Table 3.

Mode of inhibition of TbTryS by Ebselen.

Time-dependent inhibition assaya	Sample
	A	B	C
Pre-incubation (1 h)	Ebselen + TbTryS	Ebselen + MM	No
Reaction (1 h) started with	MM	TbTryS	Ebselen + TbTryS + MM
TryS inhibition ± SD (%, n = 6)	43.6 ± 4.5	−0.7 ± 2.4	1.8 ± 4.2
Irreversible inhibition assayb
Pre-incubation Ebselen + TbTryS (1 h)	Yes	Yes	Yes
Buffer exchanged	Yes	Yes	No
Incubation with DTT (5 mM, 30 min)	Yes	No	No
TryS inhibition ± SD (%, n = 6)	89 ± 8.5	97.0 ± 5.0	100 ± 9
Mass spectrometry analysisc
1. Pre-incubation with Ebselen (1 h)	Yes	No	No
2. Pre-incubation IAMd (15 mM, 1 h)	No	Yes	No
3. Desalting and IAMd treatment (15 mM, 1 h)	Yes	Yes	No
TryS inhibition ± SD (%, n = 8)	100 ± 7 (96 ± 8)e	20 ± 9

TryS activity data (% inhibition) here reported was determined according to the assay described in section Inhibition mode of candidate hits against TbTryS.

^aEbselen tested at its IC₅₀ against TbTryS (5 µM), MM: master mix.

^bEbselen added at two-fold its IC₅₀ against TbTryS (10 µM).

^cThe data reported correspond to two independent experiments.

^dIAM, iodoacetamide.

^eEbselen added at 4- (20 µM) or 10-folds (50 µM, value in parenthesis) its IC₅₀ against TbTryS.