Article Figures & Data
Figures
- Fig. 1.
KM822 binds to Caenorhabditis elegans dopamine transporter-1 at an allosteric site that closely overlaps with the KM822-binding site onto human dopamine transporter. Structural modeling and docking analysis suggest KM822 (space-filling; cyan, with N-, O-, and S-atoms in blue, red, and yellow, respectively) may bind to dopamine transporter-1 with two different poses (panels A and B), comparable to those at the allosteric site A2 previously described for hDAT (panels C and D), see (Aggarwal et al., 2021). Residues that coordinate KM822 in all four cases are shown in sticks and labeled.
- Fig. 2.
KM822 suppresses the activity of both human and Caenorhabditis elegans dopamine transporters (DATs). Radioactive dopamine transport inhibition assay using KM822 against human (hDAT) and Caenorhabditis elegans (DAT-1) DAT in transiently transfected COS-7 cells. KM822 IC50 mean values for hDAT and DAT-1 are 2.2 μM, 95% confidence interval [1.6; 3.2] and 10.4 μM, 95% confidence interval [6.3; 17.0], respectively. Nonlinear regression was used to obtain a curve consistent with a single binding site. IC50 mean values and corresponding 95% confidence intervals are calculated using an average of five independent experiments.
- Fig. 3.
KM822 affects dopamine transporter-1 interactions with amphetamine (AMPH) but has no effect on nisoxetine or cocaine actions. Two depictions of each representative dopamine uptake experiment are displayed. One of actual dopamine uptake (A, C, and E) and one with uptake values normalized relative to vehicle control (B, D, and F). (A, B) Radioactive dopamine (DA) transport inhibition assay of AMPH in the absence and presence of 20 µM of KM822 in dopamine transporter-1-transfected COS-7 cells. IC50 mean values of AMPH are 1.1 μM, 95% confidence interval (CI) [0.84; 1.4] for the vehicle and 3.4 μM, 95% CI [2.9; 3.9] in the presence of 20 µM of KM822 (**, p < 0.01). The concentration-response of AMPH-mediated inhibition was shifted to the right due to KM822 treatment. (C, D) Radioactive DA transport inhibition assay of nisoxetine in the absence and presence of 20 µM of KM822 in dopamine transporter-1-transfected COS-7 cells. IC50 mean values of nisoxetine in the absence and presence of 20 µM of KM822 are 0.04 μM, 95% CI [0.029; 0.055] and 0.03 μM, 95% CI [0.021; 0.042], respectively. No statistically significant change was observed in nisoxetine-mediated inhibition. (E, F) Radioactive DA transport inhibition assay of cocaine in the absence and presence of 20 µM of KM822 in dopamine transporter-1-transfected COS-7 cells. IC50 mean values of cocaine are 9.6 μM, 95% CI [8.1; 11] for the vehicle and 128 μM, 95% CI [61; 268] in the presence of 20 µM of KM822 (*, p < 0.05). The concentration-response of cocaine-mediated inhibition was shifted to the right due to KM822 treatment. IC50 mean values and 95% confidence intervals were calculated using the average of three independent experiments. Statistical analysis was performed using Student’s t test.
- Fig. 4.
KM288 suppresses amphetamine (AMPH)-induced swimming-induced paralysis (Swip) in wild-type Caenorhabditis elegans: (A) concentration-response profile of wild-type (N2) to KM822 treatments indicating no statistically significant change in the animals’ swimming behavior (N ≥ 240). No statistical significance was observed comparing the swimming behavior of KM822 to vehicle treated animals of the same concentration point (P value < 0.05; F-value = 0.4368). Data were analyzed by a two-way ANOVA with Bonferroni multiple comparison tests between KM822 and vehicle treatments at each concentration. (B) KM288 (250 µM) suppresses AMPH-induced Swip phenotype at various AMPH concentrations. Swip assays were performed for animals incubated in distilled water solutions containing the depicted concentrations of AMPH or AMPH + KM822 (250 µM), N ≥ 240. Data were analyzed by a two-way ANOVA with Bonferroni multiple comparison tests, comparing AMPH and AMPH + KM822 treatments at each concentration point. Statistically significant differences were observed at 250 µM, 300 µM and 500 µM AMPH concentrations (**** = P value < 0.0001; F-value = 43.05). No statistically significant changes in the swimming profile of control or vehicle groups, as indicated in A (not shown for simplicity). (C) KM822 reversal of Swip is dopamine transporter-1 dependent. A representation of the 10-min Swip assay, depicting the swimming behavior of animals after 10 min of incubation in control (vehicle) or drug conditions (N ≥ 240). No statistically significant differences were observed comparing N2 in vehicle versus 250 μM KM822 conditions (ns, P value < 0.05). Whereas N2 animals incubated in 300 μM 0f AMPH demonstrated significant Swip paralysis compared to the 250 μM of KM822 and AMPH/KM822 mixture (**** = P value < 0.0001, F-value = 192.4). A dose of 250 μM of KM822 demonstrated no statistically significant effect on the Swip paralysis of dat-1 mutants compared to the condition (ns, P value < 0.05). Data were analyzed by a one-way ANOVA with Bonferroni multiple comparison tests. (D) KM822 has no statistically significant effect on nisoxetine-induced Swip at 2.5 μM and 5 μM of nisoxetine concentration (ns, P value < 0.05). Data were evaluated using one-way ANOVA or two-way ANOVA with selected Bonferroni’s multiple comparisons tests, comparing different treatments with (**** = P value < 0.0001; F-value = 156.4). N ≥ 240. For all data, means and SDs are displayed.
- Fig 5.
KM822 suppresses dopamine transporter (DAT)-specific swimming-induced paralysis (Swip) behavior, but not LGC-55 induced Swip. A) KM822 suppresses DAT-1 dependent Swip, but not LGC-55 induced Swip, in response to amphetamine (AMPH) treatment. Depicted are 10-min Swip assays for N2, lgc-55, dat-1, and dat-1; lgc-55 animals (N ≥ 240). KM822 suppresses AMPH-induced Swip in N2 animals and lgc-55 mutants (**** P value < 0.0001; F-value = 80.82), with KM822 suppression of AMPH-induced Swip found to be significantly greater in lgc-55 than in N2 background (#### P value < 0.0001; F-value = 80.82). KM822 has no statistically significant effect on AMPH-induced Swip of dat-1 and dat-1; lgc-55 mutant backgrounds (ns, P value < 0.05). Data were analyzed using two-way ANOVA with selected Bonferroni’s multiple comparisons tests, comparing KM822 treatments over conditions and genotypes. B) KM822 demonstrates no effect on β-phenylethylamine-induced Swip of N2 animals (ns, P value < 0.05). Swip data were analyzed using ordinary one-way ANOVA, with selected Bonferroni’s multiple comparisons tests, comparing different conditions with (**** = P value < 0.0001; F-value = 151.1). N ≥ 140. For all, data means and SDs are displayed.
