Accounting for strain variations and resistance mutations in the characterization of hepatitis C NS3 protease inhibitors

Figures & data

Figure 1. Schematic of the characterized enzyme constructs, with the protease domain (black), the helicase domain (dark grey), the co-factor NS4A (medium grey) and N-terminal His-tags (light grey).

Figure 2. The four NS3 protease inhibitors evaluated and compared in this study.

Figure 3. Illustration of the relationship between the enzymatic activity of and the concentration of OGP.

Table 1. Reference and optimized buffer compositions for analysis of proteolytic activity of NS3 variants.

	Reference buffer					Optimized buffer
Enzyme variant	OGP (%)	Triton (%)	Glycerol (%)	NaCl (mM)	NS4Apep (µM)	OGP (%)	Triton (%)	Glycerol (%)	NaCl (mM)	NS4Apep (µM)
	0.1	–	40	–	25	0.7	–	50	–	25
	0.1	–	40	–	25	0.4	–	50	–	25
	0.1	–	40	–	25	0.4	–	50	–	25
NS3fl/4A^1a	–	0.4	40	50	–	–	0.4	40	50	25
	0.1	–	40	–	25	0.4	–	50	–	25
	0.1	–	40	–	25	0.3	–	50	–	25
NS3fl/4A^1b	–	0.4	40	50	–	–	0.4	40	50	25
	0.1	–	40	–	25	0.4	–	50	–	25

All buffers were based on 50 mM Hepes pH 7.5, 10 mM DTT, 3.3% DMSO. Evaluation of buffers was performed using 1 nM enzyme, except for , for which 5 nM was used.

Figure 4. Dependence of enzyme activity on detergent, glycerol and NaCl concentration for the studied enzyme variants. (a) in 0.4% OGP; (b) in 0.4% OGP; (c) in 0.4% OGP; (d) NS3_fl/4A^1a without NS4A_pep in 0.4% Triton; (f) NS3 _fl/4A^1a with NS4A_pep in 0.4% Triton; (e) NS4 _fl/4A^1b without NS4A_pep in 0.4% Triton; (g) NS3 _fl/4A^1b with NS4A_pep in 0.4% Triton; (h) in 0.7% OGP; (i) in 0.3% OGP; (j) in 0.4% OGP.

Table 2. Catalytic parameters of NS3 protease variants in reference and optimized buffers.

	kcat (s^−1)		Km (µM)		kcat/Km (µM^−1ċs^−1)
Enzyme variant	RB	OB	RB	OB	RB	OB
	1.24 ± 0.07	1.99 ± 0.07	1.73 ± 0.20	0.20 ± 0.03	0.71	10.1
	0.42 ± 0.01	1.02 ± 0.03	0.27 ± 0.03	0.17 ± 0.02	1.59	6.02
	0.79 ± 0.03	2.19 ± 0.08	0.07 ± 0.01	0.20 ± 0.03	11.67	11.03
NS3fl/4A^1a	0.88 ± 0.03	1.33 ± 0.03	1.02 ± 0.08	0.26 ± 0.02	0.86	5.1
	0.29 ± 0.04	0.68 ± 0.05	3.69 ± 0.79	2.69 ± 0.33	0.08	0.25
	1.04 ± 0.02	3.36 ± 0.05	0.17 ± 0.01	0.1 ± 0.01	6.27	33.31
NS3fl/4A^1b	0.97 ± 0.05	1.78 ± 0.02	1.04 ± 0.13	0.35 ± 0.02	0.93	5.2
	0.64 ± 0.03	1.10 ± 0.03	0.09 ± 0.02	0.10 ± 0.01	6.96	10.56

RB, Reference buffer; OB, optimized buffer.

Table 3. Inhibition constants (K_i) for NS3 protease variants in reference and optimized buffers.

	Ki (nM)
	N-1725		BILN-2061		ITMN-191		VX-950*
Enzyme variant	RB	OB	RB	OB	RB	OB	RB	OB
	3.23 ± 0.29	0.99 ± 0.06	0.32 ± 0.05	0.07 ± 0.02	0.11 ± 0.02	0.19 ± 0.04	68.0 ± 11.7	18.6 ± 2.07
	10.5 ± 2.35	3.20 ± 0.38	0.10 ± 0.01	0.05 ± 0.026	0.051 ± 0.003	0.046 ± 0.003	37.6 ± 3.16	20.2 ± 1.18
	38.5 ± 4.19	61.6 ± 9.77	23.9 ± 3.57	120 ± 17.8	3.20 ± 0.54	8.33 ± 1.09	81.9 ± 8.65	264 ± 18.7
NS3fl/4A^1a	82.8 ± 12.0	2.36 ± 0.34	0.63 ± 0.06	0.36 ± 0.02	0.11 ± 0.01	0.07 ± 0.01	86.3 ± 17.6	42.7 ± 6.15
	21.8 ± 5.10	13.5 ± 1.05	2.68 ± 0.42	2.14 ± 0.26	0.95 ± 0.20	0.95 ± 0.16	344 ± 102	391 ± 71.9
	2.42 ± 0.19	1.24 ± 0.14	0.16 ± 0.012	0.11 ± 0.01	0.11 ± 0.02	0.06 ± 0.01	71.1 ± 4.27	28.1 ± 0.99
NS3fl/4A^1b	34.7 ± 8.24	9.33 ± 1.12	0.44 ± 0.06	0.36 ± 0.04	0.13 ± 0.02	0.09 ± 0.01	65.2 ± 6.26	78.4 ± 9.24
	34.0 ± 7.17	39.1 ± 4.43	15.8 ± 3.71	29.2 ± 3.27	24.2 ± 5.91	32.9 ± 2.90	82.9 ± 8.20	102 ± 4.2

RB, Reference buffer; OB, optimized buffer.

*Apparent K_i value due to mechanism-based inhibition.

Table 4. Vitality values for the four inhibitors in this study under optimized conditions, where is used as reference.

	Vitality values
Enzyme variant	N-1725	BILN-2061	ITMN-191	VX-950*
	1	1	1	1
	35	4300	330	24
	2.1	12	7.2	7.7
	21	1000	1200	8.9

*Based on apparent K_i value due to mechanism-based inhibition.