(A) Hydrolase activity of recombinant human CD38 (rhCD38) in the presence of 78c, using 1,N6-ethenoadenine dinucleotide (ɛ-NAD+) as substrate (n = 3 experiments, half-maximal inhibitory concentration [IC 50 ] = 17.7 nM). Inset shows the structure of 78c. R 1 = H; R 2 = Me; R 3 = trans-4-OCH 2 CH 2 OMe-cyclohexyl.

(B) Effect of different substrate concentration on 78c inhibition of rhCD38 hydrolase activity. Experimental data were fitted by steady-state equations derived from the kinetic model in (C).

(C) Kinetic model of CD38 inhibition by 78c. Model depicts two interconnected cycles, one for the hydrolytic activity of rhCD38 (left side) and the other for the cyclase activity of the enzyme as well as the inhibition by its products nicotinamide and ADP-ribose (right side). In the reaction scheme, E represents the enzyme CD38; k (1–7) , the substrate binding constants; and K i , the inhibitor binding constant.

(D) NADase activity in tissue homogenates from 1-year-old wild-type (WT) and CD38 KO mice, assayed in the presence of 78c (n = 3 experiments). IC 50 : liver (3.8 nM), skeletal muscle (4.4 nM), spleen (0.7 nM), brain (14.8 nM), ileum (0.4 nM), subcutaneous fat (5.2 nM). AFU, arbitrary fluorescence units.

(E) NAD+ levels in WT and CD38 KO MEFs treated for 24 hr with 0.2 μM 78c (n = 4–6 experiments). NAD+ levels were calculated relative to control WT MEFs.

(F) NAD+ levels in tissues of 1-year-old WT and CD38 CI mice treated with 78c or vehicle (control) for 8 days (n = 4 mice per group). Spleen protein lysates of WT and CI mice were immunoblotted for CD38 and tubulin (right panel).

(G) Activity of human recombinant PARP1 in the presence of 78c or the PARP inhibitor olaparib (n = 4 experiments).

(H) NAD+ levels in A549 cells treated for 24 hr with 0.5 μM 78c and/or 5 μM olaparib (n = 3–6 experiments).

(I) Activity of human recombinant SIRT1 in the presence of 100 nM 78c or 100 μM SIRT1 inhibitor suramin (n = 3 experiments).

(J) Activity of human recombinant NAMPT in the presence of 100 nM 78c or 20 μM of NAMPT inhibitor FK866 (n = 3 experiments).