Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Pharm Sci ; 2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32980392

RESUMO

New classes of therapeutic peptides are being developed to prosecute biological targets which have been inaccessible to other modalities. Higher potency and longer half-life peptides have given rise to multiuse injectable formulations that enable convenient, low volume, and self-administered dosing; however, inclusion of antimicrobial preservatives to meet bactericidal requirements can impact other attributes of peptide formulations. Peptide-preservative interactions influencing solution-phase self-association of a non-insulin, linear, palmitoylated 31 amino acid peptide and two structurally similar peptides were assessed via turbidity, intrinsic fluorescence shifts and quenching, isothermal titration calorimetry, and 1H NMR. Meta-cresol and phenol specifically interact with the peptide, result in increased hydrophobicity near the tryptophan residue, and induce conformational changes, while benzyl alcohol does not impact tryptophan fluorescence, demonstrate any interaction enthalpy, or induce conformational changes. These same trends did not hold true for the other palmitoylated peptides evaluated, reinforcing the impacts of unique peptide sequences. Importantly, the presence of benzyl alcohol does increase the physical stability and solubility of the linear, 31 amino acid peptide under salt stress. We report new insights into the physical interactions of peptides with antimicrobial excipients, demonstrating a reversible association phenomenon and highlighting practical implications for formulation design and excipient selection.

2.
J Biomed Nanotechnol ; 16(4): 419-431, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32970975

RESUMO

Many promising pharmaceutically active compounds have low solubility in aqueous environments and their encapsulation into efficient drug delivery vehicles is crucial to increase their bioavailability. Lipodisq nanoparticles are approximately 10 nm in diameter and consist of a circular phospholipid bilayer, stabilized by an annulus of SMA (a hydrolysed copolymer of styrene and maleic anhydride). SMA is used extensively in structural biology to extract and stabilize integral membrane proteins for biophysical studies. Here, we assess the potential of these nanoparticles as drug delivery vehicles, determining their cytotoxicity and the in vivo excretion pathways of their polymer and lipid components. Doxorubicin-loaded Lipodisqs were cytotoxic across a panel of cancer cell lines, whereas nanoparticles without the drug had no effect on cell proliferation. Intracellular doxorubicin release from Lipodisqs in HeLa cells occurred in the low-pH environment of the endolysosomal system, consistent with the breakdown of the discoidal structure as the carboxylate groups of the SMA polymer become protonated. Biodistribution studies in mice showed that, unlike other nanoparticles injected intravenously, most of the Lipodisq components were recovered in the colon, consistent with rapid uptake by hepatocytes and excretion into bile. These data suggest that Lipodisqs have the potential to act as delivery vehicles for drugs and contrast agents.


Assuntos
Nanopartículas , Distribuição Tecidual , Animais , Linhagem Celular Tumoral , Doxorrubicina/toxicidade , Células HeLa , Humanos , Maleatos/toxicidade , Camundongos , Nanopartículas/toxicidade
3.
Sci Adv ; 6(33): eaav8207, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32851152

RESUMO

G protein-coupled receptors (GPCRs) are the largest and pharmaceutically most important class of membrane proteins encoded in the human genome, characterized by a seven-transmembrane helix architecture and a C-terminal amphipathic helix 8 (H8). In a minority of GPCR structures solved to date, H8 either is absent or adopts an unusual conformation. The controversial existence of H8 of the class A GPCR neurotensin receptor 1 (NTS1) has been examined here for the nonthermostabilized receptor in a functionally supporting membrane environment using electron paramagnetic resonance, molecular dynamics simulations, and circular dichroism. Lipid-protein interactions with phosphatidylserine and phosphatidylethanolamine lipids, in particular, stabilize the residues 374 to 390 of NTS1 into forming a helix. Furthermore, introduction of a helix-breaking proline residue in H8 elicited an increase in ß-arrestin-NTS1 interactions observed in pull-down assays, suggesting that the structure and/or dynamics of H8 might play an important role in GPCR signaling.

4.
Nat Biotechnol ; 37(10): 1186-1197, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31427820

RESUMO

The clinical translation of cationic α-helical antimicrobial peptides (AMPs) has been hindered by structural instability, proteolytic degradation and in vivo toxicity from nonspecific membrane lysis. Although analyses of hydrophobic content and charge distribution have informed the design of synthetic AMPs with increased potency and reduced in vitro hemolysis, nonspecific membrane toxicity in vivo continues to impede AMP drug development. Here, we analyzed a 58-member library of stapled AMPs (StAMPs) based on magainin II and applied the insights from structure-function-toxicity measurements to devise an algorithm for the design of stable, protease-resistant, potent and nontoxic StAMP prototypes. We show that a lead double-stapled StAMP named Mag(i+4)1,15(A9K,B21A,N22K,S23K) can kill multidrug-resistant Gram-negative pathogens, such as colistin-resistant Acinetobacter baumannii in a mouse peritonitis-sepsis model, without observed hemolysis or renal injury in murine toxicity studies. Inputting the amino acid sequences alone, we further generated membrane-selective StAMPs of pleurocidin, CAP18 and esculentin, highlighting the generalizability of our design platform.


Assuntos
Peptídeos Catiônicos Antimicrobianos/síntese química , Bactérias/efeitos dos fármacos , Peritonite/tratamento farmacológico , Sepse/tratamento farmacológico , Animais , Antibacterianos , Linhagem Celular , Desenho de Fármacos , Farmacorresistência Bacteriana , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/microbiologia , Sepse/microbiologia
5.
Chem Sci ; 8(2): 1105-1115, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-28451250

RESUMO

The spread of bacterial resistance to traditional antibiotics continues to stimulate the search for alternative antimicrobial strategies. All forms of life, from bacteria to humans, are postulated to rely on a fundamental host defense mechanism, which exploits the formation of open pores in microbial phospholipid bilayers. Here we predict that transmembrane poration is not necessary for antimicrobial activity and reveal a distinct poration mechanism that targets the outer leaflet of phospholipid bilayers. Using a combination of molecular-scale and real-time imaging, spectroscopy and spectrometry approaches, we introduce a structural motif with a universal insertion mode in reconstituted membranes and live bacteria. We demonstrate that this motif rapidly assembles into monolayer pits that coalesce during progressive membrane exfoliation, leading to bacterial cell death within minutes. The findings offer a new physical basis for designing effective antibiotics.

6.
Adv Drug Deliv Rev ; 112: 106-122, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28153578

RESUMO

There is an increased incidence of diabetes worldwide. The discovery of insulin revolutionized the management of diabetes, the revelation of glucagon-like peptide-1 (GLP-1) and introduction of GLP-1 receptor agonists to clinical practice was another breakthrough. Continued translational research resulted in better understanding of diabetes, which, in combination with cutting-edge biology, chemistry, and pharmaceutical tools, have allowed for the development of safer, more effective and convenient insulins and GLP-1. Advances in self-administration of insulin and GLP-1 receptor agonist therapies with use of drug-device combination products have further improved the outcomes of diabetes management and quality of life for diabetic patients. The synergies of insulin and GLP-1 receptor agonist actions have led to development of devices that can deliver both molecules simultaneously. New chimeric GLP-1-incretins and insulin-GLP-1-incretin molecules are also being developed. The objective of this review is to summarize molecular designs to improve the drug-like properties of insulin and GLP-1 and to highlight the continued advancement of drug-device combination products to improve diabetes management.


Assuntos
Diabetes Mellitus Tipo 2/terapia , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Hipoglicemiantes/uso terapêutico , Insulina/química , Insulina/farmacologia , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Composição de Medicamentos , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Humanos , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem
7.
Bioconjug Chem ; 21(3): 445-55, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-20131756

RESUMO

Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within submicrometer-sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes. In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging 30-50 nm were created, with minimal dependence of complex size on N/P ratio (PEI amines to DNA phosphates). Furthermore, PEI-DNA interactions were altered by the derivatization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison to PEI-DNA complexes. Transfection experiments in Chinese hamster ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI-DP3 complexes relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery.


Assuntos
DNA/química , Ácidos Nucleicos Peptídicos/síntese química , Transfecção/métodos , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , DNA/genética , Tamanho da Partícula , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacologia , Plasmídeos/química , Plasmídeos/genética , Polietilenoglicóis/química , Polietilenoimina/química , Polietilenoimina/farmacologia , Relação Estrutura-Atividade , Propriedades de Superfície
8.
J Med Chem ; 49(11): 3060-3, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16722624

RESUMO

Alendronate derivatives were evaluated as potential prodrugs for the osteoporosis drug alendronate sodium in an attempt to enhance the systemic exposure after oral administration. An investigation of the chemical behavior of alendronate derivatives led to development of practical synthetic strategies and prediction of each structural class's prodrug potential. Pharmacokinetic studies of N-myristoylalendronic acid revealed that 25% have been converted in vivo after i.v. administration in rat, providing an important proof-of-concept for this strategy.


Assuntos
Alendronato/análogos & derivados , Alendronato/síntese química , Conservadores da Densidade Óssea/síntese química , Pró-Fármacos/síntese química , Alendronato/farmacocinética , Animais , Densidade Óssea , Conservadores da Densidade Óssea/farmacocinética , Osteoporose/tratamento farmacológico , Pró-Fármacos/farmacocinética , Ratos , Relação Estrutura-Atividade
9.
Biochemistry ; 42(7): 1900-9, 2003 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-12590576

RESUMO

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) crystallizes from solutions containing the intermediate analogue neplanocin A with the analogue bound in its 3'-keto form at the active sites of all of its four subunits and the four tightly bound cofactors in their reduced (NADH) state. The enzyme is in the closed conformation, which corresponds to the structure in which the catalytic chemistry occurs. Examination of the structure in the light of available, very detailed kinetic studies [Porter, D. J., Boyd, F. L. (1991) J. Biol. Chem. 266, 21616-21625. Porter, D. J., Boyd, F. L. (1992) J. Biol. Chem. 267, 3205-3213. Porter, D. J. (1998) J. Biol. Chem. 268, 66-73] suggests elements of the catalytic strategy of AdoHcy hydrolase for acceleration of the reversible conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy). The enzyme, each subunit of which possesses a substrate-binding domain that in the absence of substrate is in rapid motion relative to the tetrameric core of the enzyme, first binds substrate and ceases motion. Probably concurrently with oxidation of the substrate to its 3'-keto form, the closed active site is "sealed off" from the environment, as indicated by a large (10(8)(-)(9)-fold) reduction in the rate of departure of ligands, a feature that prevents exposure of the labile 3'-keto intermediates to the aqueous environment. Elimination of the 5'-substituent (Hcy in the hydrolytic direction, water in the synthetic direction) generates the central intermediate 4',5'-didehydro-5'-deoxy-3'-ketoadenosine. Abortive 3'-reduction of the central intermediate is prevented by a temporary suspension of all or part of the redox catalytic power of the enzyme during the existence of the central intermediate. The abortive reduction is 10(4)-fold slower than the productive reductions at the ends of the catalytic cycle and has a rate constant similar to those of nonenzymic intramolecular model reactions. The mechanism for suspending the redox catalytic power appears to be a conformationally induced increase in the distance across which hydride transfer must occur between cofactor and substrate, the responsible conformational change again being that which "seals" the active site. The crystal structure reveals a well-defined chain of three water molecules leading from the active site to the subunit surface, which may serve as a relay for proton exchange between solvent and active site in the closed form of the enzyme, permitting maintenance of active-site functional groups in catalytically suitable protonation states.


Assuntos
Adenosina/análogos & derivados , Hidrolases/química , Adenosina/química , Adenosil-Homocisteinase , Sítios de Ligação , Tampões (Química) , Catálise , Cristalização , Cristalografia por Raios X , Inibidores Enzimáticos/química , Estabilidade Enzimática , Humanos , Hidrolases/antagonistas & inibidores , Hidrólise , NAD/química , Oxirredução , Proteínas da Gravidez/química , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Água/química
10.
Exp Parasitol ; 105(2): 149-58, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14969692

RESUMO

S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 degrees C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s(-1) and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s(-1) in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NAD(+) to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the K(d) of 1.3 microM for NAD(+) binding to the T. cruzi enzyme is approximately 11-fold higher than the K(d) (0.12 microM) for NAD(+) binding to the human enzyme.


Assuntos
Adenosil-Homocisteinase/genética , Clonagem Molecular , Trypanosoma cruzi/enzimologia , Adenosil-Homocisteinase/química , Adenosil-Homocisteinase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cromatografia por Troca Iônica , Sequência Consenso , Sequência Conservada , Cosmídeos/química , Cosmídeos/genética , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Humanos , Leishmania donovani/enzimologia , Leishmania donovani/genética , Dados de Sequência Molecular , Peso Molecular , NAD/metabolismo , Ratos , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Trypanosoma cruzi/genética
11.
J Org Chem ; 67(23): 8258-60, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12423165

RESUMO

Treatment of homoadenosine [9-(5-deoxy-beta-D-ribo-hexofuranosyl)adenine] with thionyl chloride and pyridine in acetonitrile gave 6'-chloro-6'-deoxyhomoadenosine, which underwent nucleophilic displacement with L-cysteine or L-homocysteine to give homologated analogues of S-adenosyl-L-homocysteine. Each amino acid in aqueous sodium hydroxide at 60 degrees C gave excellent conversion from the chloronucleoside, and adsorption on Amberlite XAD-4 resin provided more convenient isolation than prior methods. Weak binding of these non-hydrolyzed analogues to S-adenosyl-L-homocysteine hydrolase was observed.


Assuntos
Cisteína/síntese química , Cisteína/farmacologia , Inibidores Enzimáticos/síntese química , Homocisteína/síntese química , Homocisteína/farmacologia , Hidrolases/antagonistas & inibidores , Adenosil-Homocisteinase , Cisteína/análogos & derivados , Homocisteína/análogos & derivados , Humanos , Hidrolases/metabolismo , Cinética , Placenta/enzimologia , Ligação Proteica , S-Adenosilmetionina/análogos & derivados
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...