RESUMO
Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by â¼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) â¼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
Assuntos
Substâncias para a Guerra Química/intoxicação , Evolução Molecular Direcionada , Hidrolases/metabolismo , Intoxicação/prevenção & controle , Acetilcolinesterase/metabolismo , Arildialquilfosfatase/metabolismo , Biocatálise , Emulsões , Hidrolases/química , HidróliseRESUMO
BACKGROUND: Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. RESULTS: We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. CONCLUSION: The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates.
Assuntos
Bacteriófagos/metabolismo , Carcinoma/patologia , Carcinoma/prevenção & controle , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Nanomedicina/métodos , Nanoestruturas/química , Proteínas Virais/administração & dosagem , Linhagem Celular Tumoral , HumanosRESUMO
While the resistance of bacteria to traditional antibiotics is a major public health concern, the use of extremely potent antibacterial agents is limited by their lack of selectivity. As in cancer therapy, antibacterial targeted therapy could provide an opportunity to reintroduce toxic substances to the antibacterial arsenal. A desirable targeted antibacterial agent should combine binding specificity, a large drug payload per binding event, and a programmed drug release mechanism. Recently, we presented a novel application of filamentous bacteriophages as targeted drug carriers that could partially inhibit the growth of Staphylococcus aureus bacteria. This partial success was due to limitations of drug-loading capacity that resulted from the hydrophobicity of the drug. Here we present a novel drug conjugation chemistry which is based on connecting hydrophobic drugs to the phage via aminoglycoside antibiotics that serve as solubility-enhancing branched linkers. This new formulation allowed a significantly larger drug-carrying capacity of the phages, resulting in a drastic improvement in their performance as targeted drug-carrying nanoparticles. As an example for a potential systemic use for potent agents that are limited for topical use, we present antibody-targeted phage nanoparticles that carry a large payload of the hemolytic antibiotic chloramphenicol connected through the aminoglycoside neomycin. We demonstrate complete growth inhibition toward the pathogens Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli with an improvement in potency by a factor of approximately 20,000 compared to the free drug.
Assuntos
Antibacterianos/farmacologia , Bactérias , Bacteriófagos/fisiologia , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Nanopartículas , Antibacterianos/síntese química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/virologia , Infecções Bacterianas/terapia , Bacteriófagos/classificação , Cloranfenicol/síntese química , Cloranfenicol/química , Cloranfenicol/farmacologia , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Humanos , Nanomedicina/métodos , Neomicina/síntese química , Neomicina/química , Neomicina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/virologia , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/genética , Streptococcus pyogenes/crescimento & desenvolvimento , Streptococcus pyogenes/virologiaRESUMO
Bacteriophages have been used for more than a century for (unconventional) therapy of bacterial infections, for half a century as tools in genetic research, for 2 decades as tools for discovery of specific target-binding proteins, and for nearly a decade as tools for vaccination or as gene delivery vehicles. Here we present a novel application of filamentous bacteriophages (phages) as targeted drug carriers for the eradication of (pathogenic) bacteria. The phages are genetically modified to display a targeting moiety on their surface and are used to deliver a large payload of a cytotoxic drug to the target bacteria. The drug is linked to the phages by means of chemical conjugation through a labile linker subject to controlled release. In the conjugated state, the drug is in fact a prodrug devoid of cytotoxic activity and is activated following its dissociation from the phage at the target site in a temporally and spatially controlled manner. Our model target was Staphylococcus aureus, and the model drug was the antibiotic chloramphenicol. We demonstrated the potential of using filamentous phages as universal drug carriers for targetable cells involved in disease. Our approach replaces the selectivity of the drug itself with target selectivity borne by the targeting moiety, which may allow the reintroduction of nonspecific drugs that have thus far been excluded from antibacterial use (because of toxicity or low selectivity). Reintroduction of such drugs into the arsenal of useful tools may help to combat emerging bacterial antibiotic resistance.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/terapia , Sistemas de Liberação de Medicamentos , Inovirus/fisiologia , Cloranfenicol/farmacologia , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Estudos de Viabilidade , Modelos Moleculares , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/virologiaRESUMO
Amylose-conjugated linoleic acid (CLA) complexes were produced by water/dimethyl sulfoxide (DMSO) and KOH/HCl complexation methods. The formation of amylose V form was confirmed by X-ray diffraction (XRD), and complexes formed at 30, 60, and 90 degrees C exhibit melting temperatures exceeding 88 degrees C. Atomic force microscopy (AFM) images showed distinct difference in complex organization, with complexes formed in water/DMSO showing spherical shape with typical diameter of 150 nm. Complexes formed by KOH/HCl showed elongated structure with typical width of 43-160 nm. Water/DMSO complexes exhibit superior protection to CLA against oxidation. All complexes showed high retention of CLA in simulated stomach conditions, and the digestion of complexes by amylases results in high hydrolysis and CLA release by pancreatin and alpha-amylase. Only moderate release was detected following hydrolysis by amyloglucosidase and beta-amylase. It is therefore suggested that amylose-CLA complexes can serve as molecular nanocapsules for protection and delivery of CLA.