The Experts below are selected from a list of 276 Experts worldwide ranked by ideXlab platform
Bradley J. Berger - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and trypanocidal activity of the bis-carba analogue of Pentamidine
Bioorganic & Medicinal Chemistry Letters, 1996Co-Authors: Xini Zhang, Bradley J. Berger, Peter UlrichAbstract:Abstract Because toxic metabolites are formed by microsomal oxidative cleavage of the ether methylenes of the antiprotozoal drug, Pentamidine, we synthesized the previously unreported analogue of Pentamidine in which the ether oxygens are replaced by methylenes. Although this compound was about 20-fold less active than Pentamidine against Trypanosoma brucei infection in mice, it did not undergo oxidative cleavage, suggesting a new direction for design of less toxic Pentamidine analogues.
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Characterisation of Pentamidine-resistant Trypanosoma brucei brucei.
Molecular and Biochemical Parasitology, 1995Co-Authors: Bradley J. Berger, Nicola S. Carter, Alan H. FairlambAbstract:Following selection in vitro by exposure to increasing concentrations of the aromatic diamidine Pentamidine, a Trypanosoma brucei brucei clone has been characterised in vivo and in vitro. The resistant clone, designated T.b. brucei S427/118/PR32.6, was found to be less virulent than the parental clone T.b. brucei S427118, with an intraperitoneal injection of 2.5 × 106 resistant organisms required to produce a course of disease equivalent to 1 × 104 sensitive trypanosomes. This lowered virulence is not associated with an increased susceptibility to the host's immune system, and is not due to the in vitro culturing process. The Pentamidine-resistant clone was found to be 26- and 4.5-fold resistant to Pentamidine in vitro and in vivo, respectively. Although not cross-resistant in vivo to any other aromatic diamidines (stilbamidine, berenil and propamidine), a 2.4-fold increase in resistance to the melaminophenylarsine melarsoprol was observed. While Pentamidine completely inhibited uptake of 1 μM [3H]adenosine in the presence of 1 mM inosine, suggesting that Pentamidine is transported by the inosine-insensitive P2 transporter, the Pentamidine-resistant clone appeared to have a fully functional P2-adenosine transport system. Both resistant and parental cloned lines accumulated approx. 6 nmol Pentamidine (108 cells)−1 over the course of 3 h, representing an internal concentration of 0.7–1.0 mM. Thus, unlike previously characterised drug-resistant trypanosomes, T.b. brucei PR32.6 is not deficient in drug accumulation, suggesting that other resistance mechanisms are likely to be involved.
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Polyamine and Pentamidine metabolism in African trypanosomes
Acta tropica, 1993Co-Authors: Bradley J. Berger, Nicola S. Carter, Alan H. FairlambAbstract:Abstract A Pentamidine-resistant line of bloodstream Trypanosoma brucei brucei (S427/118) has been developed by stepwise selection in axenic culture in vitro. After 57 days of selection, the resistant line (S427/118/PR32) was able to grow normally in 32 ng/ml (54 pM) Pentamidine with an IC 50 value of 105 ng/ml (177 pM), which is 26-times higher than that of the parental strain. Post-mitochondrial supernatant extracts of both strains were unable to metabolize [ 3 H]Pentamidine, whereas under identical conditions rat liver microsomes were able to convert >5% of the drug to hydroxylation products. Thus metabolic conversion of Pentamidine does not appear to be involved in either the mode of action of or resistance to Pentamidine. Pentamidine-sensitive trypanosomes exposed for 4 h in vivo to therapeutic doses of Pentamidine (4 mg/kg) did not show any significant changes in either polyamine-, thiol- or S -adenosylmethionine metabolites, indicating that inhibition of S -adenosylmethionine decarboxylase is not involved in the trypanocidal action of the drug. However, a marked increase in basic amino acid content was noted. In particular, lysine content was increased 13-fold following exposure to Pentamidine.
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Primary and secondary metabolism of Pentamidine by rats.
Antimicrobial agents and chemotherapy, 1992Co-Authors: Bradley J. Berger, James Edwin Hall, N A Naiman, J Peggins, T G Brewer, R R TidwellAbstract:The antiprotozoal drug Pentamidine [1,5-bis(4'-amidinophenoxy)pentane] has been previously shown to be metabolized by rat liver microsomes, and five of the seven putative primary metabolites have been identified. With the synthesis and identification of 5-(4'-amidinophenoxy)pentanoic acid and 5-(4'-amidinophenoxy)-1-pentanol as the remaining two metabolites, the primary metabolism of Pentamidine in rats appears fully characterized. Use of [14C]Pentamidine with rat liver microsomes confirms this conclusion, since no unidentified radioactive peaks were detected by high-performance liquid chromatography (HPLC). Isolated, perfused rat livers were used with [14C]Pentamidine to identify secondary metabolites. Only two novel radioactive peaks were detected by HPLC analysis of perfused liver samples. The treatment of liver samples with sulfatase or beta-glucuronidase resulted in the reduction or elimination of these peaks and gave rise to peaks identified as para-hydroxybenzamidine and 5-(4'-amidinophenoxy)pentanoic acid. It was concluded from these results that only these two primary metabolites were conjugated with sulfate or glucuronic acid. After 4 h of incubation in the perfused liver system, approximately 15% of the recovered radiolabel was Pentamidine. These results suggest that Pentamidine metabolism can be rapid and extensive in rats.
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Problems and Pitfalls in the Assay of Pentamidine
Clinical Pharmacokinetics, 1992Co-Authors: Bradley J. Berger, Leonard Henry, James Edwin Hall, Richard R. TidwellAbstract:Of the existing techniques, HPLC analysis of Pentamidine allows for the most sensitive and rapid screening of clinical samples. However, it is important to test several extraction and chromatographic procedures to determine which system works consistently well for the particular samples being routinely analysed. Importantly, Pentamidine is metabolised in rats and may be metabolised in humans, so an HPLC assay should separate Pentamidine and its metabolites adequately. Capillary zone electrophoresis shows promise as a technique that may surpass HPLC for analysing Pentamidine, but remains, at this point, unsuitable for routine work due to insufficient validation.
Chuck Staben - One of the best experts on this subject based on the ideXlab platform.
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effects of Pentamidine isethionate on saccharomyces cerevisiae
Antimicrobial Agents and Chemotherapy, 1994Co-Authors: Gabriele Ludewig, J M Williams, Yi Li, Chuck StabenAbstract:We used Saccharomyces cerevisiae as a model system in which to examine the mechanism of action of the anti-Pneumocystis drug Pentamidine. Pentamidine at low concentrations inhibited S. cerevisiae growth on nonfermentable carbon sources (50% inhibitory concentration [IC50] of 1.25 micrograms/ml in glycerol). Pentamidine inhibited growth on fermentable energy sources only at much higher concentrations (IC50 of 250 micrograms/ml in glucose). Inhibition at low Pentamidine concentrations in glycerol was due to cytostatic activity rather than cytotoxic or mutagenic activity. Pentamidine also rapidly inhibited respiration by intact yeast cells, although inhibitory concentrations were much higher than those inhibitory to growth (IC50 of 100 micrograms/ml for respiration). Pentamidine also induced petite mutations, although only at concentrations much higher than those required for growth inhibition. These results suggest that a function essential for respiratory growth is inhibited by Pentamidine and that Pentamidine affects mitochondrial processes. We propose the hypothesis that the primary cellular target of Pentamidine in S. cerevisiae is the mitochondrion.
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Pentamidine sensitivity and resistance in Saccharomyces cerevisiae as a model for Pentamidine effects on Pneumocystis carinii.
Journal of Eukaryotic Microbiology, 1991Co-Authors: Hatfield C, Andrew Kasarskis, Chuck StabenAbstract:: Pentamidine isethionate inhibits growth of Saccharomyces cerevisiae. Inhibition requires lower drug concentrations under respiratory than fermentative growth conditions. Pentamidine-resistant strains bear heritable resistance mutations. Tested mutations are dominant to wild type sensitivity. These mutations may identify cellular targets of Pentamidine and potential mechanisms of fungal Pentamidine resistance.
Nicola Baker - One of the best experts on this subject based on the ideXlab platform.
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Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2
PLoS pathogens, 2016Co-Authors: Jie Song, Nicola Baker, Monja Rothert, Björn Henke, Laura Jeacock, David Horn, Eric BeitzAbstract:The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is Pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of Pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged Pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders Pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished Pentamidine-binding and parasites expressing this mutant were Pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for Pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that Pentamidine uptake is by endocytosis.
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Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for Pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs
Journal of Antimicrobial Chemotherapy, 2014Co-Authors: Jane Munday, Nicola Baker, Anthonius Eze, Lucy Glover, Caroline Clucas, David Aguinaga Andres, Manal Natto, Ibrahim Teka, Jennifer Mcdonald, Rebecca LeeAbstract:Objectives: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity Pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/Pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to Pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to Pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity Pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to Pentamidine and an arsenical. Conclusions: TbAQP2 mediates high-affinity uptake of Pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.
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trypanosoma brucei aquaglyceroporin 2 is a high affinity transporter for Pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs
Journal of Antimicrobial Chemotherapy, 2014Co-Authors: Jane C Munday, Nicola Baker, Lucy Glover, Caroline Clucas, Manal Natto, Ibrahim Teka, Anthonius A Eze, David Aguinaga Andres, Jennifer McdonaldAbstract:Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity Pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/Pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs.; The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants.; All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to Pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to Pentamidine and <1000-fold to melaminophenyl arsenicals and induced a high-affinity Pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to Pentamidine and an arsenical.; TbAQP2 mediates high-affinity uptake of Pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.
Alan H. Fairlamb - One of the best experts on this subject based on the ideXlab platform.
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Characterisation of Pentamidine-resistant Trypanosoma brucei brucei.
Molecular and Biochemical Parasitology, 1995Co-Authors: Bradley J. Berger, Nicola S. Carter, Alan H. FairlambAbstract:Following selection in vitro by exposure to increasing concentrations of the aromatic diamidine Pentamidine, a Trypanosoma brucei brucei clone has been characterised in vivo and in vitro. The resistant clone, designated T.b. brucei S427/118/PR32.6, was found to be less virulent than the parental clone T.b. brucei S427118, with an intraperitoneal injection of 2.5 × 106 resistant organisms required to produce a course of disease equivalent to 1 × 104 sensitive trypanosomes. This lowered virulence is not associated with an increased susceptibility to the host's immune system, and is not due to the in vitro culturing process. The Pentamidine-resistant clone was found to be 26- and 4.5-fold resistant to Pentamidine in vitro and in vivo, respectively. Although not cross-resistant in vivo to any other aromatic diamidines (stilbamidine, berenil and propamidine), a 2.4-fold increase in resistance to the melaminophenylarsine melarsoprol was observed. While Pentamidine completely inhibited uptake of 1 μM [3H]adenosine in the presence of 1 mM inosine, suggesting that Pentamidine is transported by the inosine-insensitive P2 transporter, the Pentamidine-resistant clone appeared to have a fully functional P2-adenosine transport system. Both resistant and parental cloned lines accumulated approx. 6 nmol Pentamidine (108 cells)−1 over the course of 3 h, representing an internal concentration of 0.7–1.0 mM. Thus, unlike previously characterised drug-resistant trypanosomes, T.b. brucei PR32.6 is not deficient in drug accumulation, suggesting that other resistance mechanisms are likely to be involved.
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Polyamine and Pentamidine metabolism in African trypanosomes
Acta tropica, 1993Co-Authors: Bradley J. Berger, Nicola S. Carter, Alan H. FairlambAbstract:Abstract A Pentamidine-resistant line of bloodstream Trypanosoma brucei brucei (S427/118) has been developed by stepwise selection in axenic culture in vitro. After 57 days of selection, the resistant line (S427/118/PR32) was able to grow normally in 32 ng/ml (54 pM) Pentamidine with an IC 50 value of 105 ng/ml (177 pM), which is 26-times higher than that of the parental strain. Post-mitochondrial supernatant extracts of both strains were unable to metabolize [ 3 H]Pentamidine, whereas under identical conditions rat liver microsomes were able to convert >5% of the drug to hydroxylation products. Thus metabolic conversion of Pentamidine does not appear to be involved in either the mode of action of or resistance to Pentamidine. Pentamidine-sensitive trypanosomes exposed for 4 h in vivo to therapeutic doses of Pentamidine (4 mg/kg) did not show any significant changes in either polyamine-, thiol- or S -adenosylmethionine metabolites, indicating that inhibition of S -adenosylmethionine decarboxylase is not involved in the trypanocidal action of the drug. However, a marked increase in basic amino acid content was noted. In particular, lysine content was increased 13-fold following exposure to Pentamidine.
Jennifer Mcdonald - One of the best experts on this subject based on the ideXlab platform.
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Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for Pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs
Journal of Antimicrobial Chemotherapy, 2014Co-Authors: Jane Munday, Nicola Baker, Anthonius Eze, Lucy Glover, Caroline Clucas, David Aguinaga Andres, Manal Natto, Ibrahim Teka, Jennifer Mcdonald, Rebecca LeeAbstract:Objectives: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity Pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/Pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to Pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to Pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity Pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to Pentamidine and an arsenical. Conclusions: TbAQP2 mediates high-affinity uptake of Pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.
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trypanosoma brucei aquaglyceroporin 2 is a high affinity transporter for Pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs
Journal of Antimicrobial Chemotherapy, 2014Co-Authors: Jane C Munday, Nicola Baker, Lucy Glover, Caroline Clucas, Manal Natto, Ibrahim Teka, Anthonius A Eze, David Aguinaga Andres, Jennifer McdonaldAbstract:Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity Pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/Pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs.; The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants.; All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to Pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to Pentamidine and <1000-fold to melaminophenyl arsenicals and induced a high-affinity Pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to Pentamidine and an arsenical.; TbAQP2 mediates high-affinity uptake of Pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.
