TABLE 4.

Effects of characteristic mutations at the palm and thumb binding sites of NS5B po ly mer ase on the binding affinities of NNIs

All of theK_d values shown are mean and S.D. values calculated from at least three independent experiments. TheK_d values are in boldface type when the values of compound binding to mutant proteins are more than 3-fold larger than the respective compound binding to the wild-type NS5B protein.

Compounds	Binding sites	Kd
		Wild type	M414T	C316Na	L419M	P495L
		μm
NNI-1b Benzothiadiazine	Palm 1	0.016 ± 0.01	1.3 ± 0.04	0.032 ± 0.005	0.023 ± 0.004	0.018 ± 0.005
HCV-796b Benzofuran	Palm 2	0.071 ± 0.02	0.043 ± 0.016	0.96 ± 0.23	0.064 ± 0.016	0.045 ± 0.009
NNI-3b Thiophene	Thumb 2	0.024 ± 0.015	0.048 ± 0.014	0.062 ± 0.016	0.32 ± 0.06	0.067 ± 0.013
NNI-4c Benzimidazole	Thumb 1	13 ± 2.3	14 ± 2.2	NAd	15 ± 2.9	40 ± 8.4

^a C316N is a natural polymorphism in HCV genotype 1b; the residue is C316 in Con1 strain and N316 in the BK strain.

^b TheK_d values of NNI-1, HCV-796, and NNI-3 were tested on NS5B570_Con1 (GT1b). TheK_d values for NNI-1, -3, and -4 were measured after 10 min of incubation with NS5B, except for HCV-796, which was measured after an incubation of 3 h 10 min.

^c TheK_d values of NNI-4 were tested on NS5B570-BK (GT 1b).

^d NA, not available.