Figure 1.
Characterization of the SIRT2 inhibitor AK7 in vitro and in a cell model of aSyn toxicity.
(A) Determination of AK7 IC50 on SIRT2 deacetylase activity at varying substrate (α-tubulin K40 peptide) and co-factor (NAD+) concentrations (circles 80 μM α-tubulin/1 mM NAD+; squares 80 μM α-tubulin/200 μM NAD+; triangles 400 μM α-tubulin/1 mM NAD+). Each data point is the average of at least 3 independent measurements. IC50 fits are shown as lines. (B) Docking model of a SIRT2/AK7 complex. The structure of SIRT2 (PDB ID 3zgv) is shown in gold with the catalytic His187. ADP-ribose and substrate acetylated lysine are shown in light green and deep teal, respectively. The best pose of AK7 (magenta) mainly occupies the NAD+ binding site. (C) LUHMES cells were transduced with a lentivirus encoding aSyn. 10 days after differentiation, in the presence of vehicle alone (red, 100% toxicity) or different concentrations of AK7 (black line), media were collected and AK activity was measured. (D) Percentage of AK activity in the presence of 12.5 M of AK7, compared to vehicle-treated cells (**p<0.01).
Figure 2.
Protective effects of AK7 in acute MPTP mouse model of PD.
Mice received a single injection (i.p.) of MPTP at 40 mg/kg or saline. AK7 30 mg/kg was injected i.p. 10 min before and 50 min after MPTP administration. Animals were sacrificed 7 days after the injection. Striatal DA (A) and metabolite DOPAC (B) were detected by HPLC-ECD (A&B, n = 8–10, *p<0.05 vs CON; # p<0.05 vs MPTP). (C) For determination of MPTP metabolism, mice were injected with MPTP and AK7 (10 min before and 50 min after MPTP) at indicated doses and sacrifice 90 min after MPTP treatment. MPP+ was detected in the striatum by HPLC (C, n = 5–6, ** ##, p<0.01 vs corresponding MPTP control groups).
Figure 3.
Protective effects of AK7 in subacute MPTP mouse model of PD.
Mice were treated with MPTP i.p. at 20 mg/kg once daily for 4 days and sacrificed 5 days after the last injection. AK7 was injected at 10 or 20 mg/kg i.p. 10 min before and 50 min after MPTP administration. (A) Beam test was performed 3 days after the last MPTP administration. (B) Striatal DA was detected by HPLC-ECD. TH immunohistochemistry was performed and nigral dopaminergic neurons were counted by stereological analysis of TH positive neurons (C and D). A separate experiment was performed using the same treatment regimen but mice were sacrificed 24 hr after the last MPTP injection. Acetylated α-tubulin (Ac-tubulin), total α-tubulin, and brain-predominant SIRT2.2 isofrom was detected in the striatum by Western Blotting (E) and the blot was quantified using Image J (F). (*p<0.05 vs CON; # p<0.05 vs MPTP; ** p<0.01 vs MPTP).
Figure 4.
Effects of AK7 in SOD1-G93A mouse model of ALS.
Kaplan-Meier probability curves show no significant effects of AK7 treatment on (A) symptom onset (118 ± 10.0 days for AK7, 117 ± 8.6 days for vehicle-treated controls) or (B) survival (169 ± 12.7 days for AK7, 175 ± 12.4 days for controls) of SOD1-G93A mice in this study. Values are median age ± SD; n = 23 for AK7, n = 27 for vehicle-treated controls.
Table 1.
Effects of AK7 in a mouse model of ischemic stoke.