FIGURE 3.

C-terminal hFGF21 cleavage in serum requires FAP. A, human serum immunodepleted by anti-hFAP or control IgG antibody was incubated with hFGF21 or hFGF21^R overnight at 37 °C. Immunoblot analysis was used to assess immunodepletion of FAP and FGF21 cleavage. DPPIV and GADPH served as loading controls. B, FAP activity in the immunodepleted serum samples used in A was determined by FRET-quench using wild-type (top) or cleavage-resistant peptides. C, hFGF21 was incubated with recombinant hFAP at a molar ratio of 4:1 (top) or with FBS (bottom) and the indicated concentrations of compound 60. FGF21 cleavage was visualized by immunoblot analysis with anti-hFGF21. As a control, 0.1% dimethyl sulfoxide (DMSO, vehicle) was tested. In addition, hFGF21 incubated with HI serum was loaded also as a control. D, FRET-quench was used to determine the IC₅₀ value for compound 60 for recombinant human (0.37 ± 0.02 nm), cynomolgus monkey (0.35 ± 0.02 nm), and mouse FAP (0.47 ± 0.03 nm).