A High-Throughput Chemical Screen in DJ-1β Mutant Flies Identifies Zaprinast as a Potential Parkinson's Disease Treatment

Abstract

Dopamine replacement represents the standard therapy for Parkinson's disease (PD), a common, chronic, and incurable neurological disorder; however, this approach only treats the symptoms of this devastating disease. In the search for novel disease-modifying therapies that target other relevant molecular and cellular mechanisms, Drosophila has emerged as a valuable tool to study neurodegenerative diseases due to the presence of a complex central nervous system, the blood-brain barrier, and a similar neurotransmitter profile to humans. Human PD-related genes also display conservation in flies; DJ-1β is the fly ortholog of DJ-1, a gene for which mutations prompt early-onset recessive PD. Interestingly, flies mutant for DJ-1β exhibit PD-related phenotypes, including motor defects, high oxidative stress (OS) levels and metabolic alterations. To identify novel therapies for PD, we performed an in vivo high-throughput screening assay using DJ-1β mutant flies and compounds from the Prestwick® chemical library. Drugs that improved motor performance in DJ-1ß mutant flies were validated in DJ-1-deficient human neural-like cells, revealing that zaprinast displayed the most significant ability to suppress OS-induced cell death. Zaprinast inhibits phosphodiesterases and activates GPR35, an orphan G-protein-coupled receptor not previously associated with PD. We found that zaprinast exerts its beneficial effect in both fly and human PD models through several disease-modifying mechanisms, including reduced OS levels, attenuated apoptosis, increased mitochondrial viability, and enhanced glycolysis. Therefore, our results support zaprinast as a potential therapeutic for PD in future clinical trials.

Keywords: Drosophila; GPR35 agonist; High-throughput screening; Parkinson’s disease; Phosphodiesterase inhibitor; Zaprinast.

Fig. 1

A High-throughput chemical screen and drug validation. a Scheme of the screening procedure used in the current study. Each screening stage (depicted by an arrow) led to several positive-hit compounds taken to the next stage. b Motor performance of DJ-1ß mutant flies obtained during the primary HTS for the 1120 evaluated drugs analyzed by climbing assays. Results are normalized to data obtained in DJ-1ß mutants treated with vehicle (DMSO). See “Material and Methods” section for details. c The viability of DJ-1-deficient cells was measured by MTT assays in the presence of OS (induced with 100 µM H₂O₂) and treated with the ten selected compounds. Results are normalized to data obtained in vehicle-treated DJ-1-deficient cells (DMSO). Error bars show s.d. from three independent biological replicates (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 2

Effect of zaprinast on PD model flies. a H₂O₂ levels in DJ-1β mutant flies treated with 10 μM ZAP were analyzed using the Amplex H₂O₂ Red Kit (Invitrogen). b Protein carbonylation levels in DJ-1ß mutants treated with 10 μM ZAP were analyzed by absorbance. In all cases, data were expressed as arbitrary units (a.u.) per mg of protein, and results were referred to data obtained in flies cultured in vehicle medium (DMSO). c Motor performance of y,w control flies and park mutant flies treated with vehicle (DMSO) or 1 µM ZAP was evaluated performing a climbing assay. Error bars show s.d. from at least three replicates and three independent experiments (*P < 0.05; ***P < 0.001)

Fig. 3

Effect of zaprinast on viability and Akt/JNK pathway activities in DJ-1-deficient cells. a MTT assays measured the viability of DJ-1-deficient cells in the presence of OS (induced with 100 µM H₂O₂). Cells were either treated with vehicle (DMSO) or with ZAP (0.1–80 μM). Results were normalized to data obtained in vehicle-treated mutant cells (-). b, c Antibodies against Akt, p-Akt, JNK, and p-JNK were used to detect proteins of interest in DJ-1-deficient cells subjected to OS and treated with 1 μM ZAP by Western blot (upper panels). The relative ratios of p-Akt/Akt and p-JNK/JNK were analyzed by densitometry (lower panels). Results are referred to data obtained in vehicle-treated DJ-1-deficient cells and expressed as arbitrary units (a.u.). In all cases, error bars show s.d. from three independent experiments in which three biological replicates were used (*P < 0.05; ***P < 0.001)

Fig. 4

Effect of zaprinast on mitochondrial activity in DJ-1-deficient cells. a Representative images of SH-SY5Y cells stained with MitoTracker Red FM, a specific mitochondrial dye, and the nuclear dye DAPI (blue) acquired via fluorescence microscopy. Cells stained were pLKO.1 control cells and DJ-1-deficient cells pretreated with vehicle (DMSO), and DJ-1-deficient cells treated with 1 µM ZAP. Scale bar, 50 µm. b Graphical representation of Mitotracker Red FM fluorescence quantification from a. At least ten images of each strain and condition were analyzed. Results are normalized to data obtained in vehicle-treated pLKO.1 control cells and expressed as arbitrary units (a.u.). Error bars show s.d. from three independent experiments in which three biological replicates were used (***P < 0.001)

Fig. 5

Effect of zaprinast on the activity of glycolytic enzymes and on ATP levels in DJ-1-deficient cells. a The activity of hexokinase (Hk), phosphofructokinase (Pfk), enolase (Eno), and pyruvate kinase (Pk) in DJ-1-deficient cells treated with 1 µM ZAP under OS condition induced with 50 µM H₂O₂. Results were normalized to data obtained in vehicle-treated cells (DMSO). In all cases, error bars show s.d. from three replicates and three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001). b ATP levels in DJ-1-deficient cells in the presence of OS (induced with 50 µM H₂O₂) treated with vehicle (DMSO) or with 1 μM ZAP were analyzed using the ATP Determination Kit (Invitrogen). Results were normalized to data obtained in vehicle-treated pLKO.1 cells (DMSO). Error bars show s.d. from three independent experiments in which three biological replicates were used (**P < 0.01; ***P < 0.001)

Fig. 6

Effect of kynurenic acid and CID2745687 in DJ-1-deficient cells. a MTT assays measured the viability of DJ-1-deficient cells in the presence of OS (induced by 100 µM H₂O₂). Cells were either treated with vehicle (DMSO) or with 10 µM kynurenic acid. b MTT assays measured the viability of DJ-1-deficient cells in the presence of OS (induced with 100 µM H₂O₂) treated with 1 µM ZAP. Cells were either pretreated with different concentrations of CID2745687 (0.1–10 µM) or with vehicle (-). Results were normalized to data obtained in vehicle-treated mutant cells. Error bars show s.d. from three independent biological replicates (*P < 0.05; ***P < 0.001)