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1.
Sci Rep ; 10(1): 12243, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699361

RESUMO

The development of an effective oral therapeutics is an immediate need for the control and elimination of visceral leishmaniasis (VL). We exemplify the preparation and optimization of 2-hydroxypropyl-ß-cyclodextrin (HPCD) modified solid lipid nanoparticles (SLNs) based oral combinational cargo system of Amphotericin B (AmB) and Paromomycin (PM) against murine VL. The emulsion solvent evaporation method was employed to prepare HPCD modified dual drug-loaded solid lipid nanoparticles (m-DDSLNs). The optimized formulations have a mean particle size of 141 ± 3.2 nm, a polydispersity index of 0.248 ± 0.11 and entrapment efficiency for AmB and PM was found to be 96% and 90% respectively. The morphology of m-DDSLNs was confirmed by scanning electron microscopy and transmission electron microscopy. The developed formulations revealed a sustained drug release profile upto 57% (AmB) and 21.5% (PM) within 72 h and were stable at both 4 °C and 25 °C during short term stability studies performed for 2 months. Confocal laser scanning microscopy confirmed complete cellular internalization of SLNs within 24 h of incubation. In vitro cytotoxicity study against J774A.1 macrophage cells confirmed the safety and biocompatibility of the developed formulations. Further, m-DDSLNs did not induce any hepatic/renal toxicities in Swiss albino mice. The in vitro simulated study was performed to check the stability in simulated gastric fluids and simulated intestinal fluids and the release was found almost negligible. The in vitro anti-leishmanial activity of m-DDSLNs (1 µg/ml) has shown a maximum percentage of inhibition (96.22%) on intra-cellular amastigote growth of L. donovani. m-DDSLNs (20 mg/kg × 5 days, p.o.) has significantly (P < 0.01) reduced the liver parasite burden as compared to miltefosine (3 mg/kg × 5 days, p.o.) in L. donovani-infected BALB/c mice. This work suggests that the superiority of as-prepared m-DDSLNs as a promising approach towards the oral delivery of anti-leishmanial drugs.


Assuntos
Anfotericina B/química , Anfotericina B/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Nanopartículas/química , Paromomicina/química , Paromomicina/farmacologia , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular , Emulsões/química , Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão/métodos , Tamanho da Partícula , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacologia
2.
Exp Parasitol ; 212: 107873, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32165146

RESUMO

Ginsenoside-Rh2 and cucurbitacin-B (CuB) are secondary metabolites of Ginseng (Panax ginseng) and Cucurbitaceae plants respectively. We assessed the anticryptosporidial activity of these two functional compounds in a cell culture model of cryptosporidiosis. The highest concentration of each compound that was not toxic to the host cells was used to assess the activity against C. parvum during infection/invasion and growth in HCT-8 cell monolayers. Monolayers were infected with pre-excysted C. parvum oocysts. Infected monolayers were incubated at 37 °C for 24 h and 48 h in the presence of different concentrations of each test compound. A growth resumption assay was performed by incubating infected monolayers in the presence of compounds for 24 h followed by a second 24-h incubation in the absence of compound. To screen for invasion inhibiting activity, freshly excysted C. parvum sporozoites were pre-treated with different concentrations of compounds prior to adding them to the cell monolayers. Paromomycin, a known inhibitor of C. parvum, and DMSO were used as positive and negative control, respectively. The level of infection was initially assessed using an immunofluorescent assay and quantified by real-time PCR. Both compounds were found to strongly inhibit C. parvum intracellular development in a dose-dependent manner. IC50 values of 25 µM for a 24 h development period and 5.52 µM after 48 h development were measured for Rh2, whereas for CuB an IC50 value of 0.169 µg/ml and 0.118 µg/ml were obtained for the same incubation periods. CuB also effectively inhibited resumption of growth, an activity that was not observed with Rh2. CuB was more effective at inhibiting excystation and/or host cell invasion, indicating that this compound also targets extracellular stages of the parasite.


Assuntos
Coccidiostáticos/farmacologia , Cryptosporidium parvum/efeitos dos fármacos , Cucurbitacinas/farmacologia , Ginsenosídeos/farmacologia , Extratos Vegetais/farmacologia , Triterpenos/farmacologia , Animais , Linhagem Celular , Cryptosporidium parvum/citologia , Cryptosporidium parvum/crescimento & desenvolvimento , Cucurbitaceae/química , Dimetil Sulfóxido , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Camundongos , Panax/química , Paromomicina/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Solventes
3.
Int J Mol Sci ; 21(3)2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31979077

RESUMO

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3'-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.


Assuntos
Microalgas/genética , Plasmídeos/genética , Transgenes/genética , Antibacterianos/farmacologia , Marcadores Genéticos/genética , Canamicina Quinase/genética , Paromomicina/farmacologia , Regiões Promotoras Genéticas/genética , Streptomyces/efeitos dos fármacos , Streptomyces/genética , Transformação Genética/genética
4.
Nat Commun ; 10(1): 5627, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31819054

RESUMO

Current genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. Here, we use chemical mutagenesis, drug selection and next generation sequencing to characterize miltefosine and paromomycin resistant clones of the parasite Leishmania. We highlight several genes involved in drug resistance by sequencing the genomes of 41 resistant clones and by concentrating on recurrent SNVs. We associate genes linked to lipid metabolism or to ribosome/translation functions with miltefosine or paromomycin resistance, respectively. We prove by allelic replacement and CRISPR-Cas9 gene-editing that the essential protein kinase CDPK1 is crucial for paromomycin resistance. We have linked CDPK1 in translation by functional interactome analysis, and provide evidence that CDPK1 contributes to antimonial resistance in the parasite. This screen is powerful in exploring networks of drug resistance in an organism with diploid to mosaic aneuploid genome, hence widening the scope of its applicability.


Assuntos
Resistência a Medicamentos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leishmania/genética , Mutagênese , Mutação/genética , Paromomicina/farmacologia , Fosforilcolina/análogos & derivados , Fosforilação/efeitos dos fármacos , Fosforilcolina/farmacologia
5.
Artigo em Inglês | MEDLINE | ID: mdl-31658971

RESUMO

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 µM at the promastigote stage and 32 and 195 µM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.


Assuntos
Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Paromomicina/farmacologia , Animais , Resistência a Medicamentos/genética , Feminino , Genômica , Humanos , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Lipidômica , Macrófagos/parasitologia , Metabolômica , Camundongos , Camundongos Endogâmicos BALB C , Nepal , Testes de Sensibilidade Parasitária , Polimorfismo Genético
6.
ACS Infect Dis ; 5(10): 1718-1730, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31436080

RESUMO

A series of derivatives of the 4,5-disubstituted class of 2-deoxystreptamine aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin was prepared and assayed for (i) their ability to inhibit protein synthesis by bacterial ribosomes and by engineered bacterial ribosomes carrying eukaryotic decoding A sites, (ii) antibacterial activity against wild type Gram negative and positive pathogens, and (iii) overcoming resistance due to the presence of aminoacyl transferases acting at the 2'-position. The presence of five suitably positioned residual basic amino groups was found to be necessary for activity to be retained upon removal or alkylation of the 2'-position amine. As alkylation of the 2'-amino group overcomes the action of resistance determinants acting at that position and in addition results in increased selectivity for the prokaryotic over eukaryotic ribosomes, it constitutes an attractive modification for introduction into next generation aminoglycosides. In the neomycin series, the installation of small (formamide) or basic (glycinamide) amido groups on the 2'-amino group is tolerated.


Assuntos
Aminoglicosídeos/síntese química , Aminoglicosídeos/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Sítios de Ligação , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Hexosaminas , Humanos , Testes de Sensibilidade Microbiana , Neomicina/química , Neomicina/farmacologia , Paromomicina/química , Paromomicina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/metabolismo , Relação Estrutura-Atividade
7.
Artigo em Inglês | MEDLINE | ID: mdl-31160283

RESUMO

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative ß-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.


Assuntos
Antiprotozoários/farmacologia , Trypanosoma brucei brucei/metabolismo , Adenosina Trifosfatases/metabolismo , Anfotericina B/farmacologia , Gluconato de Antimônio e Sódio/farmacologia , Leishmania/parasitologia , Paromomicina/farmacologia , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Proteínas R-SNARE/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
8.
Acta Parasitol ; 64(4): 710-719, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30941668

RESUMO

BACKGROUND: Leishmania donovani (L. donovani) is one of the parasites that cause leishmaniasis. The mechanisms by which L. donovani fights against adverse environment and becomes resistant to drugs are not well understood yet. OBJECTIVE: The present study was designed to evaluate the effects of different regulators on the modulation of Transplasma Membrane Electron Transport (transPMET) systems of susceptible and resistant L. donovani cells. MATERIALS AND METHODS: Effects of UV, different buffers, and electron transport inhibitors and stimulators on the reduction of α-lipoic acid (ALA), 1,2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide were determined. RESULTS AND DISCUSSION: ALA reductions were inhibited in susceptible, sodium antimony gluconate (SAG)-resistant and paromomycin (PMM)-resistant AG83 amastigote cells, and stimulated in susceptible and SAG-resistant AG83 promastigote cells upon UV exposure. The results indicate that UV irradiation almost oppositely affect ALA reductions in amastigotes and promastigotes. ALA reductions were stimulated in sensitive and inhibited in resistant GE1 amastigotes upon UV exposure. Susceptible amastigotes and promastigotes inhibited, and resistant amastigotes and promastigotes stimulated NQSA reduction under UV irradiation. Thus, susceptible and drug-resistant amastigotes and promastigotes are different in the reduction of ALA. Susceptible and resistant AG83 amastigotes and promastigotes inhibited the ferricyanide reductions upon UV exposure, which indicates, there is no such difference in ferricyanide reductions among susceptible as well as resistant AG83 amastigotes and promastigotes. The reductions of extracellular electron excerptors in susceptible promastigotes requires the availability of Na+ and Cl- ions for maximal activity but susceptible amastigotes are mostly not dependent on the availability of Na+ and Cl- ions. Both in promastigotes and amastigotes, reductions of electron acceptors were strongly inhibited by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Furthermore, antimycin A, rotenone and capsaicin markedly inhibited the reductions of electron acceptors in promastigotes, but not in amastigotes. CONCLUSION: Results of this study suggest that the transPMET system is functionally different in wild and resistant strains of L. donovani.


Assuntos
Resistência a Medicamentos , Transporte de Elétrons , Leishmania donovani/fisiologia , Ferricianetos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/efeitos da radiação , Estágios do Ciclo de Vida , Naftoquinonas/farmacologia , Oxirredução , Paromomicina/farmacologia , Ácidos Sulfônicos/farmacologia , Ácido Tióctico/metabolismo , Raios Ultravioleta
9.
J Biomol Struct Dyn ; 37(6): 1582-1596, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29633917

RESUMO

The bacterial ribosome is an established target for anti-bacterial therapy since decades. Several inhibitors have already been developed targeting both defined subunits (50S and 30S) of the ribosome. Aminoglycosides and tetracyclines are two classes of antibiotics that bind to the 30S ribosomal subunit. These inhibitors can target multiple active sites on ribosome that have a complex structure. To screen putative inhibitors against 30S subunit of the ribosome, the crystal structures in complex with various known inhibitors were analyzed using pharmacophore modeling approach. Multiple active sites were considered for building energy-based three-dimensional (3D) pharmacophore models. The generated models were validated using enrichment factor on decoy data-set. Virtual screening was performed using the developed 3D pharmacophore models and molecular interaction towards the 30S ribosomal unit was analyzed using the hits obtained for each pharmacophore model. The hits that were common to both streptomycin and paromomycin binding sites were identified. Further, to predict the activity of these hits a robust 2D-QSAR model with good predictive ability was developed using 16 streptomycin analogs. Hence, the developed models were able to identify novel inhibitors that are capable of binding to multiple active sites present on 30S ribosomal subunit.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Paromomicina/química , Subunidades Ribossômicas Menores de Bactérias/química , Estreptomicina/química , Sítios de Ligação , Domínio Catalítico , Descoberta de Drogas , Ligantes , Testes de Sensibilidade Microbiana , Estrutura Molecular , Paromomicina/farmacologia , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estreptomicina/farmacologia
10.
Exp Parasitol ; 195: 59-65, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30385266

RESUMO

Members of the genus Cryptosporidium are frequent protozoan pathogens in humans and a wide range of animals. There is no consistently effective treatment against cryptosporidiosis, especially in immunodeficient patients. The present study was carried out to study the therapeutic effects of curcumin against cryptosporidiosis in immunosuppressed BALB/c mice. Mice were divided into five groups and immunosuppressed by dexamethasone. Three groups were inoculated with C. parvum oocysts, administered with curcumin, paromomycin, and without treatment. The reminders were regarded as controls. The oocysts in the fecal smear were counted daily. At days 0, 3, 7, and 11 post-treatment, the mice were sacrificed, and the efficacy of drugs was evaluated by comparing the histopathological alterations in jejunum and ileum, measuring the total antioxidant capacity, and malondialdehyde in the affected tissues. The infection was completely eliminated in the curcumin-treated group, and oocyst shedding stopped with no recurrence after drug withdrawal. On the contrary, paromomycin was unable to eliminate C. parvum infection completely, and oocyst shedding continued even 10 days after the drug withdrawal. Based on these findings, curcumin can be a trustworthy compound for the elimination of infection in immunosuppressed hosts. Further evaluation to find its accurate mechanism of action should be considered.


Assuntos
Antiprotozoários/uso terapêutico , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Curcumina/uso terapêutico , Animais , Antioxidantes/metabolismo , Antiprotozoários/farmacologia , Bovinos , Criptosporidiose/imunologia , Criptosporidiose/patologia , Cryptosporidium parvum/crescimento & desenvolvimento , Cryptosporidium parvum/fisiologia , Curcumina/farmacologia , Modelos Animais de Doenças , Fezes/parasitologia , Feminino , Íleo/parasitologia , Íleo/patologia , Imunossupressão , Jejuno/parasitologia , Jejuno/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microvilosidades/parasitologia , Microvilosidades/patologia , Oocistos/fisiologia , Oxidantes/metabolismo , Paromomicina/farmacologia , Paromomicina/uso terapêutico , Distribuição Aleatória
11.
Ann Parasitol ; 64(3): 181-187, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30316208

RESUMO

Cutaneous leishmaniosis (CL) is treated with pentavalent antimony (SbV) as a first-line drug, while amphotericin B and paromomycin are potential alternatives in antimonial- resistant isolates. However, the mechanisms of drug resistance remain unclear. The present study analyses the gene expression of RNA polymerase II (RNAP II) and J-binding protein 1 (JBP1), and J-binding protein 2 (JBP2) in Leishmania major after exposure to drugs in vitro. L. major (MRHO/IR/75/ER) promastigotes were exposed to various concentrations of glucantime, paromomycin and amphotericin B for 72 hours. The RNA was then extracted and used for cDNA synthesis. The expressions of JBP1, JBP2 and RNAP II were analysed using SYBR Green real-time PCR. No change in JBP2 or RNAP II expression was associated with amphotericin B, but JBP1 expression decreased with increasing drug concentration. Paromomycin had no effect on JBP2 expression, but a 13.5-fold increase in JBP1 was observed at 100 µg/ml, and a decrease in RNAP II expression at 25 and 50 µg/ml. Exposure to glucantime resulted in 1.4-fold lower JBP1 expression at 5 µg/ml, and 333.33- to 500-fold lower RNAP II at concentrations of 5 to 15 µg/ml. As Base J synthesis requires both JBP1 and JBP2, RNAP II (encoding RNA polymerase II) could reduce expression. However, RNAP II was not expressed in all groups, indicating that the genes associated with drug resistance may be regulated in other ways.


Assuntos
Antiprotozoários , Proteínas de Transporte , Leishmania major , Paromomicina , RNA Polimerase II , Anfotericina B , Antiprotozoários/farmacologia , Proteínas de Transporte/metabolismo , Resistência a Medicamentos , Leishmania major/efeitos dos fármacos , Leishmania major/genética , Paromomicina/farmacologia , RNA Polimerase II/metabolismo
12.
Plant Physiol ; 178(4): 1436-1447, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30206105

RESUMO

Insertional mutagenesis, in which a piece of exogenous DNA is integrated randomly into the genomic DNA of the recipient cell, is a useful method to generate new mutants with phenotypes of interest. The unicellular green alga Chlamydomonas reinhardtii is an outstanding model for studying many biological processes. We developed a new computational algorithm, MAPINS (mapping insertions), to efficiently identify insertion sites created by the integration of an APHVIII (aminoglycoside 3'-phosphotransferase VIII) cassette that confers paromomycin resistance. Using whole-genome sequencing data, this method eliminates the need for genomic DNA manipulation and retains all the sequencing information provided by paired-end sequencing. We experimentally verified 38 insertion sites out of 41 sites (93%) identified by MAPINS from 20 paromomycin-resistant strains. Using meiotic analysis of 18 of these strains, we identified insertion sites that completely cosegregate with paromomycin resistance. In six of the seven strains with a mutant phenotype, we demonstrated complete cosegregation of the mutant phenotype and the verified insertion site. In addition, we provide direct evidence of complex rearrangements of genomic DNA in five strains, three of which involve the APHVIII insertion site. We suggest that strains obtained by insertional mutagenesis are more complicated than expected from previous analyses in Chlamydomonas To map the locations of some complex insertions, we designed 49 molecular markers based on differences identified via whole-genome sequencing between wild-type strains CC-124 and CC-125. Overall, MAPINS provides a low-cost, efficient method to characterize insertional mutants in Chlamydomonas.


Assuntos
Chlamydomonas reinhardtii/genética , Biologia Computacional/métodos , Análise Mutacional de DNA/métodos , Rearranjo Gênico , Mutagênese Insercional , Mapeamento Cromossômico , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Vetores Genéticos , Canamicina Quinase/genética , Paromomicina/farmacologia , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes
13.
Nucleic Acids Res ; 46(19): 9960-9970, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30239867

RESUMO

A synthetic riboswitch N1, inserted into the 5'-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6' hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6'-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.


Assuntos
Aminoglicosídeos/farmacologia , Simulação de Dinâmica Molecular , Riboswitch/efeitos dos fármacos , Sítios de Ligação , Conformação Molecular/efeitos dos fármacos , Neomicina/farmacologia , Paromomicina/farmacologia , Ribostamicina/farmacologia , Riboswitch/fisiologia
14.
Exp Parasitol ; 194: 1-8, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30237052

RESUMO

Cryptosporidiosis is a zoonotic disease caused by species in the genus Cryptosporidium. In young ruminants, Cryptosporidium parvum causes economically significant disease with mild to severe clinical signs and occasional death. The typical clinical course in animals aged 1-3 weeks old is acute diarrhoea. Currently there are no available treatments that are fully effective against cryptosporidiosis in either humans or animals. Therefore there is a critical need for the development of new therapeutic agents. We adapted two in vitro culture systems (HCT-8 and Caco-2 cell lines) for C. parvum infection to investigate the "anticryptosporidial" activity of two chitosans; Chitosan NAG and Chitosan Mix. Chitosan-a naturally-occurring polysaccharide compound-has been found to be active against a variety of diseases, possessing both antimicrobial and anticancer properties. We investigated both chitosan's toxicity and effects on C. parvum in the two in vitro models. To evaluate chitosan's effects on oocyst shedding in vivo, CD-1 neonate mice were orally inoculated with C. parvum oocysts (Iowa strain), treated with chitosan, and compared to infected non-treated animals. Paromomycin, a classical drug used in veterinary medicine, was used as a reference compound. Immunofluorescence techniques were used to analyse the parasites. Our results showed significant reductions in Cryptosporidium oocyst viability (>95%) after oocyst pre-incubation with either paromomycin (P < 0.001), Chitosan Mix or Chitosan NAG (P < 0.001), for 24 h at 37 °C. Additionally, paromomycin, Chitosan Mix, and Chitosan NAG significantly inhibited C. parvum multiplication in HCT-8 and Caco-2 cell lines (P < 0.005). These effects were dose-dependent. In in vivo studies, treatment with both chitosans (Chitosan NAG, Chitosan Mix) or paromomycin sulfate significantly reduced parasite shedding in infected treated newborn mice (-56%, -34.5% and -58%, respectively). In conclusion, these findings provide the first in vitro and in vivo evidence of the anticryptosporidial activities of this natural polysaccharide.


Assuntos
Antiprotozoários/farmacologia , Quitosana/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Células CACO-2 , Bovinos , Linhagem Celular Tumoral , Quitosana/uso terapêutico , Quitosana/toxicidade , Modelos Animais de Doenças , Humanos , Enteropatias Parasitárias/tratamento farmacológico , Enteropatias Parasitárias/parasitologia , Camundongos , Paromomicina/farmacologia , Paromomicina/uso terapêutico , Paromomicina/toxicidade
15.
J Dermatol Sci ; 92(1): 78-88, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30037731

RESUMO

BACKGROUND: Cutaneous leishmaniasis (CL) skin lesions are the result of a deregulated immune response, which is unable to eliminate Leishmania parasites. The control of both, parasites and host immune response, is critical to prevent tissue destruction. The skin ulceration has been correlated with high TNF-α level. OBJECTIVE: Because human anti-TNF-α antibodies (Ab) have been successfully assayed in several mice inflammatory diseases, we hypothesized that their anti-inflammatory effect could optimize the healing of CL lesions achieved after topical application of paromomycin (PM), the current chemotherapy against CL. METHODS AND RESULTS: We first compared the in vitro efficacy of PM and Ab alone and the drug given in combination with Ab to assess if the Ab could interfere with PM leishmanicidal activity in L. major-infected bone marrow-derived macrophages. The combination therapy had similar antileishmanial activity to the drug alone and showed no influence on NO production, which allows macrophage-mediated parasite killing. Next, we demonstrated in an in vivo model of Imiquimod®-induced inflammation that topical Ab and PM inhibit the infiltration of inflammatory cells in the skin. In the efficacy studies in L. major-infected BALB/c mice, PM combined with Ab led to a sharp infection reduction and showed a stronger anti-inflammatory activity than PM alone. This was confirmed by the down-regulation of TNF-α, IL-1ß, iNOS, IL-17, and CCL3 as well as by a decrease of the neutrophilic infiltrate during infection upon treatment with the Ab. CONCLUSIONS: In terms of parasite elimination and inflammation reduction, topical application of Ab in combination with PM was more effective than the drug alone.


Assuntos
Anticorpos/farmacologia , Antiprotozoários/farmacologia , Dermatite/tratamento farmacológico , Mediadores da Inflamação/antagonistas & inibidores , Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Paromomicina/farmacologia , Pele/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Células Cultivadas , Dermatite/etiologia , Dermatite/imunologia , Dermatite/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Interações Hospedeiro-Patógeno , Imiquimode , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Leishmania major/imunologia , Leishmania major/patogenicidade , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Pele/parasitologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Eur J Pharm Biopharm ; 129: 162-174, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29857136

RESUMO

The goal of this study was to create a mass transport model (MTM) model for gastric emptying and upper gastrointestinal (GI) appearance that can capture the in vivo concentration-time profiles of the nonabsorbable drug phenol red in solution in the stomach and upper small intestine by direct luminal measurement while simultaneously recording the contractile activity (motility) via manometry. We advanced from a one-compartmental design of the stomach to a much more appropriate, multi-compartmental 'mixing tank' gastric model that reflects drug distribution along the different regions of the stomach as a consequence of randomly dosing relative to the different contractile phases of the migrating motor complex (MMC). To capture the intraluminal phenol red concentrations in the different segments of the GI tract both in fasted and fed state conditions, it was essential to include a bypass flow compartment ('magenstrasse') to facilitate the transport of the phenol red solution directly to the duodenum (fasted state) or antrum (fed state). The fasted and fed state models were validated with external reference data from an independent aspiration study using another nonabsorbable marker (paromomycin). These results will be essential for the development and optimization of computational programs for GI simulation and absorption prediction, providing a realistic gastric physiologically-based pharmacokinetic (PBPK) model based on direct measurement of gastric concentrations of the drug in the stomach.


Assuntos
Esvaziamento Gástrico/efeitos dos fármacos , Absorção Intestinal , Intestino Delgado/efeitos dos fármacos , Modelos Biológicos , Estômago/fisiologia , Administração Oral , Adulto , Jejum , Feminino , Voluntários Saudáveis , Humanos , Intestino Delgado/fisiologia , Masculino , Pessoa de Meia-Idade , Paromomicina/farmacologia , Fenolsulfonaftaleína/farmacologia , Período Pós-Prandial , Solubilidade , Estômago/efeitos dos fármacos , Adulto Jovem
17.
Comput Biol Chem ; 76: 1-16, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29857255

RESUMO

A series of 2-Cl-benzimidazole derivatives was synthesized and assessed for antibacterial activity. Antibacterial results indicated that compounds 2d, 2e, 3a, 3b, 3c, 4d and 4e showed promising activity against B. cerus, S. aureus and P. aeruginosa (MIC: 6.2 µg/mL) and excellent efficacy against E. coli (MIC: 3.1 µg/mL). Furthermore, compounds 3d and 3e displayed better activity (MIC: 3.1 µg/mL) than the reference drugs chloramphenicol and cycloheximide against gram positive and gram negative bacterial strains. The compounds 3d-e also showed better activity than the reference drug paromomycin against B. cerus and P. aeruginosa and showed similar inhibition pattern against S. aureus and E. coli. (MIC: 3.1 µg/mL). Studies on fractional inhibitory concentration (FIC) determination of compounds 1a-e, 2a-c, 4a-c and the reference antibiotic via combination approach revealed a synergistic effect as the MIC values were lowered up to 1/8th to 1/33rd of the original MIC. In-vitro cytotoxicity study indicated that 2-Cl-benzimidazole derivatives showed less toxicity than the reference used against PBM, CEM and Vero cell lines. Docking studies and MD simulations of compounds on bacterial protein (eubacterial ribosomal decoding A site, PDB: 1j7t) have been conducted to find the possible mode of action of the molecules. In silico ADMET evaluations of compounds 3d and 3e showed promising results comparable to the reference drugs used in this study.


Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/metabolismo , Antibacterianos/toxicidade , Bacillus cereus/efeitos dos fármacos , Benzimidazóis/síntese química , Benzimidazóis/metabolismo , Benzimidazóis/toxicidade , Linhagem Celular , Cloranfenicol/farmacologia , Chlorocebus aethiops , Cicloeximida/farmacologia , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Humanos , Ligantes , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Paromomicina/farmacologia , Ligação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , RNA Ribossômico 16S/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
18.
Int J Parasitol Drugs Drug Resist ; 8(2): 246-264, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29689531

RESUMO

Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (SbIII, S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D-LDH-BCAT, the amplification of which being related to PMM resistance.


Assuntos
Antiprotozoários/farmacologia , Resistência a Múltiplos Medicamentos/genética , Genômica , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Transcriptoma , Antimônio/farmacologia , Leishmania donovani/enzimologia , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Leishmania major/efeitos dos fármacos , Leishmania major/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Testes de Sensibilidade Parasitária , Paromomicina/farmacologia , Fenótipo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia
19.
Vet Parasitol ; 250: 7-14, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29329627

RESUMO

Cryptosporidium is a ubiquitous protozoan parasite causing gastrointestinal disorder in various hosts worldwide. The disease is self-limiting in the immunocompetent but life-threatening in immunodeficient individuals. Investigations to find an effective drug for the complete elimination of the Cryptosporidium infection are ongoing and urgently needed. The current study was undertaken to examine the anti-cryptosporidial efficacy of curcumin in experimentally infected mice compared with that of paromomycin. Oocysts were isolated from a pre-weaned dairy calf and identified as Cryptosporidium parvum using a nested- polymerase chain reaction (PCR) on Small subunit ribosomal ribonucleic acid (SSU rRNA) gene and sequencing analysis. One hundred and ten female BALB/c mice were divided into five groups. Group 1 was infected and treated with curcumin; Group 2 infected and treated with paromomycin; Group 3 infected without treatment; Group 4 included uninfected mice treated with curcumin, and Group 5 included uninfected mice treated with distilled water for 11 successive days, starting on the first day of oocyst shedding. The oocyst shedding was recorded daily. At days 0, 3, 7, and 11 of post treatments, five mice from each group were killed humanly; jejunum and ileum tissue samples were processed for histopathological evaluation and counting of oocyst on villi, simultaneously. Furthermore, total antioxidant capacity (TAC) and malondialdehyde (MDA) concentrations in affected tissues were also measured in different groups. By treatments, tissue lesions and the number of oocyst on villi of both jejunum and ileum were decreased with a time-dependent manner. In comparison with Group 3, oocyst shedding was stopped at the end of treatment period in both groups 1 and 2 without recurrence at 10days after drug withdrawal. Also, TAC was increased and the MDA concentrations were decreased in Group 1. Moreover, paromomycin showed acceptable treatment outcomes during experiment and its anti-cryptosporidial activity was faster than curcumin. The results confirmed the anti-cryptosporidial and antioxidant activity of curcumin against C. parvum and further evaluation of immunosuppressed animal models needs to be carried out.


Assuntos
Criptosporidiose/tratamento farmacológico , Curcumina/uso terapêutico , Paromomicina/uso terapêutico , Animais , Cryptosporidium parvum/efeitos dos fármacos , Cryptosporidium parvum/fisiologia , Curcumina/farmacologia , Modelos Animais de Doenças , Fezes/parasitologia , Feminino , Intestino Delgado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Oocistos , Paromomicina/farmacologia
20.
Nucleic Acids Res ; 46(3): 1362-1374, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29267976

RESUMO

We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.


Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Código Genético , Magnésio/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Cátions Bivalentes , Códon , Escherichia coli/genética , Escherichia coli/metabolismo , Gentamicinas/farmacologia , Cinética , Neomicina/farmacologia , Paromomicina/farmacologia , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Frações Subcelulares/química , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo
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