Table 2.
IPI and DVT competitive binding analysis of CHO expressed SHBG mutants of key binding interaction residues identified from the structure model
For WT SHBG and different SHBG mutants, IC50 values for IPI and DVT were calculated from competitive displacement of [3H]DHT using unlabeled DHT as the reference. Errors were established using the 95% confidence interval of the IC50 values produced from nine concentration point displacement curves. The RBA values were calculated by expressing the DHT IC50 values of IPI or DVT as a percentage of the DHT IC50s for WT SHBG or given mutant, thereby allowing comparisons of mutants where DHT affinity was also changed relative to WT SHBG. NCD, no competitive displacement.
| SHBG mutant | DHT IC50 (nm) | IPI IC50 (nm) | DVT IC50 (nm) | IPI (RBA%) | DVT (RBA%) |
|---|---|---|---|---|---|
| Wildtype | 4.7 ± 0.7 | 55 ± 7 | 2500 ± 370 | 8.5 | 0.19 |
| D65A | 4.0 ± 1.8 | 430 ± 160 | NCD | 0.93 | NCD |
| M107V | 3.6 ± 1.0 | 7.6 ± 1.3 | 690 ± 350 | 47 | 0.52 |
| M139V | 4.6 ± 1.7 | 41 ± 27 | 590 ± 370 | 11 | 0.78 |
| F67A | 5.1 ± 1.6 | 390 ± 240 | 820 ± 320 | 1.3 | 0.62 |
| R135L | 3.0 ± 0.6 | 30 ± 13 | 3100 ± 1600 | 10 | 0.10 |
| N82A | 3.3 ± 1.2 | 23 ± 7 | 310 ± 180 | 14 | 1.1 |
| E176K | 14 ± 2 | 150 ± 16 | 4200 ± 790 | 9.3 | 0.33 |
| S42A | 42 ± 9 | NCD | 4100 ± 950 | NCD | 1.0 |