Design, synthesis and biological characterization of novel inhibitors of CD38

Abstract

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.

Figure 1

Inhibitors of CD38

Figure 2

NAD⁺ and N-aromatic ether substituted nicotinamides.

Figure 2

NAD⁺ and N-aromatic ether substituted nicotinamides.

Figure 3

Biological effects of compounds 4, 7 and 9 on agonist-induced muscle contraction. A. Concentration–response curves for solvent (MQ water) control, compound 4 (active) and compound 9 (inactive), on the phenylephrine-induced vascular contraction in isolated rat aortic ring preparations. B. Nicotinamide, a commonly used inhibitor of CD38 induced similar vascular relaxation. Results are means±S.E.M. using tissues from three to five rats in each group. C. The relaxing effects of Compounds 4 and 7 on the acetylcholine-induced contraction in isolated tracheal strips of guinea pigs. Data represent the mean ± SEM (n= 7). *P<0.05, statistically significant as compared with Krebs-Henseleit solution group.

Figure 4. Structural alignment between CD38-Compound 4 and CD38-NAD complexes

(A) Surface presentation of the active pocket of CD38 (palegreen). NAD (sticks presentation in magentas) penetrated to the bottom of the active pocket of CD38, while compound 4 (sticks presentation in grey) floated over the active site. (B) The nicotinamide group of both compound 4 (grey) and NAD (magentas) is similarly positioned and stabilized by the interactions with residue Glu146, Asp155 and Trp189 of CD38.

Figure 5

a) The molecular dynamics simulation binding mode of compound 7 in the active pocket of human CD38. The carbon atoms of 7 are indicated in cyan. All the nitrogen atoms are blue; oxygen atoms are red. Hydrogen bonds are represented by blue dotted lines. Corresponding residues are labeled and shown in blue lines. b) Superimposition the binding poses of compound 4 in the enzyme-inhibitor cyrastal with molecular dynamics simulation result of 7. 4 is shown as magenta stick; 7 is shown as cyan stick.

Scheme 1

Synthesis of analogues 1 and 2.

Scheme 2

Synthesis of analogues 3–7.

Scheme 3

Synthesis of analogues 12

Scheme 4

Synthesis of 8–11.

Scheme 5

Synthesis of analogues 13 and 14.