Table 3.

Relative affinity of genotype 1 NS3-NS4A variant enzymes to BI 201335

NS3/4A varianta	Change in the relative affinity of the enzyme (fold change in Ki [app]) tob:
	BI 201335	Telaprevir	Boceprevir	TMC435	ITMN-191
GT 1a H77
R155K	420 ± 30	24 ± 6	14 ± 6
D168V	>690	0.34 ± 0.02	0.37 ± 0.09
V36M/R155K	530 ± 80	264 ± 105	126 ± 54
GT 1b CON-1
R155K	210 ± 52	20 ± 9	6.5 ± 1.6	44c	31 ± 2
R155Q	430 ± 20			13c	18 ± 1
A156T	710c	>416	540c	21c	4.0 ± 0.2
A156V	1100 ± 43	>398	>1,041		7.3 ± 0.8
D168V	830 ± 106	0.3 ± 0.1	0.6 ± 0.1	>52	5.2 ± 0.2
D168A	236 ± 27			>68	15 ± 2
V36M/A156T	462 ± 40	>459	1,080 ± 34
V36 M	1c	18 ± 2	6.3 ± 0.5		1.6c
Q41R	2.2 ± 0.1	1.5 ± 0.2	2.5 ± 0.7	1.7 ± 0.2
T54A	1.3 ± 0.3	9.8 ± 2.9	6.6 ± 2.7	0.9 ± 0.2
F43S	1.3 ± 0.3	7.2 ± 0.0	4.4 ± 0.5	8.0 ± 1.9
F43V	1.6 ± 0.6	6.9 ± 0.2	8.5 ± 0.3	>34
Q80K	1.9 ± 0.1	1.0 ± 0.0		2.8 ± 0.4	1.3 ± 0.1
Q80R	1.7 ± 0.1	1.1 ± 0.1		4.0 ± 0.2	1.3 ± 0.2

^aReplicon variants in boldface type represent those selected in replicon colonies resistant to BI 201335.

^bKinetic parameters for NS3-NS4A proteins and inhibition by PIs were determined using the depsipeptide fluorogenic substrate derived from the NS5A-NS5B cleavage site: anthraniloyl-DDIVP-Abu[C(O)-O] AMY(3-NO₂)-TW-OH. The assay conditions are similar to those previously described (17, 25). For boceprevir and telaprevir, the compound was preincubated with enzyme for 60 min prior to the addition of substrate. The calculated percentage of inhibition at each inhibitor concentration was used to determine the median effective concentration (IC₅₀) by nonlinear regression routine Symyx Assay Explorer v3.2 SP1 of Symyx Technologies. K_i (apparent [app]) values were calculated from the IC₅₀s using the equation IC₅₀ = K_i (1 + [S]/K_m) where S is substrate. Fold change = K_i of the NS3 mutant/K_i for the WT NS3. For most of the data, the fold change value is the mean ± SD, which was calculated from at least 3 experiments.

^cn < 3 experiments.