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1.
Drug Des Devel Ther ; 14: 757-771, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158194

RESUMO

BACKGROUND: Antiviral actions of tetrapyrroles have been described in a number of systems. Our goal was to evaluate antagonism of the HCV NS3-4A protease by a variety of common porphyrins and characterize structure-activity relationships that may be useful for future drug design of HCV and related Flaviviruses. METHODS: Using fluorometric assays, common metalloprotoporphyrins (MPP) all inhibited NS3-4A protease with IC50 values in low micromolar ranges [CoPP (1.4 µM) < ZnPP = MnPP = SnPP < CuPP < FePP (6.5 µM) = protoporphyrin]. RESULTS: Lineweaver-Burk plots confirmed that MPP: NS3 inhibition was basically competitive. All tested MPPs inhibited HCV genotype 1A, 1B, 2A and 3A recombinant proteases with the same fidelity suggesting wide antagonistic capabilities. However, when the MPPs were tested in cellular incubations with HCV replicons only Zn, Fe and free-base protoporphyrin showed comparable EC50 and IC50 values suggesting that there may be critical differences in MPP uptake and intracellular availability. Meso, deutero, and isohematoporphyrin derivatives, with or without metal substitution, all showed less anti-protease and antiviral activities as compared to protoporphyrins, suggesting that the planar, vinyl side chains are important for protease active site binding. MPPs were also active against three common protease mutants (T54A, A156T, and V36M) with equivalent or better IC50 values as compared to wild type enzyme. CONCLUSION: These findings document the versatility of MPPs as antiviral agents with an expanded sensitivity for HCV genotypes and resistance to some common viral mutations. The results also suggest that further study of MPP structure and function will be useful for the development of new antiviral agents.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Células Cultivadas , Relação Dose-Resposta a Droga , Hepacivirus/genética , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Inibidores de Proteases/química , Serina Proteases/genética , Serina Proteases/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
2.
J Biophotonics ; 12(8): e201800318, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30667177

RESUMO

Antimicrobial photodynamic inactivation (aPDI) employs the combination of nontoxic photosensitizing dyes and visible light to kill pathogenic microorganisms regardless of drug-resistance, and can be used to treat localized infections. A meso-substituted tetra-methylpyridinium porphyrin with one methyl group replaced by a C12 alkyl chain (FS111) and its Pd-derivative (FS111-Pd) were synthesized and tested as broad-spectrum antimicrobial photosensitizers when excited by blue light (5 or 10 J/cm2 ). Both compounds showed unprecedented activity, with the superior FS111-Pd giving 3 logs of killing at 1 nM, and eradication at 10 nM for Gram-positive methicillin-resistant Staphylococcus aureus. For the Gram-negative Escherichia coli, both compounds produced eradication at 100 nM, while against the fungal yeast Candida albicans, both compounds produced eradication at 500 nM. Both compounds could be categorized as generators of singlet oxygen (ΦΔ = 0.62 for FS111 and 0.71 for FS111-Pd). An in vivo study was carried out using a mouse model of localized infection in a partial thickness skin abrasion caused by bioluminescent Gram-negative uropathogenic E. coli. Both compounds were effective in reducing bioluminescent signal in a dose-dependent manner when excited by blue light (405 nm), but aPDI with FS111-Pd was somewhat superior both during light and in preventing recurrence during the 6 days following PDT.


Assuntos
Quelantes/química , Interações Hidrofóbicas e Hidrofílicas , Paládio/química , Porfirinas/química , Porfirinas/farmacologia , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/efeitos da radiação , Escherichia coli/efeitos dos fármacos , Escherichia coli/efeitos da radiação , Feminino , Camundongos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Oxigênio Singlete/metabolismo , Superóxidos/metabolismo
3.
BMC Biotechnol ; 12: 89, 2012 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-23176158

RESUMO

BACKGROUND: Biliverdin IXα is produced when heme undergoes reductive ring cleavage at the α-methene bridge catalyzed by heme oxygenase. It is subsequently reduced by biliverdin reductase to bilirubin IXα which is a potent endogenous antioxidant. Biliverdin IXα, through interaction with biliverdin reductase, also initiates signaling pathways leading to anti-inflammatory responses and suppression of cellular pro-inflammatory events. The use of biliverdin IXα as a cytoprotective therapeutic has been suggested, but its clinical development and use is currently limited by insufficient quantity, uncertain purity, and derivation from mammalian materials. To address these limitations, methods to produce, recover and purify biliverdin IXα from bacterial cultures of Escherichia coli were investigated and developed. RESULTS: Recombinant E. coli strains BL21(HO1) and BL21(mHO1) expressing cyanobacterial heme oxygenase gene ho1 and a sequence modified version (mho1) optimized for E. coli expression, respectively, were constructed and shown to produce biliverdin IXα in batch and fed-batch bioreactor cultures. Strain BL21(mHO1) produced roughly twice the amount of biliverdin IXα than did strain BL21(HO1). Lactose either alone or in combination with glycerol supported consistent biliverdin IXα production by strain BL21(mHO1) (up to an average of 23. 5mg L(-1) culture) in fed-batch mode and production by strain BL21 (HO1) in batch-mode was scalable to 100L bioreactor culture volumes. Synthesis of the modified ho1 gene protein product was determined, and identity of the enzyme reaction product as biliverdin IXα was confirmed by spectroscopic and chromatographic analyses and its ability to serve as a substrate for human biliverdin reductase A. CONCLUSIONS: Methods for the scalable production, recovery, and purification of biliverdin IXα by E. coli were developed based on expression of a cyanobacterial ho1 gene. The purity of the produced biliverdin IXα and its ability to serve as substrate for human biliverdin reductase A suggest its potential as a clinically useful therapeutic.


Assuntos
Biliverdina/biossíntese , Escherichia coli/metabolismo , Técnicas de Cultura Celular por Lotes , Biliverdina/genética , Reatores Biológicos , Heme Oxigenase (Desciclizante)/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo
4.
Int J Biochem Cell Biol ; 37(2): 306-19, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15474977

RESUMO

In this paper we present a study on the intracellular localisation and the efficiency of cell photoinactivation of a series of derivatives of 5,10,15,20-tetrakis-(4-N-methylpyridyl)-porphine (C1), whose degree of lipophilicity was varied through replacement of one methyl group with an alkyl chain of various length. Human HT1080 fibrosarcoma cells exposed to the various C1 derivatives (0.25 microM) for 24 h and irradiated with increasing doses of red-light (0.45-27 J/cm2) were inactivated with different efficiencies. The efficiency of cell photoinactivation increased with the increasing length of the hydrocarbon tail and lipophilicity and correlated with the efficiency of the porphyrin accumulation into the cells. Despite the presence of positive charges, these porphyrins did localise rather selectively in lysosomes while mitochondrial localisation was not evident, as demonstrated by fluorescence microscopy studies. Studies on isolated mitochondria provided evidence that the porphyrin uptake and distribution in these organelles were not modulated by the transmembrane potential but were exclusively controlled by partitioning phenomena which might have prevented mitochondria localization in whole cells. Our findings demonstrated that these porphyrins entered the cells through the endocytotic pathway and were transported to lysosomes whose pH increased rapidly upon irradiation. Lysosomal damage did not cause any intracellular redistribution of the porphyrin and represented the primary event causing cell death, very likely via necrosis.


Assuntos
Sistemas de Liberação de Medicamentos , Fibrossarcoma/metabolismo , Lisossomos/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Porfirinas/metabolismo , Cátions/química , Cátions/metabolismo , Cátions/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Linhagem Celular Tumoral , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/patologia , Fibrossarcoma/radioterapia , Humanos , Hidrocarbonetos/química , Lisossomos/efeitos da radiação , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Porfirinas/farmacologia
5.
Photochem Photobiol ; 75(5): 462-70, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12017471

RESUMO

A series of derivatives of 5,10,15,20-tetrakis-(4-N-methylpyridyl)-porphine, where one N-methyl group was replaced by a hydrocarbon chain ranging from C6 to C22, were characterized for their photophysical and photosensitizing properties. The absorption and fluorescence features of the various compounds in neutral aqueous solutions were typical of largely monomeric porphyrins, with the exception of the C22 derivative, which appeared to be extensively aggregated. This was confirmed by the very low triplet quantum yield and lifetime of the C22 derivative as compared with 0.2-0.7 quantum yields and 88-167 micros lifetimes for the other porphyrins. The photophysical properties and photosensitizing activity toward N-acetyl-L-tryptophanamide of the C22 porphyrin became comparable to those typical of the other derivatives in 2% aqueous sodium dodecyl sulfate, where the C22 compound is fully monomerized. All the porphyrin derivatives exhibited at micromolar concentrations photoinactivation activity against both Staphylococcus aureus and Escherichia coli, even though the gram-negative bacteria were markedly less photosensitive. The photosensitizing efficiency was influenced by (1) the amount of cell-bound porphyrin, which increased with increasing length of the hydrocarbon chain; and (2) the tendency to undergo partial aggregation in the cell, which seems to be especially important for the C22 derivative.


Assuntos
Antibacterianos/farmacologia , Porfirinas/química , Porfirinas/farmacologia , Escherichia coli/efeitos dos fármacos , Estrutura Molecular , Espectrometria de Fluorescência
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