Figure 2. Purified Compound Z Binds PrP^C Reversibly, Inhibiting Aβo/PrP^C Interaction via Competition for PrP^C Aβo-Binding Domains.

(A) Biolayer interferometric association (60–180 s) and dissociation (180–300 s) traces of 10–20 kDa Z with PrP^C-coated sensor in 4-fold dilution steps from 1 μM top concentration, indicating a dissociation constant of 1.7 nM (top graph). Aβo-coated sensor detects soluble full-length PrP^C interaction but not compound Z (middle and bottom graphs, respectively).

(B) PLISA measurement of 10–20 kDa Z Aβo/PrP^C inhibitory activity, indicating an IC₅₀ of 910 pM. Data are mean ± SEM, n = 3 replicates per sample.

(C) PrP^C immunoblot of non-denaturing gel-shift assay of full-length PrP^C incubated with 10–40 kDa Z. Laddering indicates multiple PrP^C molecules bound per Z molecule. Incubation at 65°C after co-incubation shows reversible association of Z and PrP^C.

(D) Biotinylated 10–20 kDa Z binds full-length PrP^C-coated plate concentration-dependently. Data are mean ± SEM, n = 3 replicates per sample.

(E) Binding of biotinylated Z to PrP^C-coated plate is inhibited by antibodies directed against either of the two Aβo-binding domains on PrP^C and not by antibodies against other regions of PrP, indicating direct and selective Z interaction with PrP^C Aβo-binding domains. Data are mean ± SEM, n = 3 replicates per sample. *p <0.05, Student’s t test.