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

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 μM, compared with a substrate K_m value of 30 μM) 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-(N¹-acrylamidospermidyl)-1,2,3,4,4a,9b- hexahydrobenzofuran (PK43), is also described using these procedures.