RESUMO
Antimicrobial preservatives are used as functional excipients in multidose formulations of biological therapeutics to destroy or inhibit the growth of microbial contaminants, which may be introduced by repeatedly administering doses. Antimicrobial agents can also induce the biophysical instability of proteins and peptides, which presents a challenge in optimizing the drug product formulation. Elucidating the structural basis for aggregation aids in understanding the underlying mechanism and can offer valuable knowledge and rationale for designing drug substances and drug products; however, this remains largely unexplored due to the lack of high-resolution characterization. In this work, we utilize solution nuclear magnetic resonance (NMR) as an advanced biophysical tool to study an acylated 31-residue peptide, acyl-peptide A, and its interaction with commonly used antimicrobial agents, benzyl alcohol and m-cresol. Our results suggest that acyl-peptide A forms soluble octamers in the aqueous solution, which tumble slowly due to an increased molecular weight as measured by diffusion ordered spectroscopy and 1H relaxation measurement. The addition of benzyl alcohol does not induce aggregation of acyl-peptide A and has no chemical shift perturbation in 1H-1H NOESY spectra, suggesting no detectable interaction with the peptide. In contrast, the addition of 1% (w/v) m-cresol results in insoluble aggregates composed of 25% (w/w) peptides after a 24-hour incubation at room temperature as quantified by 1H NMR. Interestingly, 1H-13C heteronuclear single-quantum coherence and 1H-1H total correlation experiment spectroscopy have identified m-cresol and peptide interactions at specific residues, including Met, Lys, Glu, and Gln, suggesting that there may be a combination of hydrophobic, hydrogen bonding, and electrostatic interactions with m-cresol driving this phenomenon. These site-specific interactions have promoted the formation of higher-order oligomerization such as dimers and trimers of octamers, eventually resulting in insoluble aggregates. Our study has elucidated a structural basis of m-cresol-induced self-association that can inform the optimized design of drug substances and products. Moreover, it has demonstrated solution NMR as a high-resolution tool to investigate the structure and dynamics of biological drug products and provide an understanding of excipient-induced peptide and protein aggregation.
Assuntos
Anti-Infecciosos , Excipientes , Antibacterianos , Anti-Infecciosos/química , Álcool Benzílico/química , Excipientes/química , Peptídeos , Conservantes Farmacêuticos/químicaRESUMO
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 SuperfícieRESUMO
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-AtividadeRESUMO
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/genéticaRESUMO
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.