Table 1 Screening up-mutants for GFRAL binding affinity and p-ERK signaling activity.

From: Fc-GDF15 glyco-engineering and receptor binding affinity optimization for body weight regulation

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

  1. 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.
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