Table 1.
Screening up-mutants for GFRAL binding affinity and p-ERK signaling activity.
| Fc-GDF15 variant | NxT mutation | Up-mutation | KD analysis (N = 2) | Functional activity (N = 3) | ||
|---|---|---|---|---|---|---|
| hGFRAL ECD KD (nM) |
mGFRAL ECD KD (nM) |
% Intrinsic activity (Ymax) | EC50 (pM) | |||
| Fc-GDF15 WT | – | – | 11.0 ± 8.2E−10 | 230.0 ± 4.2E−08 | 100 | 177 ± 95 |
| Mutant 2 | A3N/N5T | – | 10.0 ± 4.0E−11 | 245.0 ± 3.3E−08 | 64 ± 17 | 114 ± 19 |
| Mutant 2A | A3N/N5T | L36H | 6.03 ± 2.3E−11 | 186.0 ± 1.3E−08 | 66 ± 18 | 312 ± 61 |
| Mutant 2C | A3N/N5T | V98I | 5.52 ± 4.0E−10 | 95.8 ± 3.2E−09 | 85 ± 16 | 436 ± 277 |
| Mutant 3 | R4N/G6T | – | 11.2 ± 3.7E−10 | 300.0 ± 8.1E−08 | 59 ± 18 | 298 ± 95 |
| Mutant 3A | R4N/G6T | L36H | 6.09 ± 4.5E−12 | 178.0 ± 2.8E−09 | 67 ± 15 | 4838 ± 2165 |
| Mutant 3B | R4N/G6T | L36R | 3.68 ± 8.9E−11 | 82.8 ± 2.0E−09 | 47 ± 14 | 4225 ± 3773 |
| Mutant 3C | R4N/G6T | V98I | 5.44 ± 4.0E−11 | 124.0 ± 4.2E−08 | 45 ± 12 | 642 ± 210 |
Table summarizing in vitro characteristics of Fc-GDF15 up-mutants compared to a wildtype (WT) Fc-GDF15 control. Binding affinity (KD) was measured for recombinant human and mouse GFRAL extra-cellular domain (ECD) using surface plasmon resonance (N = 2). Functional activity was measured using p-ERK HTRF assay and reported as EC50 and % intrinsic activity (Ymax) normalized to Fc-GDF15 WT (N = 3). Data are shown as mean ± SD.