ABSTRACT
Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb) and is ranked as the second killer infectious disease after COVID-19. Proteasome accessory factor A (PafA) is considered an attractive target because of its low sequence conservation in humans and its role in virulence. In this study, we designed a mutant of Mtb PafA that enabled large-scale purification of active PafA. Using a devised high-throughput screening assay, two PafA inhibitors were discovered. ST1926 inhibited Mtb PafA by binding in the Pup binding groove, but it was less active against Corynebacterium glutamicum PafA because the ST1926-binding residues are not conserved. Bithionol bound to the conserved ATP-binding pocket, thereby, inhibits PafA in an ATP-competitive manner. Both ST1926 and bithionol inhibited the growth of an attenuated Mtb strain (H37Ra) at micromolar concentrations. Our work thus provides new tools for tuberculosis research and a foundation for future PafA-targeted drug development for treating tuberculosis.
Subject(s)
Mycobacterium tuberculosis , Proteasome Inhibitors , Adenosine Triphosphate/metabolism , Bacterial Proteins/metabolism , Bithionol/metabolism , Mycobacterium tuberculosis/drug effects , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/chemistry , Proteasome Inhibitors/pharmacologyABSTRACT
By searching the mouse EST database, we identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (RIKEN cDNA 2410078J06). Sequence analysis revealed that this new SULT belongs to the cytosolic SULT6 gene family. The recombinant form of this newly identified SULT, designated SULT6B1, was expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Purified mouse SULT6B1 exhibited sulfonating activity toward thyroxine and bithionol among a variety of endogenous and xenobiotic compounds tested as substrates. pH optimum of purified mouse SULT6B1 was determined to be 8.0. Tissue-specific expression of mouse and human SULT6B1 was examined by RT-PCR. While human SULT6B1 was specifically expressed in kidney and testis, mouse SULT6B1 was detected in brain, heart, kidney, thymus, lung, liver and testis. Further studies are needed in order to clarify the role of SULT6B1 in the metabolism of thyroxine and possibly some xenobiotics in mouse.
Subject(s)
Recombinant Fusion Proteins/metabolism , Sulfotransferases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Bithionol/metabolism , Cloning, Molecular , DNA, Complementary/metabolism , Expressed Sequence Tags , Female , Gene Expression , Gene Library , Humans , Hydrogen-Ion Concentration , Kinetics , Male , Mice , Molecular Sequence Data , Myocardium/enzymology , Organ Specificity , Phosphoadenosine Phosphosulfate/metabolism , Phylogeny , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Sulfotransferases/chemistry , Sulfotransferases/genetics , Thyroxine/metabolismABSTRACT
A sensitive and specific method is described for determination of 5 fasciolicides in milk. The drugs are used to control liver flukes in cattle. The milk sample was homogenized with acetone and acetonitrile, sonicated, and centrifuged. The supernatant was extracted with dichloromethane. The extract was evaporated to dryness, dissolved in 1% sodium hydrogen carbonate, and purified on a C18 cartridge. The 5 drugs were separated from the matrix by reversed-phase liquid chromatography (LC) and determined by dual-electrode coulometric detection on a Kaseisorb LC ODS-300-5 column. The mobile phase was acetonitrile-0.05M potassium dihydrogen phosphate (55 + 45) at pH 3.0. The flow rate was 1 mL/min at 40 degrees C. The applied potentials of detectors 1 and 2 were set at 0.20 and 0.55 V, respectively. The average recovery of the drugs added to milk at 0.01 and 0.1 micrograms/mL was 89.6%, and the coefficient of variation was 4.7%. The detection limits of the drugs in milk were 4-20 ng/mL. The method is used to monitor commercial milk samples and to determine the residual levels of these drugs in milk from cows treated with a fasciolicide.
Subject(s)
Anthelmintics/analysis , Drug Residues/analysis , Milk/chemistry , Acetone/chemistry , Acetonitriles/chemistry , Animals , Anthelmintics/metabolism , Bithionol/analysis , Bithionol/metabolism , Cattle , Chlorophenols/analysis , Chlorophenols/metabolism , Chromatography, High Pressure Liquid , Chromatography, Liquid , Drug Residues/metabolism , Electrochemistry , Gas Chromatography-Mass Spectrometry , Milk/metabolism , Oxyclozanide/analysis , Oxyclozanide/metabolism , Polybrominated Biphenyls/analysis , Polybrominated Biphenyls/metabolism , Reference Standards , Salicylanilides/analysis , Salicylanilides/metabolism , Sensitivity and Specificity , Sodium Bicarbonate/chemistryABSTRACT
The mutagenic effects of bithionol sulfoxide and its two major metabolites, bithionol and bithionol sulfone, on 4 Salmonella typhimurium strains (TA97, TA98, TA100 and TA102) were investigated. Bithionol sulfoxide was found to be mutagenic to TA98 and TA100. However, mutagenicity was abolished in the presence of rat-liver S9 fractions.
Subject(s)
Bithionol/pharmacology , Phenols/pharmacology , Salmonella typhimurium/drug effects , Animals , Biotransformation , Bithionol/analogs & derivatives , Bithionol/metabolism , Inactivation, Metabolic , Male , Microsomes, Liver/metabolism , Mutagenicity Tests , Rats , Rats, Inbred StrainsABSTRACT
The distribution of a single oral dose of 14C bithionol sulfoxide in mice was determined by whole body autoradiography. The bithionol sulfoxide was found in the whole organism mainly in richly vascularized organs, liver, kidney, lung. It did not pass through the blood-brain barrier and did not accumulate in any particular organ but persisted in the blood. Observations were performed in 18-day pregnant mice in order to investigate an eventual placental transfer. Six hours after the administration, radioactivity was detected in blood and fetal vascularized tissues.
Subject(s)
Bithionol/metabolism , Phenols/metabolism , Animals , Autoradiography , Bithionol/analogs & derivatives , Female , Kinetics , Male , Maternal-Fetal Exchange , Mice , Pregnancy , Time Factors , Tissue DistributionABSTRACT
The metabolic detoxication of bis(2-hydroxy-3, 5-dichlorophenyl)sulfoxide (BTS) in man was investigated. Bis(2-hydroxy-3, 5-dichlorophenyl)sulfide (BT) was identified in beta-glucuronidase treated urine following the administration of BTS by thin-layer chromatography, gas chromatography, ultraviolet spectrum and quantitative analysis. No other metabolites were detectable. BT-glucuronide was also identified in urine. It was assumed that BTS was reduced to BT and successively conjugated with glucuronide in man, and excreted as BT-glucuronide in the urine.
