Fig. 7: EGCG provides a basis for generating novel NAD-producing compounds.

A An NMNAT2 homology model using NMNAT1 and NMNAT3 was further refined using loop-modelling software (DaReUS-Loop) to refine a low homology domain in the central region of the protein (green). Molecular dynamic simulations demonstrated that the protein reached a stable conformation after 100 ns. The final generated protein conformations demonstrated greater reliability (ERRAT, VERIFY 3D, PROVE, and Z-score). B Three potential druggable binding pockets (independent of the NMN catalytic pocket) were identified. The docking pose of EGCG was well accommodated in these three different druggable pockets with corresponding ΔG scores < −40 kcal/mol (left). Only one ligand-protein complex (Site 1; orange) could maintain a stable conformation over a 500 ns molecular dynamic simulation (right), demonstrating in silico evidence that EGCG can directly bind to NMNAT2. C Given its poor drug-like properties, EGCG was used as a tool compound to identify novel NAD-producing compounds. The EGCG structure was truncated in series to identify a biologically active core which was then used in a scaffold-hopping strategy. D An iterative synthesis and NAD-testing (luminometry of dissociated cortex neurons) pipeline was established to identify and test NAD-producing compounds. A number of compounds with greater efficacy than EGCG at producing NAD were identified (n = 4/condition; statistical testing in Supplementary Data 1). Outliers denoted by black circles. *P < 0.05, **P < 0.01, ***P < 0.001, NS non-significant (P > 0.05). For box plots, the centre hinge represents the median with upper and lower hinges representing the first and third quartiles; whiskers represent 1.5 times the interquartile range.