the low/submicromolar ranges have been identified. Here, we investigated the binding mechanism of the most potent and selective inhibitor, N-benzyl-2-(2-oxopyridin-1(2H)-yl)propanamide (1), to elucidate the steps from the ligand entrance into the binding pocket to the ligand-induced conformational changes. We carried out a total of 400 ns of MD-binding analyses, followed by 200 ns of plain MD. The trajectories clustering allowed identifying three representative poses evidencing new key interactions with Phe31 and Lys33 together in a flipped orientation of a representative pose. Further, Binding Pose MetaDynamics (BPMD) studies were performed to evaluate the binding stability, comparing 1 with four other inhibitors of the ß1i subunit: N-benzyl-2-(2-oxopyridin-1(2H)-yl)acetamide (2), N-cyclohexyl-3-(2-oxopyridin-1(2H)-yl)propenamide (3), N-butyl-3-(2-oxopyridin-1(2H)-yl)propanamide (4), and (S)-2-(2-oxopyridin-1(2H)-yl)-N,4-diphenylbutanamide (5). The obtained results in terms of free binding energy were consistent with the experimental values of inhibition, confirming 1 as a lead compound of this series. The adopted methods provided a full dynamic description of the binding events, and the information obtained could be exploited for the rational design of new and more active inhibitors.


Assuntos
Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Sítios de Ligação , Dipeptídeos/química , Dipeptídeos/farmacologia , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Compostos de Organossilício/química , Compostos de Organossilício/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Ligação Proteica
12.
Molecules ; 26(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299591

RESUMO

A series of tripeptides TrpTrpPhe (1), TrpTrpTyr (2), and TrpTrpTyr[CH2N(CH3)2] (3) were synthesized, and their photophysical properties and non-covalent binding to polynucleotides were investigated. Fluorescent Trp residues (quantum yield in aqueous solvent ΦF = 0.03-0.06), allowed for the fluorometric study of non-covalent binding to DNA and RNA. Moreover, high and similar affinities of 2×HCl and 3×HCl to all studied double stranded (ds)-polynucleotides were found (logKa = 6.0-6.8). However, the fluorescence spectral responses were strongly dependent on base pair composition: the GC-containing polynucleotides efficiently quenched Trp emission, at variance to AT- or AU-polynucleotides, which induced bisignate response. Namely, addition of AT(U) polynucleotides at excess over studied peptide induced the quenching (attributed to aggregation in the grooves of polynucleotides), whereas at excess of DNA/RNA over peptide the fluorescence increase of Trp was observed. The thermal denaturation and circular dichroism (CD) experiments supported peptides binding within the grooves of polynucleotides. The photogenerated quinone methide (QM) reacts with nucleophiles giving adducts, as demonstrated by the photomethanolysis (quantum yield ΦR = 0.11-0.13). Furthermore, we have demonstrated photoalkylation of AT oligonucleotides by QM, at variance to previous reports describing the highest reactivity of QMs with the GC reach regions of polynucleotides. Our investigations show a proof of principle that QM precursor can be imbedded into a peptide and used as a photochemical switch to enable alkylation of polynucleotides, enabling further applications in chemistry and biology.


Assuntos
Indolquinonas/química , Oligopeptídeos/química , Processos Fotoquímicos , Poli A-U/química , Desaminação , Triptofano/química
13.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207233

RESUMO

Multiple sources of evidence suggest that soluble amyloid ß (Aß)-oligomers are responsible for the development and progression of Alzheimer's disease (AD). In order to specifically eliminate these toxic Aß-oligomers, our group has developed a variety of all-d-peptides over the past years. One of them, RD2, has been intensively studied and showed such convincing in vitro and in vivo properties that it is currently in clinical trials. In order to further optimize the compounds and to elucidate the characteristics of therapeutic d-peptides, several rational drug design approaches have been performed. Two of these d-peptides are the linear tandem (head-to-tail) d-peptide RD2D3 and its cyclized form cRD2D3. Tandemization and cyclization should result in an increased in vitro potency and increase pharmacokinetic properties, especially crossing the blood-brain-barrier. In comparison, cRD2D3 showed a superior pharmacokinetic profile to RD2D3. This fact suggests that higher efficacy can be achieved in vivo at equally administered concentrations. To prove this hypothesis, we first established the in vitro profile of both d-peptides here. Subsequently, we performed an intraperitoneal treatment study. This study failed to provide evidence that cRD2D3 is superior to RD2D3 in vivo as in some tests cRD2D3 failed to show equal or higher efficacy.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Oligopeptídeos/química , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinética
14.
Molecules ; 26(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34208805

RESUMO

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together In Vivo at neuromuscular junctions and in motor neurons.


Assuntos
Glicoproteínas de Membrana/química , Metaloendopeptidases/química , Fármacos Neuroprotetores/química , Oligopeptídeos/química , Receptor trkB/química , Toxina Tetânica/química , Animais , Cristalografia por Raios X , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Metaloendopeptidases/metabolismo , Metaloendopeptidases/farmacologia , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , Receptor trkB/metabolismo , Receptor trkB/farmacologia , Toxina Tetânica/metabolismo , Toxina Tetânica/farmacologia
15.
Nat Commun ; 12(1): 4047, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193851

RESUMO

The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains. NPCs contain several distinct FG domains, each comprising variable repeats. Nevertheless, we now found that sequence heterogeneity is no fundamental requirement for barrier function. Instead, we succeeded in engineering a perfectly repeated 12mer GLFG peptide that self-assembles into a barrier of exquisite transport selectivity and fast transport kinetics. This barrier recapitulates RanGTPase-controlled importin- and exportin-mediated cargo transport and thus represents an ultimately simplified experimental model system. An alternative proline-free sequence forms an amyloid FG phase. Finally, we discovered that FG phases stain bright with 'DNA-specific' DAPI/ Hoechst probes, and that such dyes allow for a photo-induced block of nuclear transport.


Assuntos
Núcleo Celular/metabolismo , Carioferinas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Oligopeptídeos/química , Engenharia de Proteínas/métodos , Transporte Ativo do Núcleo Celular , Fenômenos Biofísicos , Células Cultivadas , Sequência Consenso , Humanos , Cinética , Permeabilidade , Sequências Repetitivas de Aminoácidos
16.
Int J Mol Sci ; 22(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205705

RESUMO

From a previous collection of lipopeptides derived from BP100, we selected 18 sequences in order to improve their biological profile. In particular, analogues containing a D-amino acid at position 4 were designed, prepared, and tested against plant pathogenic bacteria and fungi. The biological activity of these sequences was compared with that of the corresponding parent lipopeptides with all L-amino acids. In addition, the influence of the length of the hydrophobic chain on the biological activity was evaluated. Interestingly, the incorporation of a D-amino acid into lipopeptides bearing a butanoyl or a hexanoyl chain led to less hemolytic sequences and, in general, that were as active or more active than the corresponding all L-lipopeptides. The best lipopeptides were BP475 and BP485, both incorporating a D-Phe at position 4 and a butanoyl group, with MIC values between 0.8 and 6.2 µM, low hemolysis (0 and 24% at 250 µM, respectively), and low phytotoxicity. Characterization by NMR of the secondary structure of BP475 revealed that the D-Phe at position 4 disrupts the α-helix and that residues 6 to 10 are able to fold in an α-helix. This secondary structure would be responsible for the high antimicrobial activity and low hemolysis of this lipopeptide.


Assuntos
Anti-Infecciosos/síntese química , Lipopeptídeos/síntese química , Testes de Sensibilidade Microbiana , Oligopeptídeos/química , Doenças das Plantas/terapia , Doenças das Plantas/microbiologia
17.
Int J Nanomedicine ; 16: 4495-4513, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34239301

RESUMO

Purpose: Hepatocellular carcinoma (HCC) is a common malignant tumor that seriously threatens human life and health. Currently, the majority of antitumor drugs are administered in an injectable manner, which can cause pain and side effects to patients. Objective of this study is to establish an effective oral drug delivery system for anti hepatoma drugs. Methods: In this study, intestinal targeting cell penetrating peptide (R6LRVG) was obtained by binding cell penetrating peptide (R6) with the polypeptide of LRVG (targeting intestinal epithelial cells). Next, R6LRVG-modified tyroserleutide-poly(lactic-co-glycolic acid) (PLGA) nanoparticles (YSL-PLGA/R6LRVG NPs) were prepared. After that, the nanoparticles were characterized and their stability was evaluated. The cellular uptake, in vitro bioactivity and in vivo antitumor activity of the nanoparticles were investigated. In addition, the mechanism, including the endocytic pathway and respiratory rate detection of mitochondria, was further investigated. Results: YSL-PLGA/R6LRVG NPs were successfully prepared. Characterization revealed YSL-PLGA/R6LRVG NPs to be globular particles with smooth surfaces and an average diameter of 222.6 nm. The entrapment efficiency and drug loading of tyroserleutide were 70.27% and 19.69%, respectively. Furthermore, the YSL-PLGA/R6LRVG NPs group exhibited the largest amount of YSL uptake. We also found that cell uptake of YSL-PLGA/R6LRVG NPs could be related to the endocytosis pathways mediated by reticulin and caveolae/lipid rafts. Additionally, the YSL-PLGA/R6LRVG NPs could interfere with mitochondrial function. In vivo experiments revealed that orally administered YSL-PLGA/R6LRVG NPs exerted excellent anticancer effects in tumor-bearing mice. Hematoxylin-eosin staining did not show any histological changes in the major organs. Conclusion: To summarize, YSL-PLGA/R6LRVG NPs could be a useful oral delivery system of YSL and may provide a new platform for the oral delivery of anticancer drugs.


Assuntos
Antineoplásicos/química , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas/química , Oligopeptídeos/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Humanos , Camundongos
18.
Bioconjug Chem ; 32(6): 1167-1174, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34060308

RESUMO

Multiple, site-specific protein conjugation is increasingly attractive for the generation of antibody-drug conjugates (ADCs). As it is important to control the number and position of cargoes in an ADC, position-selective generation of reactive sites in the protein of interest is required. Formylglycine (FGly) residues are generated by enzymatic conversion of cysteine residues embedded in a certain amino acid sequence motif with a formylglycine-generating enzyme (FGE). The addition of copper ions increases FGE activity leading to the conversion of cysteines within less readily accepted sequences. With this tuned enzyme activity, it is possible to address two different recognition sequences using two aerobic formylglycine-generating enzymes. We demonstrate an improved and facile strategy for the functionalization of a DARPin (designed ankyrin repeat protein) and the single-chain antibody scFv425-Fc, both directed against the epidermal growth factor receptor (EGFR). The single-chain antibody was conjugated with monomethyl auristatin E (MMAE) and carboxyfluorescein (CF) and successfully tested for receptor binding, internalization, and cytotoxicity in cell culture, respectively.


Assuntos
Enzimas/metabolismo , Glicina/análogos & derivados , Imunoconjugados/química , Imunoconjugados/metabolismo , Aerobiose , Repetição de Anquirina , Cobre/química , Fluoresceínas/química , Glicina/metabolismo , Oligopeptídeos/química
19.
Biomed Pharmacother ; 141: 111722, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34144455

RESUMO

Coronavirus disease 2019 is associated with clinical symptoms including severe inflammatory syndrome and a higher expression of angiotensin II. As a pro-inflammatory mediator, the physiologic effects of angiotensin II are mediated by a G-protein coupled receptor, termed AT1R. Following binding, AT1R initiates the process of signal desensitization necessary to maintain cellular homeostasis. At the cellular level, this function occurs via the G protein-dependent signaling and the phosphorylation. We describe amino acids similarities between SARS COV-2 nonstructural protein (NSP8) which is associated with intracellular membranes and AT1R key sites. Since abnormal activation of AT1R receptor leads to a number of physiological disorders, we hypothesize that SARS COV-2 might further interfere with the angiotensin II receptor functions.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/genética , Oligopeptídeos/genética , Receptor Tipo 1 de Angiotensina/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , COVID-19/genética , RNA-Polimerase RNA-Dependente de Coronavírus/química , Humanos , Oligopeptídeos/química , Receptor Tipo 1 de Angiotensina/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química
20.
Sci Rep ; 11(1): 13208, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168183

RESUMO

Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.


Assuntos
Antivirais/química , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Reposicionamento de Medicamentos , Oligopeptídeos/química , Linhagem Celular , Humanos , Serpinas/química , Proteínas Virais/química
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