Multifunctional D2/D3 agonist D-520 with high in vivo efficacy: modulator of toxicity of alpha-synuclein aggregates

Abstract

We have developed a series of dihydroxy compounds and related analogues based on our hybrid D2/D3 agonist molecular template to develop multifunctional drugs for symptomatic and neuroprotective treatment for Parkinson's disease (PD). The lead compound (-)-24b (D-520) exhibited high agonist potency at D2/D3 receptors and produced efficacious activity in the animal models for PD. The data from thioflavin T (ThT) assay and from transmission electron microscopy (TEM) analysis demonstrate that D-520 is able to modulate aggregation of alpha-synuclein (αSN). Additionally, coincubation of D-520 with αSN is able to reduce toxicity of preformed aggregates of αSN compared to control αSN alone. Finally, in a neuroprotection study with dopaminergic MN9D cells, D-520 clearly demonstrated the effect of neuroprotection from toxicity of 6-hydroxydopamine. Thus, compound D-520 possesses properties characteristic of multifunctionality conducive to symptomatic and neuroprotective treatment of PD.

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Figure 1

Molecular structures of (S)-5-OH-DPAT, D-264, and rifampicin.

Figure 2

Effect of different drugs upon reserpine-induced (5.0 mg/kg, s.c.) hypolocomotion in rats. Data are means ± SEM, n = 3–4 rats per value. Horizontal activity was measured as described in Methods. The plots are the representation of horizontal locomotor activity at discrete 30 min intervals after the administration of D-520 (i.p.) and ropinirole (i.p.) at the dose of 10 μmol/kg compared to control reserpine treated rats in 18 h post reserpine treatment. One-way ANOVA analysis demonstrates significant effect among treatments F(2,35) = 10.06 (P < 0.0001). Dunnett’s analysis following ANOVA showed that the effects of D-520 (P < 0.01) and ropinirole (P < 0.01) were significantly different compared to reserpine control.

Figure 3

Effect on turning behavior of two different doses of D-520 (i.p.), ropinirole, and vehicle in lesioned rats studied for maximum 12 h. Each point is the mean ± SEM of three to four rats. The drugs were administered i.p. One-way ANOVA analysis demonstrates significant effect among treatments: F(3,62) = 16.79 (P < 0.0001). Dunnett’s analysis showed that the effect of two doses of D-520, and ropinirole on rotations was significantly different compared to vehicle (P < 0.01).

Figure 4

Inhibition of αSN fibrillization by D-520 and the reference drug rifampicin. The drugs were incubated with αSN over a period of 10 days and the fibrilization was measured at different time intervals by ThT fluorescence assay. Data values shown are means ± SEM of three independent experiments.

Figure 5

Effect of treatment of time dependent prefabricated αSN aggregates as well as αSN aggregates formed in the presence of drugs on cell viability of PC12 cells. (A) PC12 cells were treated with 10 μM prefabricated αSN aggregates collected at different time points. (B) PC12 cells were treated with 10 μM prefabricated αSN aggregates formed in the presence of drugs collected at day 0 and day 6. Values shown are means ± SEM of three independent experiments performed in four to six replicates. (C) PC12 cells were treated with 10 μM prefabricated αSN aggregates formed at day 6 and 20 μM D-520 at day 0 as well as with 20 μM D-520 after shaking for 6 days. One-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test was performed. (**p < 0.01 and ***p < 0.001 compared to the control; ^##p < 0.01 compared to the αSN D0.)

Figure 6

TEM analysis of αSN. (A) αSN at day 0, (B) 60 μM αSN after 6 days, (C) 60 μM αSN shaken in the presence of 120 μM rifampicin after 6 days, and (D) 60 μM αSN shaken in the presence of 120 μM D-520 after 6 days. Scale bar = 100 nm.

Figure 7

Dose dependent effect of combination of pretreatment followed by cotreatment of D-520 with 75 μM 6-OHDA on cell viability of MN9D cells from toxicity of 75 μM 6-OHDA. MN9D cells were pretreated with different doses of D-520 for 1 h followed by cotreatment with 75 μM 6-OHDA for 24 h. The values shown are mean ± SEM of three independent experiments performed in four to six replicates. One-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test was performed. (**p < 0.001 compared to the 6-OHDA control. ^##p < 0.001 compared to the control.)

Figure 8

Schematic description of development of a multifunctional neuroprotective drug.