Fig. 8: Generation of NAD-salvage pathway specific compounds that drive NAD production and provide neuroprotection.

A We selected 10 compounds for further testing based on their NAD-producing capacity (structures shown in comparison to EGCG). B For 9 of the 10 top compounds, FK866 suppressed the NAD-boosting effect, demonstrating that these drugs retained a mechanism of action through the NAD-salvage pathway (n = 4 cortex for all conditions; fold change in NAD comparable to normal controls, denoted by red space; fold change comparable to FK866 treated normal controls, denoted by black space; statistical testing in Supplementary Data 2). C Compounds were tested for NAD modifying capacity in dissociated cortex, liver, muscle, and spleen (n = 4 for all conditions). An NAD-boosting effect in neuron-low tissue was only demonstrated for 2 compounds (51, 56) with both increasing NAD in muscle by <1.2 fold relative to untreated controls. A reduction in NAD relative to control was identified for 3 compounds in neuron-low tissue, with compound 21 and 55 reducing NAD in spleen to ~0.8 fold, and compound 54 reducing NAD in muscle to ~0.9 fold. Results are normalized to untreated controls of matched tissue type. D The compounds were used to generate a structure-activity relationship model which identified favourable (green, at C2) and unfavourable (purple) hydrophobic regions which affect the activity, and negative electrostatics regions (blue, at C1) which are crucial for NAD-boosting activity. E Five of the top ten compounds were tested for neuroprotective capacity in a retinal explant model. Compounds 54 and 55 (from group 5) demonstrated a significant protection of RGCs, demonstrating the potential of these compounds to provide neuroprotection (n = 6 retina/condition). Scale bar = 20 µm. For B, C, and E, *P < 0.05, **P < 0.01, ***P < 0.001, NS non-significant (P > 0.05), Student’s t-test to control. 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.