Figure 3.

CZ-48 Mimics Endogenous Metabolite NMN in Activating SARM1

(A) HEK-293T cells were treated with 100 μM of different compounds for 24 h, and cADPR contents were measured. The structures of the compounds are shown in the right panel.

(B) SARM1-FLAG-overexpressing HEK-293 cells were permeabilized by 100 μM digitonin, and the supernatant was incubated with 100 μM of different compounds, together with 50 μM ɛNAD, and the activities of SARM1 (slopes of the fluorescence production) were analyzed and plotted.

(C) The dose-response curves of CZ-48 and NMN on SARM1-FLAG in the cell lysate. Lysate of SARM1-FLAG cells, with HEK-293 as a control, were incubated with different doses of NMN or CZ-48, and NADase activities were measured as (B).

(D) The proteins SARM1-FLAg were immunoprecipitated with anti-FLAg beads, eluted with 3× FLAG peptide, and the NADase activities were measured as in (B) in the presence of 100 μM NMN or CZ-48.

(E–G) Cellular levels of NMN (E), cADPR (F), and NAD (G) were measured by cycling assays in wild-type, NMNAT1-knockout, and NMNAT1/SARM1 double knockout (DKO) HEK-293T cells. All the above experiments were repeated at least three times (means ± SDs; n ≥ 3; Student's t test, **p < 0.01, ***p < 0.001, ****p < 0.0001).