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ADP-ribosylation is a highly dynamic posttranslational modification frequently studied in stress response pathways with recent attention given to its role in response to viral infection. Notably, the alphaviruses encode catalytically active macrodomains capable of ADP-ribosylhydrolase (ARH) activities, implying a role in remodeling the cellular ADP-ribosylome. This report decouples mono- and poly-ARH contributions to macrodomain function using a newly engineered Sindbis virus (SINV) mutant with attenuated poly-ARH activity. Our findings indicate that viral poly-ARH activity is uniquely required for high titer replication in mammalian systems. Despite translating incoming genomic RNA as efficiently as WT virus, mutant viruses have a reduced capacity to establish productive infection, offering a more complete understanding of the kinetics and role of the alphavirus macrodomain with important implications for broader ADP-ribosyltransferase biology. IMPORTANCE Viral macrodomains have drawn attention in recent years due to their high degree of conservation in several virus families (e.g., coronaviruses and alphaviruses) and their potential druggability. These domains erase mono- or poly-ADP-ribose, posttranslational modifications written by host poly-ADP-ribose polymerase (PARP) proteins, from undetermined host or viral proteins to enhance replication. Prior work determined that efficient alphavirus replication requires catalytically active macrodomains; however, which form of the modification requires removal and from which protein(s) had not been determined. Here, we present evidence for the specific requirement of poly-ARH activity to ensure efficient productive infection and virus replication.
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Coronavirus , Hidrolases , RNA Viral , Sindbis virus , Animais , Coronavirus/genética , Hidrolases/metabolismo , Mamíferos/genética , Poli Adenosina Difosfato Ribose/metabolismo , RNA Viral/genética , Sindbis virus/enzimologia , Sindbis virus/genética , Replicação ViralRESUMO
The cyclic GMP-AMP synthase (cGAS)-cGAMP-STING pathway plays a key role in innate immunity, with cGAS sensing both pathogenic and mislocalized DNA in the cytoplasm. Human cGAS (h-cGAS) constitutes an important drug target for control of antiinflammatory responses that can contribute to the onset of autoimmune diseases. Recent studies have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGASCD-DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGASCD-DNA complex, with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation, as monitored by cGAMP formation, gel shift, spin-down, and turbidity assays, as well as time-lapse imaging of liquid droplet formation. We expand on an earlier ladder model of cGAS dimers bound to a pair of parallel-aligned DNAs to propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGAS-DNA interface. We also report the crystal structure of the h-cGASCD-DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development at the DNA-bound h-cGAS level. Finally, we solved the structure of RU.521 bound in two alternate alignments to apo h-cGASCD, thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site-binding inhibitors.
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Domínio Catalítico/fisiologia , DNA/metabolismo , Nucleotidiltransferases/metabolismo , Sequência de Aminoácidos , Humanos , Imunidade Inata/fisiologia , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologiaRESUMO
Measurements of fluorescence intensity of the hydrophobic pyridinium salt (DTPSH) remaining in the organic phase after partition experiments in the DCM/H2O system allowed an approximate method to be developed to estimate the mean number of molecules (N = 942) on the surface of 22.8 nm gold nanoparticles and the separation (1.89 nm) between these organic molecules. This protocol is based on the ability that the organic molecules possess to coat the surface of the nanoparticle, which can migrate from the organic to the aqueous phase as a result of the driving force of the strong binding of sulfur to gold. To validate our estimation, we used a projection of the results obtained by Wales and Ulker to solve the Thomson problem, a mathematicians' challenge, used as a model to calculate the mean distance (1.82 nm) separating particles on the surface, in excellent agreement with the results obtained by our method. The quality of results, the simplicity of calculations, the low fluorescence detection limit, and the inexpensive materials, recommend this procedure for rapid estimates of the mean number of molecules on the surface of nanoparticles.
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We engineered and employed a chaperone-like amyloid-binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross-reacted with amyloid beta-peptide (Aß42) protofibrils, but not with Aß40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1-hIAPP complex cross-react with Aß42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation-specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation-sensitive and sequence-independent and can target more than one type of protofibril species.
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Peptídeos beta-Amiloides/imunologia , Amiloide/imunologia , Anticorpos Monoclonais/imunologia , Fragmentos de Peptídeos/imunologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Camundongos , Nucleobindinas/imunologia , Nucleobindinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Células Piramidais/imunologia , Células Piramidais/metabolismoRESUMO
During host infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress that impair protein integrity, including reactive oxygen and nitrogen species and chemotherapy. The resulting protein aggregates can be resolved or degraded by molecular machinery conserved from bacteria to eukaryotes. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is essential in Mycobacterium smegmatis, and ClpB is involved in asymmetrically distributing damaged proteins during cell division as a mechanism of survival in Mtb, commending both proteins as potential drug targets. However, their molecular partners in protein reactivation have not been characterized in mycobacteria. Here, we reconstituted the activities of the Mtb ClpB/DnaK bichaperone system with the cofactors DnaJ1, DnaJ2, and GrpE and the small heat shock protein Hsp20. We found that DnaJ1 and DnaJ2 activate the ATPase activity of DnaK differently. A point mutation in the highly conserved HPD motif of the DnaJ proteins abrogates their ability to activate DnaK, although the DnaJ2 mutant still binds to DnaK. The purified Mtb ClpB/DnaK system reactivated a heat-denatured model substrate, but the DnaJ HPD mutants inhibited the reaction. Finally, either DnaJ1 or DnaJ2 is required for mycobacterial viability, as is the DnaK-activating activity of a DnaJ protein. These studies lay the groundwork for strategies to target essential chaperone-protein interactions in Mtb, the leading cause of death from a bacterial infection.
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Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteostase , Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismoRESUMO
The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.
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Inibidores de Adenilil Ciclases/farmacologia , Adenilil Ciclases/metabolismo , Pirimidinas/farmacologia , Tiofenos/farmacologia , Inibidores de Adenilil Ciclases/química , Adenilil Ciclases/química , Regulação Alostérica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Pirimidinas/química , Solubilidade , Relação Estrutura-Atividade , Tiofenos/químicaRESUMO
Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia. Prior to the discovery of TDI-11055, existing inhibitors of ENL YEATS showed in vitro potency, but had not shown efficacy in in vivo animal models. During the course of the medicinal chemistry campaign described here, we identified ENL YEATS inhibitor TDI-11055 that has an improved pharmacokinetic profile and is appropriate for in vivo evaluation of the ENL YEATS inhibition mechanism in AML.
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OBJECTIVE: Thioesterase superfamily member 1 (Them1) is a long chain acyl-CoA thioesterase comprising two N-terminal HotDog fold enzymatic domains linked to a C-terminal lipid-sensing steroidogenic acute regulatory transfer-related (START) domain, which allosterically modulates enzymatic activity. Them1 is highly expressed in thermogenic adipose tissue, where it functions to suppress energy expenditure by limiting rates of fatty acid oxidation, and is induced markedly in liver in response to high fat feeding, where it suppresses fatty acid oxidation and promotes glucose production. Them1-/- mice are protected against non-alcoholic fatty liver disease (NAFLD), suggesting Them1 as a therapeutic target. METHODS: A high-throughput small molecule screen was performed to identify promising inhibitors targeting the fatty acyl-CoA thioesterase activity of purified recombinant Them1.Counter screening was used to determine specificity for Them1 relative to other acyl-CoA thioesterase isoforms. Inhibitor binding and enzyme inhibition were quantified by biophysical and biochemical approaches, respectively. Following structure-based optimization, lead compounds were tested in cell culture. RESULTS: Two lead allosteric inhibitors were identified that selectively inhibited Them1 by binding the START domain. In mouse brown adipocytes, these inhibitors promoted fatty acid oxidation, as evidenced by increased oxygen consumption rates. In mouse hepatocytes, they promoted fatty acid oxidation, but also reduced glucose production. CONCLUSION: Them1 inhibitors could prove attractive for the pharmacologic management of NAFLD.
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Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Ensaios de Triagem em Larga Escala , Glucose/metabolismo , Ácidos Graxos/metabolismoRESUMO
DnaK is the bacterial homolog of Hsp70, an ATP-dependent chaperone that helps cofactor proteins to catalyze nascent protein folding and salvage misfolded proteins. In the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), DnaK and its cofactors are proposed antimycobacterial targets, yet few small-molecule inhibitors or probes exist for these families of proteins. Here, we describe the repurposing of a drug called telaprevir that is able to allosterically inhibit the ATPase activity of DnaK and to prevent chaperone function by mimicking peptide substrates. In mycobacterial cells, telaprevir disrupts DnaK- and cofactor-mediated cellular proteostasis, resulting in enhanced efficacy of aminoglycoside antibiotics and reduced resistance to the frontline TB drug rifampin. Hence, this work contributes to a small but growing collection of protein chaperone inhibitors, and it demonstrates that these molecules disrupt bacterial mechanisms of survival in the presence of different antibiotic classes.
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Proteínas de Escherichia coli , Mycobacterium tuberculosis , Tuberculose , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/metabolismo , Dobramento de ProteínaRESUMO
INTRODUCTION: Gold nanorods are highly reactive, have a large surface-to-volume ratio, and can be functionalized with biomolecules. Gold nanorods can absorb infrared electromagnetic radiation, which is subsequently dispersed as local heat. Gold nanoparticles can be used as powerful tools for the diagnosis and therapy of different diseases. To improve the biological barrier permeation of nanoparticles with low cytotoxicity, in this study, we conjugated gold nanorods with cell-penetrating peptides (oligoarginines) and with the amphipathic peptide CLPFFD. METHODS: We studied the interaction of the functionalized gold nanorods with biological membrane models (liposomes) by dynamic light scattering, transmission electron microscopy and the Langmuir balance. Furthermore, we evaluated the effects on cell viability and permeability with an MTS assay and TEM. RESULTS AND DISCUSSION: The interaction study by DLS, the Langmuir balance and cryo-TEM support that GNR-Arg7CLPFFD enhances the interactions between GNRs and biological membranes. In addition, cells treated with GNR-Arg7CLPFFD internalized 80% more nanoparticles than cells treated with GNR alone and did not induce cell damage. CONCLUSION: Our results indicate that incorporation of an amphipathic sequence into oligoarginines for the functionalization of gold nanorods enhances biological membrane nanoparticle interactions and nanoparticle cell permeability with respect to nanorods functionalized with oligoarginine. Overall, functionalized gold nanorods with amphipathic arginine rich peptides might be candidates for improving drug delivery by facilitating biological barrier permeation.
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Peptídeos Penetradores de Células/química , Lipossomos/farmacocinética , Nanotubos/química , Arginina/química , Linhagem Celular Tumoral , Sobrevivência Celular , Peptídeos Penetradores de Células/farmacocinética , Sistemas de Liberação de Medicamentos , Difusão Dinâmica da Luz , Ouro/química , Humanos , Lipossomos/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Peptídeos/químicaRESUMO
Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.
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Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Células Cultivadas , Cristalografia por Raios X , DNA/imunologia , DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferons/imunologia , Interferons/metabolismo , Macrófagos , Modelos Moleculares , Nucleotídeos Cíclicos/imunologia , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/isolamento & purificação , Nucleotidiltransferases/metabolismo , Cultura Primária de Células , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08-2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI's oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.
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Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Experimental/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Pteridinas/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Flavinas , Perfilação da Expressão Gênica , Humanos , Leucemia Experimental/sangue , Leucemia Mieloide Aguda/sangue , Masculino , Camundongos , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pteridinas/uso terapêutico , RNA/metabolismo , Motivo de Reconhecimento de RNA/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Cyclic GMP-AMP synthase is essential for innate immunity against infection and cellular damage, serving as a sensor of DNA from pathogens or mislocalized self-DNA. Upon binding double-stranded DNA, cyclic GMP-AMP synthase synthesizes a cyclic dinucleotide that initiates an inflammatory cellular response. Mouse studies that recapitulate causative mutations in the autoimmune disease Aicardi-Goutières syndrome demonstrate that ablating the cyclic GMP-AMP synthase gene abolishes the deleterious phenotype. Here, we report the discovery of a class of cyclic GMP-AMP synthase inhibitors identified by a high-throughput screen. These compounds possess defined structure-activity relationships and we present crystal structures of cyclic GMP-AMP synthase, double-stranded DNA, and inhibitors within the enzymatic active site. We find that a chemically improved member, RU.521, is active and selective in cellular assays of cyclic GMP-AMP synthase-mediated signaling and reduces constitutive expression of interferon in macrophages from a mouse model of Aicardi-Goutières syndrome. RU.521 will be useful toward understanding the biological roles of cyclic GMP-AMP synthase and can serve as a molecular scaffold for development of future autoimmune therapies.Upon DNA binding cyclic GMP-AMP synthase (cGAS) produces a cyclic dinucleotide, which leads to the upregulation of inflammatory genes. Here the authors develop small molecule cGAS inhibitors, functionally characterize them and present the inhibitor and DNA bound cGAS crystal structures, which will facilitate drug development.
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Doenças Autoimunes/imunologia , Autoimunidade/efeitos dos fármacos , Benzofuranos/farmacologia , Inibidores Enzimáticos/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Doenças Autoimunes do Sistema Nervoso/imunologia , Autoimunidade/imunologia , DNA/metabolismo , Ensaios de Triagem em Larga Escala , Imunidade Inata/imunologia , Inflamação , Macrófagos/imunologia , Espectrometria de Massas , Camundongos , Malformações do Sistema Nervoso/imunologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
The previously published version of this Article contained errors in Fig. 6. In panel h the units of the x axis were incorrectly given as mM and should have been given as µM. Also, the IC50s for RU.365, RU.332 and RU.521 within panel h were incorrectly given as mM and should have been given as µM. These errors have been corrected in both the PDF and HTML versions of the Article.
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Gold nanorods (AuNRs) stabilized by cetyltrimethylammonium bromide (CTAB) were deposited onto crystals of α-cyclodextrin (α-CD) inclusion compounds (ICs) that contained octanethiol (OT) as guest molecules. The nanodecoration was produced specifically at the {001} crystal planes through interaction between the -SH groups of the ICs and the AuNRs.
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Ouro/química , Nanotubos/química , alfa-Ciclodextrinas/química , Cetrimônio , Compostos de Cetrimônio/química , Cristalização , Nanotubos/ultraestrutura , Compostos de Sulfidrila/químicaRESUMO
Gold nanorods used in therapy and diagnosis must be nontoxic and stable in biological media and should be specific for the target. The complete combination of these three factors has hindered the use of gold nanorods as carriers in biological and biomedical applications. In this study, we produced a conjugate of gold nanorods with the peptide CLPFFD that recognizes toxic ß-amyloid aggregates present in Alzheimer's disease, demonstrates colloidal stability, maintains plasmonic properties, and shows no effects on cell viability in the SH-SY5Y cell line. Furthermore, the irradiation of ß-amyloid in the presence of the conjugate with near-infrared region irradiation energy reduces the amyloidogenic process reducing also its cytotoxicity. The nanorods were synthesized following the seed-mediated method in cetyltrimethylammonium bromide (CTAB) and were conjugated with the N-terminal cysteine peptide, CLPFFD. The conjugate was exhaustively characterized using different techniques (Absorption spectroscopy, X-ray photoelectron spectroscopy, electron energy loss spectroscopy, and zeta potential). The effects on cell viability and cell penetration by transmission electron microscopy of the conjugate were evaluated. The chemisorption of the peptide on the surface of gold nanorods increases their stability and reduces their effects on cell viability.
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Ouro/química , Nanotubos , Peptídeos/química , Sobrevivência Celular , Microscopia Eletrônica de Transmissão e Varredura , Espectroscopia Fotoeletrônica , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
BACKGROUND & AIMS: Gold nanoparticles (GNPs) have promising applications for drug delivery as well as for the diagnosis and treatment of several pathologies, such as those related to the CNS. However, GNPs are retained in a number of organs, such as the liver and spleen. Owing to their negative charge and/or processes of opsonization, GNPs are retained by the reticuloendothelial system, thereby decreasing their delivery to the brain. It is therefore crucial to modify the nanoparticle surface in order to increase its lipophilicity and reduce its negative charge, thus achieving enhanced delivery to the brain. RESULTS: In this article, we have shown that conjugation of 12 nm GNPs with the amphipathic peptide CLPFFD increases the in vivo penetration of these particles to the rat brain. The C(GNP)-LPFFD conjugates showed a smaller negative charge and a greater hydrophobic character than citrate-capped GNPs of the same size. We administered intraperitoneal injections of citrate GNPs and C(GNP)-LPFFD in rats, and determined the gold content in the tissues by neutron activation. Compared with citrate GNPs, the C(GNP)-LPFFD conjugate improved the delivery to the brain, increasing the concentration of gold by fourfold, while simultaneously reducing its retention by the spleen 1 and 2 h after injection. At 24 h, the conjugate was partially cleared from the brain, and mainly accumulated in the liver. The C(GNP)-LPFFD did not alter the integrity of the blood-brain barrier, and had no effect on cell viability.