High-throughput screening affords novel and selective trypanothione reductase inhibitors with anti-trypanosomal activity

Bioorganic and Medicinal Chemistry Letters

Volumn 17, Issue 5, 2007, Pages 1280-1283

  Martyn, Derek C ^a   Jones, Deuan C ^b   Fairlamb, Alan H ^b   Clardy, Jon ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

^b UNIVERSITY OF DUNDEE   (United Kingdom)

Author keywords

High throughput screening; Trypanosomiasis; Trypanothione reductase

Indexed keywords

ENZYME INHIBITOR; TRYPANOTHIONE REDUCTASE; TRYPANOTHIONE REDUCTASE INHIBITOR; UNCLASSIFIED DRUG;

ANTIPROTOZOAL ACTIVITY; ARTICLE; DRUG ACTIVITY; DRUG IDENTIFICATION; DRUG SCREENING; DRUG SELECTIVITY; ENZYME ACTIVITY; ENZYME INHIBITION; HIGH THROUGHPUT SCREENING; IN VITRO STUDY; NONHUMAN; OXIDATIVE STRESS; TRYPANOSOMA BRUCEI;

ANIMALS; DRUG EVALUATION, PRECLINICAL; ENZYME INHIBITORS; GLUTATHIONE REDUCTASE; HUMANS; NADH, NADPH OXIDOREDUCTASES; STRUCTURE-ACTIVITY RELATIONSHIP; TRYPANOCIDAL AGENTS; TRYPANOSOMA; TRYPANOSOMA BRUCEI BRUCEI;

TRYPANOSOMA BRUCEI; TRYPANOSOMATIDAE;

EID: 33846933089     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2006.12.016     Document Type: Article

References (13)

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8. An assay solution containing potassium HEPES (40 mM, pH 7.4), EDTA (1 mM), trypanothione disulfide (4.22 μM), DTNB (70.3 μM), and TR (2.81 mU/mL) was prepared. The positive control contained the above reagents plus the TR inhibitor clomipramine hydrochloride (200 μM). Eighty microliter per well of assay solution or positive control is added to the appropriate columns of clear Nunc 384-well Polysorb polystyrene plates using a BioTek μFill liquid handler. A CyBio liquid handler was used to transfer 200 nL of library compound, dissolved in DMSO at a concentration of 5 mg/mL, to each well. Enzymatic activity is initiated by the addition of 10 μL of an NADPH solution (135 μM in potassium HEPES, 40 mM, pH 7.4, and EDTA, 1 mM). The NADPH solution is added to four plates at a time, with 4 min 20 s delay between each set of plates. Plates are incubated for 60 min at 30 °C, before being removed and read in batches of four on a Perkin-Elmer Envision plate reader at 405 nm. Raw data are processed using Microsoft Excel.
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10. An assay solution containing potassium HEPES (40 mM, pH 7.4), EDTA (1 mM), NADPH (130 μM), and TR (10 mU/mL) was prepared. The positive control does not use enzyme. Twenty microliter per well of reagent A or positive control is added to the appropriate columns of clear Nunc 384-well Polysorb polystyrene plates. One-hundred nanoliter of library compound, dissolved in DMSO at a concentration of 5 mg/mL, was added to each well. Enzymatic activity is initiated by the addition of 20 μL of a trypanothione disulfide solution (110 μM in potassium HEPES, 40 mM, pH 7.4, and EDTA, 1 mM). The trypanothione disulfide is added to four plates at a time, with 4 min 20 s delay between each set of plates. Incubation, plate reading, and data analysis are performed as in Ref. 9, except that the plates are read at 355 nm.
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13. 2 humidified atmosphere for 72 h, then 45 μM resazurin (Sigma) added. After a further 4-h incubation, fluorescence due to formation of resorufin was measured at λ excitation 528 nm, λ emission 590 nm. Each inhibitor set was tested in triplicate and the experiments repeated on three separate occasions. Data are presented as weighted means and standard errors of the mean.