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. 2016 Sep 26;45(2):e10. doi: 10.1093/nar/gkw853

Figure 4.

Figure 4.

Validation of single-well determination of inhibition constants of PSMA and CAIX inhibitors using DIANA. (A) Plot of Ki values of 41 PSMA inhibitors determined by titrating recombinant purified PSMA with inhibitor and measuring kinetics (x-axis) versus Ki values determined by DIANA from a single well containing 100 μM inhibitor and recombinant purified PSMA (y-axis). (B) Plot of Ki values of 41 PSMA inhibitors determined by DIANA from a single well containing 100 μM inhibitor with recombinant purified PSMA (x-axis) versus Ki values determined by DIANA from two measurements with unpurified endogenous PSMA in 1 μl of human serum in the presence of either 100 μM or 100 nM inhibitor (y-axis). (C) Plot of Ki values of 10 CAIX inhibitors determined by titrating recombinant purified CAIX with inhibitor and measuring kinetics (x-axis) versus Ki values determined by DIANA from two measurements with unpurified CAIX in cell lysate in the presence of either 100 μM or 1 μM inhibitor (y-axis). (D) Plot of Ki values of 10 CAIX inhibitors determined by DIANA from two measurements with unpurified CAIX in cell lysate in the presence of either 100 μM or 1 μM inhibitor (x-axis) and by DIANA from two measurements with unpurified endogenous CAIX in 10 μl of human serum in the presence of either 100 μM or 1 μM inhibitor (y-axis). (A–D) Lines show linear regression of log-transformed values; dashed lines indicate values that are 1.5-fold (PSMA) or 2-fold (CAIX) higher or lower than the linear fit. Error bars show s.d. of duplicate measurements in the case of DIANA, or s.e. of the titration in the case of enzyme kinetics.