Table 1. Summary of results of five rounds of mutagenesis and screening, based on Y2H and HEC-1 transactivation profiles.

		Yeast				HEC-1
Round	Best variant	EC50, DHB, nm	EC50, E2, nm	Selectivity	Fold improvement	EC50, DHB, nm	EC50, E2, nm	Selectivity	Fold improvement
0	wt	500 ± 200	0.5 ± 0.3	1.0 × 10-3	1.0	66 ± 19	0.012	1.8 × 10-4	1.0
1-S	A350M	25 ± 20	3.0 ± 2.2	0.1	1.0 ± 102	ND	ND	ND	ND
2-S	A350M, L346I	10 ± 5	70 ± 30	7.0	7.0 × 103	ND	ND	ND	ND
3-S	A350M, L346I, M388Q	100 ± 80	≥5,000	≥50	≥5.0 × 104	ND	ND	ND	ND
4-S	A350M, L346I, M388Q, G521S, Y526D	65 ± 40	≥65,000*	≥1.0 × 103†	≥1.0 × 106†	0.37 ± 0.02	≥1.0 × 104	≥2.7 × 104	≥1.5 × 108
5-E	A350M, L346I, M388Q, G521S, Y526D, F461L, V560M	100 ± 40	≥106*	≥1.0 × 104†	≥1.0 × 107†	0.38 ± 0.17	≥1.0 × 104	≥2.6 × 104	≥1.4 × 108

ND, not determined.

^*Calculated from the estimated selectivity (^†) and EC₅₀ values for DHB.

^†Estimated based on incubation of yeast two-hybrid ligand response microtiter plates at room temperature for 3-4 days, after which time mutants responded to high concentrations (≥1 μM) of E₂.