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. 2016 Apr 22;291(27):13974–13986. doi: 10.1074/jbc.M116.725978

FIGURE 2.

FIGURE 2.

A, potency scatter plot (IC50 Nav1. 7 versus IC50 counter-screening channel Nav1.2) of molecules generated by directed evolution experiments. B, same plot as in A, but with Nav1.6 as counter-screening channel. The starting molecule (CcoTx1) and the molecule selected for saturation mutagenesis and optimization (2670) are depicted in yellow. Increased potency and selectivity are highlighted by black arrows. The three rounds of directed evolution are color-coded black (round 1), blue (round 2), and red (round 3). C–F, certain amino acids that increase selectivity are preferentially found in some positions. Position-specific potency scatter plots are shown (Nav1.7 versus Nav1.2), where each compound's potency on the two channels is represented with a letter corresponding to the amino acid at that specific position. Positions 5, 12, and 20 (C and D) show good separation between amino acids, suggesting clear selectivity preferences. The plots indicate that a methionine achieves better selectivity than tryptophan at position 5; glutamate achieves better selectivity than lysine at position 12, and arginine achieves better selectivity than tyrosine at position 20. In contrast, at position 32 (F), no strong separation is observed between aspartate and lysine.