Ellman's-reagent-mediated regeneration of trypanothione in situ: substrate-economical microplate and time-dependent inhibition assays for trypanothione reductase

Abstract

Trypanothione reductase (TryR) is a key enzyme involved in the oxidative stress management of the Trypanosoma and Leishmania parasites, which helps to maintain an intracellular reducing environment by reduction of the small-molecular-mass disulphide trypanothione (T[S](2)) to its di-thiol derivative dihydrotrypanothione (T[SH](2)). TryR inhibition studies are currently impaired by the prohibitive costs of the native enzyme substrate T[S](2). Such costs are particularly notable in time-dependent and high-throughput inhibition assays. In the present study we report a protocol that greatly decreases the substrate quantities needed for such assays. This is achieved by coupling the assay with the chemical oxidant 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), which can rapidly re-oxidize the T[SH](2) product back into the disulphide substrate T[S](2), thereby maintaining constant substrate concentrations and avoiding deviations from rate linearity due to substrate depletion. This has enabled the development of a continuous microplate assay for both classical and time-dependent TryR inhibition in which linear reaction rates can be maintained for 60 min or more using minimal substrate concentrations (<1 microM, compared with a substrate K (m) value of 30 microM) that would normally be completely consumed within seconds. In this manner, substrate requirements are decreased by orders of magnitude. The characterization of a novel time-dependent inhibitor, cis -3-oxo-8,9b-bis-(N(1)-acrylamidospermidyl)-1,2,3,4,4a,9b-hexahydrobenzofuran (PK43), is also described using these procedures.

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

“…The colorimetric microtitre plate assay was adapted from that described by Hamilton et al (2003). It was run in 40 mM HEPES (pH 7.5), 1 mM EDTA, 0.12 mM NADPH and 0.8 µM trypanothione TS 2 in a total assay volume of 250 µl.…”

Discovery of 2-iminobenzimidazoles as a new class of trypanothione reductase inhibitor by high-throughput screening

“…The 100,000 compounds in our 'lead discovery library' represent a diverse set of molecules as judged by Tanimoto dissimilarity analysis (T ≤ 0.85), and although simple filters based on the Lipinski criteria were not used in the selection process, 89% of the compounds in the library are Lipinski compliant 14 and 81% conform with Oprea's criteria for 'lead-likeness'. 15 An automated screening protocol for TR was developed using the photometric assay described by Hamilton et al 16,17 The assay was adapted for operation in 384-well microtitre plates and was found to be exceptionally robust and reproducible. The average Z′ and Z values 18 achieved over the entire primary screen were 0.72 and 0.54, respectively.…”

“…9, 11 and 20 (n = 4)). With n = 2, the 3,4-dichloro aryl group was tolerated regardless of linker composition (15,16,22). The 4-methoxyphenyl substituent was distinctly less favoured (5).…”

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

“…After this period, 17.5 nmol of DTNB [5,5'-dithiobis(2-nitrobenzoic acid), Ellman's reagent] was added, and the absorbance (Abs) was measured at 412 nm in the kinetic mode every 10 s for 5 min. The resulting slope of the δAbs/δt plot is proportional to DTNB reduction and the enzyme activity (Hamilton et al 2003). The enzyme inhibition was calculated as the ratio between (δAbs/δt) of the experimental wells and that of the controls without drug, that is, percent inhibition = (1 -(δAbs) exp /(δAbs) contr ) x 100.…”