RESUMO
Glioblastoma (GB) is one of the most lethal types of neoplasms with unique anatomic, physiologic, and pathologic features that usually persist after exposure to standard therapeutic modalities. It is biologically aggressive, and the existence of the blood-brain barrier (BBB) limits the efficacy of standard therapies. In this work, we hypothesize the potential of surface-functionalized ultra-small nanostructured lipid carriers (usNLCs) with charge-switchable cell-penetrating peptides (CPPs) to overcome this biological barrier and improve targeted delivery to brain tumor tissues. The big question is: what is the potential of CPPs in directing nanoparticles toward brain tumor tissue? To answer this question, the usNLCs were functionalized with distinct biomolecules [five CPPs, c(RGDfK) and transferrin, Tf] through electrostatic interaction and its ability as a targeting approach to BBB (HBMEC) and glioma cells (U87 cells) evaluated in terms of physicochemical properties, cellular uptake, permeability in a 2D-BBB model, and tumor growth inhibition. Monte Carlo simulations elucidated CPP adsorption patterns. The permeability studies revealed that targeted usNLCs, especially usNLCsTf and usNLCsCPP4, exhibited an increased permeability coefficient compared to the non-targeted usNLCs. Functionalized usNLCs evidenced enhanced uptake in BBB cells, with smaller CPPs showing higher internalization (CPP1 and CPP2). Similarly, functionalized usNLCs exhibited more significant cytotoxicity in glioma cells, with specific CPPs promoting favorable internalization. Analysis of the endocytic pathway indicated that usNLCsCPPs were mainly internalized by direct translocation and caveolae-mediated endocytosis. Optimal usNLCs with dual targeting capabilities to both BBB and GB cells provide a promising therapeutic strategy for GB.
Assuntos
Barreira Hematoencefálica , Peptídeos Penetradores de Células , Glioblastoma , Nanopartículas , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Humanos , Nanopartículas/química , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Propriedades de Superfície , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Tamanho da Partícula , Eletricidade Estática , Método de Monte Carlo , Sobrevivência Celular/efeitos dos fármacos , Lipídeos/química , Sistemas de Liberação de Medicamentos , Portadores de Fármacos/químicaRESUMO
Mucus is a complex biological hydrogel that acts as a barrier for almost everything entering or exiting the body. It is therefore of emerging interest for biomedical and pharmaceutical applications. Besides water, the most abundant components are the large and densely glycosylated mucins, glycoproteins of up to 20 MDa and carbohydrate content of up to 80 wt%. Here, we designed and explored a library of glycosylated peptides to deconstruct the complexity of mucus. Using the well-characterized hFF03 coiled-coil system as a hydrogel-forming peptide scaffold, we systematically probed the contribution of single glycans to the secondary structure as well as the formation and viscoelastic properties of the resulting hydrogels. We show that glycan-decoration does not affect α-helix and coiled-coil formation while it alters gel stiffness. By using oscillatory macrorheology, dynamic light scattering microrheology, and fluorescence lifetime-based nanorheology, we characterized the glycopeptide materials over several length scales. Molecular simulations revealed that the glycosylated linker may extend into the solvent, but more frequently interacts with the peptide, thereby likely modifying the stability of the self-assembled fibers. This systematic study highlights the interplay between glycan structure and hydrogel properties and may guide the development of synthetic mucus mimetics.
Assuntos
Glicopeptídeos , Hidrogéis , Polissacarídeos , Hidrogéis/química , Glicopeptídeos/química , Polissacarídeos/química , Elasticidade , Viscosidade , Simulação de Dinâmica Molecular , ReologiaRESUMO
Decellularized extracellular matrix (dECM) is an excellent natural source for 3D bioprinting materials due to its inherent cell compatibility. In vat photopolymerization, the use of dECM-based bioresins is just emerging, and extensive research is needed to fully exploit their potential. In this study, two distinct methacryloyl-functionalized, photocrosslinkable dECM-based bioresins were prepared from digested porcine liver dECM through functionalization with glycidyl methacrylate (GMA) or conventional methacrylic anhydride (MA) under mild conditions for systematic comparison. Although the chemical modifications did not significantly affect the structural integrity of the dECM proteins, mammalian cells encapsulated in the respective hydrogels performed differently in long-term culture. In either case, photocrosslinking during 3D (bio)printing resulted in transparent, highly swollen, and soft hydrogels with good shape fidelity, excellent biomimetic properties and tunable mechanical properties (~ 0.2-2.5 kPa). Interestingly, at a similar degree of functionalization (DOF ~ 81.5-83.5 %), the dECM-GMA resin showed faster photocrosslinking kinetics in photorheology resulting in lower final stiffness and faster enzymatic biodegradation compared to the dECM-MA gels, yet comparable network homogeneity as assessed via Brillouin imaging. While human hepatic HepaRG cells exhibited comparable cell viability directly after 3D bioprinting within both materials, cell proliferation and spreading were clearly enhanced in the softer dECM-GMA hydrogels at a comparable degree of crosslinking. These differences were attributed to the additional hydrophilicity introduced to dECM via methacryloylation through GMA compared to MA. Due to its excellent printability and cytocompatibility, the functional porcine liver dECM-GMA biomaterial enables the advanced biofabrication of soft 3D tissue analogs using vat photopolymerization-based bioprinting.
Assuntos
Matriz Extracelular , Hidrogéis , Metacrilatos , Polimerização , Animais , Metacrilatos/química , Suínos , Hidrogéis/química , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fígado , Humanos , Impressão Tridimensional , Processos Fotoquímicos , Bioimpressão/métodos , Materiais Biocompatíveis/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Reagentes de Ligações Cruzadas/química , Compostos de Epóxi/químicaRESUMO
Biocompatible and functionalizable hydrogels have a wide range of (potential) medicinal applications. The hydrogelation process, particularly for systems with very low polymer weight percentages (<1 wt %), remains poorly understood, making it challenging to predict the self-assembly of a given molecular building block into a hydrogel. This severely hinders the rational design of self-assembled hydrogels. In this study, we demonstrate the impact of an N-terminal group on the self-assembly and rheology of the peptide hydrogel hFF03 (hydrogelating, fibril forming peptide 03) using molecular dynamics simulations, oscillatory shear rheology, and circular dichroism spectroscopy. We find that the chromophore and even its specific regioisomers have a significant influence on the microscopic structure and dynamics of the self-assembled fibril, and on the macroscopic mechanical properties. This is because the chromophore influences the possible salt bridges, which form and stabilize the fibril formation. Furthermore, we find that the solvation shell fibrils by itself cannot explain the viscoelasticity of hFF03 hydrogels. Our atomistic model of the hFF03 fibril formation enables a more rational design of these hydrogels. In particular, altering the N-terminal chromophore emerges as a design strategy to tune the mechanic properties of these self-assembled peptide hydrogels.
Assuntos
Hidrogéis , Peptídeos , Hidrogéis/química , Peptídeos/química , Polímeros , ReologiaRESUMO
This study explores the effectiveness of the antineoplastic agent 5-FU in cancer cells by leveraging the unique properties of cationic antimicrobial peptides (CAMPs) and cell-penetrating peptides (CPPs). Traditional anticancer therapies face substantial limitations, including unfavorable pharmacokinetic profiles and inadequate specificity for tumor sites. These drawbacks often necessitate higher therapeutic agent doses, leading to severe toxicity in normal cells and adverse side effects. Peptides have emerged as promising carriers for targeted drug delivery, with their ability to selectively deliver therapeutics to cells expressing specific receptors. This enhances intracellular drug delivery, minimizes drug resistance, and reduces toxicity. In this research, we comprehensively evaluate the ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of various AMPs and CPPs to gain insights into their potential as anticancer agents. The peptide synthesis involved a solid-phase synthesis using a Liberty Microwave Peptide Synthesizer. The peptide purity was confirmed via LC-MS and HPLC methods. For the ADMET screening, computational tools were employed, assessing parameters like absorption, distribution, metabolism, excretion, and toxicity. The cell lines A549 and UM-UC-5 were cultured and treated with 5-FU, CAMPs, and CPPs. The cell viability was measured using the MTT assay. The physicochemical properties analysis revealed favorable drug-likeness attributes. The peptides exhibited potential inhibitory activity against CYP3A4. The ADMET predictions indicated variable absorption and distribution characteristics. Furthermore, we assessed the effectiveness of these peptides alone and in combination with 5-FU, a widely used antineoplastic agent, in two distinct cancer cell lines, UM-UC-5 and A549. Our findings indicate that CAMPs can significantly reduce the cell viability in A549 cells, while CPPs exhibit promising results in UM-UC-5 cells. Understanding these multifaceted effects could open new avenues for antiviral and anticancer research. Further, experimental validation is necessary to confirm the mechanism of action of these peptides, especially in combination with 5-FU.
RESUMO
Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.
Assuntos
Matriz Extracelular , Papaína , Ratos , Suínos , Humanos , Animais , Matriz Extracelular/química , Papaína/metabolismo , Matriz Extracelular Descelularizada , Pepsina A/análise , Pepsina A/metabolismo , Pepsina A/farmacologia , Proteômica , Hidrogéis/química , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.
Assuntos
Neuropeptídeos , Transdução de Sinais , Ligantes , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Neuropeptídeos/metabolismo , PeptídeosRESUMO
Phytochromes are bistable red/far-red light-responsive photoreceptor proteins found in plants, fungi, and bacteria. Light-activation of the prototypical phytochrome Cph1 from the cyanobacterium Synechocystis sp. PCC 6803 allows photoisomerization of the bilin chromophore in the photosensory module and a subsequent series of intermediate states leading from the red absorbing Pr to the far-red-absorbing Pfr state. We show here via osmotic and hydrostatic pressure-based measurements that hydration of the photoreceptor modulates the photoconversion kinetics in a controlled manner. While small osmolytes like sucrose accelerate Pfr formation, large polymer osmolytes like PEG 4000 delay the formation of Pfr. Thus, we hypothesize that an influx of mobile water into the photosensory domain is necessary for proceeding to the Pfr state. We suggest that protein hydration changes are a molecular event that occurs during photoconversion to Pfr, in addition to light activation, ultrafast electric field changes, photoisomerization, proton release and uptake, and the major conformational change leading to signal transmission, or simultaneously with one of these events. Moreover, we discuss this finding in light of the use of Cph1-PGP as a hydration sensor, e.g., for the characterization of novel hydrogel biomaterials.
Assuntos
Materiais Biocompatíveis , Fitocromo , Osmose , Transporte Biológico , EletricidadeRESUMO
The α-helical coiled coil (CC) is one of the best-characterized folding motifs in the protein world. In this context, fluorinated amino acids have been shown to be capable of tuning the properties of CC assemblies, and especially fluorinated derivatives of aliphatic amino acids can significantly increase the stability of this folding motif when placed in the hydrophobic a and d positions. However, it has not been shown yet whether fluorinated amino acids, by means of rational design, can be used as an orthogonal tool to control CC assembly processes. In the current work, we approached this question by creating a combinatorial peptide library based on a VPE/VPK heteromeric CC system previously established and characterized in our group. This CC model allowed us to screen fluorinated amino acids for interaction with different potential binding partners in position a of the VPE/VPK model with a particular emphasis on studying the impact of stereochemistry within the side chain of α-branched aliphatic fluorinated amino acids on CC properties such as oligomerization state, thermodynamic stability, and orientation. 28 combinations of library members were characterized regarding structure, oligomerization, and thermal stability utilizing circular dichroism, size exclusion chromatography, and Förster resonance energy transfer measurements. This detailed approach showed that the stability and oligomerization state of the motif were not only dependent on the steric demand and the fluorination of corresponding amino acids but also on the stereochemistry within the side chain. The results were applied for a rational design of the fluorine-driven orthogonal assembly, and we could show that CC dimer formation occurred based on specific interactions between fluorinated amino acids. These results demonstrate the potential of fluorinated amino acids as an orthogonal tool besides classical electrostatic and hydrophobic interactions for the fine-tuning and direction of peptide-peptide interactions. Furthermore, within the space of fluorinated amino acids, we could demonstrate the specificity of interactions between differently fluorinated side chains.
Assuntos
Aminoácidos , Flúor , Aminoácidos/química , Flúor/química , Sequência de Aminoácidos , Proteínas/química , Peptídeos/química , Dicroísmo CircularRESUMO
Three peptides comprising mono-, di-, and tri-fluoroethylglycine (MfeGly, DfeGly, and TfeGly) residues alternating with lysine were digested by readily available proteases (elastase, bromelain, trypsin, and proteinase K). The degree of degradation depended on the enzyme employed and the extent of fluorination. Incubation of the peptides with a microbial consortium from garden soil resulted in degradation, yielding fluoride ions. Further biodegradation studies conducted with the individual fluorinated amino acids demonstrated that the degree of defluorination followed the sequence MfeGly > DfeGly > TfeGly. Enrichment of the soil bacteria employing MfeGly as a sole carbon and energy source resulted in the isolation of a bacterium, which was identified as Serratia liquefaciens. Cell-free extracts of this bacterium enzymatically defluorinated MfeGly, yielding fluoride ion and homoserine. In silico analysis of the genome revealed the presence of a gene that putatively codes for a dehalogenase. However, the low overall homology to known enzymes suggests a potentially new hydrolase that can degrade monofluorinated compounds. 19F NMR analysis of aqueous soil extracts revealed the unexpected presence of trifluoroacetate, fluoride ion, and fluoroacetate. Growth of the soil consortium in tryptone soya broth supplemented with fluoride ions resulted in fluoroacetate production; thus, bacteria in the soil produce and degrade organofluorine compounds.
Assuntos
Bactérias , Fluoretos , Fluoretos/análise , Fluoretos/metabolismo , Bactérias/genética , Fluoracetatos/análise , Fluoracetatos/metabolismo , Peptídeos/metabolismo , Biodegradação AmbientalRESUMO
A de novo designed class of peptide-based fluoropolymers composed of fluorinated aliphatic amino acids as main components is reported. Structural characterization provided insights into fluorine-induced alterations on ß-strand to α-helix transition upon an increase in SDS content and revealed the unique formation of PPII structures for trifluorinated fluoropeptides. A combination of circular dichroism, fluorescence-based leaking assays and surface enhanced infrared absorption spectroscopy served to examine the insertion and folding processes into unilamellar vesicles. While partitioning into lipid bilayers, the degree of fluorination conducts a decrease in α-helical content. Furthermore, this study comprises a report on the proteolytic stability of peptides exclusively built up by fluorinated amino acids and proved all sequences to be enzymatically degradable despite the degree of fluorination. Herein presented fluoropeptides as well as the distinctive properties of these artificial and polyfluorinated foldamers with enzyme-degradable features will play a crucial role in the future development of fluorinated peptide-based biomaterials.
Assuntos
Aminoácidos , Peptídeos , Peptídeos/química , Aminoácidos/química , Peptídeo Hidrolases , Bicamadas Lipídicas/química , Proteólise , Dicroísmo Circular , Dobramento de ProteínaRESUMO
Structural waters in the S1 binding pocket of ß-trypsin are critical for the stabilization of the complex of ß-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific noncovalent interactions with the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly-BPTI-ß-trypsin complex (pdb: 7PH1). Both experimental and computational data show a stepwise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regard to energetic properties that could explain the increase in inhibitor strength. The analysis of the energy and the entropy of the water molecules in the S1 binding pocket using grid inhomogeneous solvation theory confirms this result. Overall, fluorination systematically changes the binding affinity, but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly.
Assuntos
Aprotinina , Água , Tripsina , AminoácidosRESUMO
Synthetic multichromophore systems are of great importance in artificial light harvesting devices, organic optoelectronics, tumor imaging and therapy. Here, we introduce a promising strategy for the construction of self-assembled peptide templated dye stacks based on coupling of a de novo designed pH sensitive peptide with a cyanine dye Cy5 at its N-terminus. Microscopic techniques, in particular cryogenic TEM (cryo-TEM) and cryo-electron tomography technique (cryo-ET), reveal two types of highly ordered three-dimensional assembly structures on the micrometer scale. Unbranched compact layered rods are observed at pHâ 7.4 and two-dimensional membrane-like assemblies at pHâ 3.4, both species displaying spectral features of H-aggregates. Molecular dynamics simulations reveal that the coupling of Cy5 moieties promotes the formation of both ultrastructures, whereas the protonation states of acidic and basic amino acid side chains dictates their ultimate three-dimensional organization.
Assuntos
Corantes , Peptídeos , Carbocianinas/química , Corantes/química , Peptídeos/química , Concentração de Íons de HidrogênioRESUMO
Fluorinated amino acids play an important role in the field of peptide and protein engineering. Although numerous syntheses have been published in recent decades, strategies that allow routine access to fluorinated amino acids on a gram-scale have been poorly described. Furthermore, the described pathways that gain fluorinated amino acids are based on different synthetic strategies, making a uniform approach that uses similar starting materials highly beneficial. Chiral Ni(II) complexes were introduced as powerful tools in the synthesis of noncanonical amino acids. In this work, we present a strategy for the synthesis of a diverse range of fluorinated amino acids based on the corresponding Ni(II) complex from which the products can be obtained in enantiopure form (99% ee) on a gram-scale. In addition, we describe an optimized procedure for the synthesis of alkyl iodide building blocks that are required for the alkylation reactions with the corresponding Ni(II) complex. Finally, we characterized the synthesized fluorinated amino acids with regard to their hydrophobicity and α-helix propensity.
Assuntos
Aminoácidos , Níquel , Alquilação , Aminoácidos/química , Níquel/química , EstereoisomerismoRESUMO
Advanced peptide-based nanomaterials composed of self-assembling peptides (SAPs) are of emerging interest in pharmaceutical and biomedical applications. The introduction of fluorine into peptides, in fact, offers unique opportunities to tune their biophysical properties and intermolecular interactions. In particular, the degree of fluorination plays a crucial role in peptide engineering as it can be used to control the characteristics of fluorine-specific interactions and, thus, peptide conformation and self-assembly. Here, we designed and explored a series of amphipathic peptides by incorporating the fluorinated amino acids (2S)-4-monofluoroethylglycine (MfeGly), (2S)-4,4-difluoroethylglycine (DfeGly) and (2S)-4,4,4-trifluoroethylglycine (TfeGly) as hydrophobic components. This approach enabled studying the impact of fluorination on secondary structure formation and peptide self-assembly on a systematic basis. We show that the interplay between polarity and hydrophobicity, both induced differentially by varying degrees of side chain fluorination, does affect peptide folding significantly. A greater degree of fluorination promotes peptide fibrillation and subsequent formation of physical hydrogels in physiological conditions. Molecular simulations revealed the key role played by electrostatically driven intra-chain and inter-chain contact pairs that are modulated by side chain fluorination and give insights into the different self-organization behaviour of selected peptides. Our study provides a systematic report about the distinct features of fluorinated oligomeric peptides with potential applications as peptide-based biomaterials.
Assuntos
Flúor , Hidrogéis , Flúor/química , Hidrogéis/química , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Estrutura Secundária de ProteínaRESUMO
Substituting the P1 position in bovine pancreatic trypsin inhibitor (BPTI) is known to heavily influence its inhibitory activity towards serine proteases. Side-chain fluorinated aliphatic amino acids have been shown to alter numerous properties of peptides and proteins and thus are of interest in the context of BPTI. In our study, we systematically investigated the site-specific incorporation of non-canonical amino acids into BPTI by microwave-assisted solid-phase peptide synthesis (SPPS). Inhibitor activity of the variants was tested towards the serine protease α-chymotrypsin. We observed enhanced inhibition of two fluorinated BPTIs compared to wild type and hydrocarbon variants. To further investigate the complexes, we performed X-ray structure analysis. Our findings underline the power fluorine offers as a tool in protein engineering to beneficially alter the effects on phenomena as protein-protein interactions.
RESUMO
The pH low insertion peptide (pHLIP) is a pH-sensitive cell penetrating peptide that transforms from an unstructured coil on the membrane surface at pH > 7, to a transmembrane (TM) α-helix at pH < 5. By exploiting this unique property, pHLIP attracts interest as a potential tool for drug delivery and visualisation of acidic tissues produced by various maladies such as cancer, inflammation, hypoxia etc. Even though the structures of initial and end states of pHLIP insertion have been widely accepted, the intermediate structures in between these two states are less clear. Here, we have applied in situ Surface-Enhanced Infrared Absorption spectroscopy to examine the pH-induced insertion and folding processes of pHLIP into a solid-supported lipid bilayer. We show that formation of partially helical structure already takes place at pH only slightly below 7.0, but with the helical axis parallel to the membrane surface. The peptide starts to reorientate its helix from horizontal to vertical direction, accompanied by the insertion into the TM region at pH < 6.2. Further insertion into the TM region of the peptide results in an increase of inherent α-helical structure and complete secondary structure formation at pH 5.3. Analysis of the changes of the carboxylate vibrational bands upon pH titration shows two distinctive groups of aspartates and glutamates with pKa values of 4.5 and 6.3, respectively. Comparison to the amide bands of the peptide backbone suggests that the latter Asp/Glu groups are directly involved in the conformational changes of pHLIP in the respective intermediate states.
Assuntos
Peptídeos Penetradores de Células , Bicamadas Lipídicas , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Conformação Proteica em alfa-HéliceRESUMO
With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp41 do Envelope de HIV/antagonistas & inibidores , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Peptídeos/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/química , Infecções por HIV/metabolismo , HIV-1/metabolismo , Halogenação , Humanos , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Peptídeos/químicaRESUMO
Organofluorine compounds are known to be toxic to a broad variety of living beings in different habitats, and chemical fluorination has been historically exploited by mankind for the development of therapeutic drugs or agricultural pesticides. On the other hand, several studies so far have demonstrated that, under appropriate conditions, living systems (in particular bacteria) can tolerate the presence of fluorinated molecules (e.g., amino acids analogues) within their metabolism and even repurpose them as alternative building blocks for the synthesis of cellular macromolecules such as proteins. Understanding the molecular mechanism behind these phenomena would greatly advance approaches to the biotechnological synthesis of recombinant proteins and peptide drugs. However, information about the metabolic effects of long-term exposure of living cells to fluorinated amino acids remains scarce. Hereby, we report the long-term propagation of Escherichia coli (E. coli) in an artificially fluorinated habitat that yielded two strains naturally adapted to live on fluorinated amino acids. In particular, we applied selective pressure to force a tryptophan (Trp)-auxotrophic strain to use either 4- or 5-fluoroindole as essential precursors for the in situ synthesis of Trp analogues, followed by their incorporation in the cellular proteome. We found that full adaptation to both fluorinated Trp analogues requires a low number of genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity, and quality control of protein folding. These findings highlight the cellular mechanisms behind the adaptation to unnatural amino acids and provide the molecular foundation for bioengineering of novel microbial strains for synthetic biology and biotechnology.