LRRK2 Kinase Inhibitor PF-06447475 Protects Drosophila melanogaster against Paraquat-Induced Locomotor Impairment, Life Span Reduction, and Oxidative Stress

Parkinson’s disease (PD) is a complex multifactorial progressive neurodegenerative disease characterized by locomotor alteration due to the specific deterioration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). Mounting evidence shows that human LRRK2 (hLRRK2) kinase activity is involved in oxidative stress (OS)-induced neurodegeneration, suggesting LRRK2 inhibition as a potential therapeutic target. We report that the hLRRK2 inhibitor PF-06447475 (PF-475) prolonged lifespan, increased locomotor activity, maintained DAergic neuronal integrity, and reduced lipid peroxidation (LPO) in female Drosophila melanogaster flies chronically exposed to paraquat (PQ), a redox cycling compound, compared to flies treated with vehicle only. Since LRRK2 is an evolutionary conserved kinase, the present findings reinforce the idea that either reduction or inhibition of the LRRK2 kinase might decrease OS and locomotor alterations associated with PD. Our observations highlight the importance of uncovering the function of the hLRRK2 orthologue dLrrk2 in D. melanogaster as an excellent model for pharmacological screenings. Supplementary Information The online version contains supplementary material available at 10.1007/s11064-024-04141-9.


Introduction
Parkinson's disease (PD) is a neurological condition characterized by the progressive loss of 50-70% of dopaminergic (DAergic) neurons from the substantia nigra, leading to movement alterations [1].PD is now considered a pandemic disease [2].Therefore, one of the major goals of PD research is to protect this group of neuronal cells from structural and functional deterioration or the dying process.Unfortunately, elucidation of the molecular mechanisms of neuronal loss or damage has been elusive until now [3,4].Despite this drawback, genetic and environmental factors have been However, no data are available to establish the definitive role played by LRRK2 in OS-stimuli in vivo.
Drosophila melanogaster (referred to here as D. melanogaster or Drosophila) is one of the most widely used experimental organisms for neurodegenerative disorders [20].Specifically, the fly provides an excellent toolkit for the modeling of environmental and genetic aspects of PD [21][22][23].Indeed, D. melanogaster reproduces many features of the disease, such as loss of DAergic neurons, reduced movement, mitochondrial abnormalities, LPO, and OS [24][25][26].Human LRRK2 (hLRRK2) kinase is a highly conserved protein at a structural and functional level across species [27].D. melanogaster has a single orthologue, dLrrk2, and the kinase domain is 31% identical and 52% similar between dLrrk2 and hLRRK2 [28].Interestingly, studies have shown that genetic ablation of dLrrk2 in Drosophila [29,30] or pharmacological inhibition in rats and worms [31,32] provide resistance to OS-stimuli.Despite these advances, it is not yet established whether pharmacological inhibition offers the most effective and promising approach to PD management.Since Drosophila presents a high percentage identity and similarity of disease-related genetic variants (approximately 75%; [33]) and has deep homology with brain structures involved in PD neuropathology [34,35], the fly might be an excellent model organism for drug screening [36].
The aim of the present investigation was to assess the effect of the hLRRK2 kinase inhibitor PF-475 in female D. melanogaster flies (w[1118]).To this end, flies were chronically exposed to PQ for 15 days in the presence or absence of the inhibitor PF-475 to evaluate lifespan, locomotor activity, integrity of DAergic neurons in terms of TH expression level, and lipid peroxidation (LPO), as indicative of OS.Our findings suggest that dLrrk2 (or hLRRK2) work as regulator of OS and that their action could be blocked by specific inhibitor LRRK2 PF-475.The high similarity between hLRRK2 and dLrrk2 highlights the importance of uncovering the function of the LRRK2 orthologue dLrrk2 in Drosophila as an invaluable model for pharmacological screenings.

Protection Assay
A protection assay was performed on virgin 2-to 3-day-old female flies collected overnight and kept on regular food medium.Subsequently, 60 separated adult female flies were starved in empty vials for 3 h at 25 °C.Then, groups of six flies were placed in ten vials containing a filter paper (Bio Rad Mini Trans-Blot 1,703,932) saturated with 1% glucose (1G, 55.5 mM glucose) in distilled water (dW) for 24 h.After this time, flies were starved in empty vials for 3 h at 25 °C and transferred to vials with a filter paper saturated with 200 µl paraquat (PQ 1 mM, MP Biomedicals cat 02195323) and/or hLRRK2 selective inhibitor PF-475 at 0.05, 0.075, and 0.1 µM (Sigma cat PZ0248) in 1G for 15 days.The PQ feeding schedule was adjusted according to the protection assay.Red food dye (8 µl/1 ml) (Red food color McCormick) was added to ensure homogeneity and food intake.Living flies were counted daily.

Locomotion Assay
The movement deficit assay was performed on treated flies, according to Ortega-Arellano et al. [39].Briefly, treated flies were placed in empty plastic vials.After a 10-minute rest period, the flies were tapped to the bottom of the vials, and the number of flies able to climb 5 cm in 6 s was recorded at each interval of time.The assays were repeated three times at 1-minute intervals.For each experiment, a climbing percent (%) was calculated as ½ [(n tot + n top -n bot )/n tot ] x 100.Data were shown as a mean ± standard deviation of the mean (SD).The Chi Square (c 2 ) statistic was performed to compare proportions of percentages between independent groups.Differences were considered statistically significant at p < 0.05.

Survival Test
Flies were treated chronically with paraquat (PQ; 1 mM, MP Biomedicals cat 02195323) and the selective inhibitor PF-475 (0.05, 0.075, and 0.1 mM, Sigma cat PZ0248) as described above for 15 days.Live flies were counted in groups of six per vial daily.Sixty flies per treatment were used.Survival curves were plotted using the Kaplan-Meier estimator.The statistical significance was calculated using the log rank test within the portable IBM SPSS Statistics 22 package program.The null hypothesis in all survival assays was that the exposure of PQ and/or PF-475 to female Drosophila made no difference in the survival of the flies in the absence of those reagents.Differences were considered statistically significant at p < 0.05.

Lipid Peroxidation (LPO) Assay
Quantification of lipid peroxidation involving TBARS (thiobarbituric acid reactive substance) was performed according [40].Briefly, 30 heads (approx.30 mg) from untreated or PQ (1 mM) treated flies with or without PF-475 were homogenized in a 0.6 mL solution containing 50 mM sodium phosphate buffer, pH 6.0, and 10% trichloroacetic acid (TCA).Then, samples were centrifuged at 10,000 rpm for 10 min.The supernatant was divided into two aliquots.The first supernatant (0.3 mL) was mixed with 0.1 mL of 0.1 M EDTA and 0.6 mL of a solution containing 1% thiobarbituric acid in 0.05 M NaOH, and then incubated at 100 ºC for 15 min.The second aliquot (0.3 mL) was mixed with 0.7 mL of H 2 O and incubated under the same conditions as described above.This sample was used as an internal absorbance control to avoid artifacts in the LPO measurement.After cooling on ice, the samples were centrifuged at 10,000 rpm for 1 min.The malondialdehyde (MDA) product was measured at 535 nm.The molar absorptivity of MDA (1.56 × 10 5 M − 1 cm − 1 ) was used to express lipid peroxidation levels as MDA (nMol per mg of fly heads).To compare the differences between two or more groups, a one-way ANOVA followed by the Bonferroni post hoc comparison calculated with SPSS 25 software was performed.

PF-06447475 Increases Lifespan and Reduces Motor Impairment in Flies Exposed to Paraquat
Previously, it has been demonstrated that PQ (1 mM) induced a significant reduction in survival and locomotor activity in TH/+ flies and such noxious effects were importantly diminished in TH > Lrrk2-RNAi/+ flies [30].Effectively, w[1118] flies fed with PQ (1 mM) had a significantly decreased lifespan (Fig. 2A) and locomotor activity (Fig. 2B) compared to untreated flies.Noticeably, 50% of w[1118] flies exposed to PQ perished at day 5 and reduced locomotor activity at the same day, but survival and locomotor activity in flies treated with vehicle only were extended beyond day 15 (Table 2, track 1 vs. 2, Fig. 2A and  B).We confirmed that TH > Lrrk2-RNAi/+ flies exposed to PQ showed a prolonged survival (Fig. 2C) and increased percentage of climbing activity (Fig. 2D).Of note, whereas 50% of TH > Lrrk2-RNAi/+ flies treated with vehicle only extended the survival and locomotor activity beyond day 15 (Table 2, track 11, Fig. 2C and D), the knockdown flies exposed to PQ perished at day 10 and reduced locomotor activity at the same day (Table 2, track 12, Fig. 2C and D).

Molecular Docking
We used X-ray crystal structure of hLRRK2 (protein data bank code: 7LI4) and dLrrk2 (code AF-A0A0B4KHT3, created with Alphafold, https://alphafold.ebi.ac.uk/) for molecular docking experiments.The blind molecular docking was performed with CB-Dock version 2 [41], a cavity detection-guided protein-ligand blind docking web server that uses Autodock Vina (version 1.1.2,Scripps Research Institute, La Jolla, USA) or FitDock: protein-ligand docking by template fitting.The SDF structure files of the tested compounds (ATP, compound CID: 5957, and PF06447475, compound CID: 72,706,840) were downloaded from Pub-Chem (https://pubchem.ncbi.nlm.nih.gov/;available in March 2024).The molecular blind docking was performed by uploading the 3D structure PDB file of hLRRK2 and dLrrk2 into the server with the SDF file of each compound.For analysis, we selected the docking poses with the strongest Vina score or fitDock score in the catalytical pocket.The generated PDB files of the molecular docking of each compound were visualized with the CB-Dock2 interphase or through BIOVIA Discovery Studio Visualizer (https:// discover.3ds.com/discovery-studio-visualizer-download;available in March 2024) and compared against the experimentally validated X-ray structures of the interaction of ATP with hLRRK2 [42].

PF06447475 (PF-475) Binds with High Affinity to the ATP-Binding Pocket of the dLrrk2 Kinase in D. melanogaster
We first investigated whether PF-475 might bind to the ATP pocket of the dLrrk2 kinase in D. melanogaster, as it has previously been reported in the hLRRK2 kinase [11].As a validation procedure, we performed an in silico molecular docking analysis using CB-Dock2, an accurate proteinligand blind docking tool [41], to analyze the ATP-binding and PF-475-binding pockets in hLRRK2.As expected, docking analysis reveals that ATP and PF-475 are bound to hLRRK2 with high affinity, albeit with different strengths.ATP bound to the dLRRK2 pocket with an affinity of -5.8 kCal/mol (fitDock score), involving meanly 21 aa (Table 1, Suppl Fig. S1A-B), whereas PF-475 inhibitor bound to the hLRRK2 pocket with an affinity of -6.9 kCal/mol (fit-Dock score), involving nearly the same aa in the catalytical pocket, 17/21 (81%) (Table 1, Suppl Fig. S1C-D).Indeed,  The fitDock score is based on Template-based docking [63] Bold letters represent important amino acid residue in the (catalytic) protein pocket that interact with ATP or hLRRK2 inhibitor PF-475

LRRK2 Inhibitor PF-06447475 Reduced the Lipid Peroxidation (LPO) Index in Flies Exposed to Paraquat
The above observations prompted us to assess whether PF-475 could reduce PQ-induced OS in flies.As shown in Fig. 4 and Table 3, WT Drosophila (w[1118]) flies exposed to PQ produced a statistically increase (9-fold) in MDA compared to flies treated with vehicle only (Table 3, track 1 vs. 2).Remarkably, PF-475 significantly reduced the levels of MDA in flies treated with PQ to a comparable level to flies treated with inhibitor alone or with vehicle only (Table 3, track 1, 3 and 4).Of note, untreated knockdown flies (Table 3, track 5) or treated with PQ (Table 3, track 6), PF-475 only (Table 3, track 7), or PQ and PF-475 (Table 3, track 8) showed similar MDA values (Fig. 4).
and climbing activity to flies treated with PQ only (Table 2, track 12).

PF-06447475 Does Not Modify the Expression Level of Tyrosine Hydroxylase (TH) or dLrrk2 Protein in Wild-Type Flies Exposed to Paraquat
We wanted to evaluate whether inhibitor PF-475 affected the expression of enzyme TH or dLrrk2 kinase in the absence or presence of PQ.Western blot analysis (Fig. 3A) revealed no statistically significant differences in the expression levels of TH or dLrrk2 (Fig. 3B and C) in flies treated with vehicle only (Fig. 3A, track 1), treated with PQ only (Fig. 3A, track 2), with inhibitor only (Fig. 3A, track 3), or flies treated with both inhibitor and PQ (Fig. 3A, track 4).model Drosophila PD-like symptoms [19].In fact, the PQ-Drosophila model has been used to interrogate potential neuroprotective compounds from natural resources (e.g., [46][47][48][49]).We also confirmed that TH > Lrrk-RNAi/+ flies treated with PQ showed an extended lifespan and locomotor activity by 100% and 125%, respectively compared to w[1118] flies treated with PQ.We conclude that reduced expression of Lrrk2 in the transgenic TH > Lrrk-RNAi/+ flies conferred resistance PQ stimuli [30].
Given that LRRK2 kinase increases the generation of ROS and causes enhanced neurotoxicity under stress stimuli in dopaminergic SN4741 cells [10] and in neuron-like cells [12], it is therefore reasonable to think that its specific inhibition might alleviate DAergic neurons from OS-induced neurodegeneration in vivo.In the present study, we show for the first time that the inhibitor LRRK2 kinase PF-06447475 (PF-475) attenuates chronic PQ-induced neurotoxicity in D. melanogaster by increasing life span, improving climbing ability, and decreasing OS.Our observations suggest that the dLrrk2 kinase modulates neurodegeneration in PD.Although the underlying mechanisms by which dLrrk2 might contribute to PQ-induced neurodegeneration are not yet fully described in the fly, we speculate that it most probably involves ROS-signaling mechanisms [12,48,50] and/ or alterations of neurocytoskeletal proteins (e.g., [51][52][53]).Therefore, PF-475 might block dLrrk2, thereby maintaining locomotor activity, reducing OS (LPO index), and prolonging life span, indicative of normal DAergic neuronal functionality.Interestingly, neither PQ alone nor in combination with PF-475 affected the expression levels of TH or dLrrk2.Taken together, these results imply that PF-475 is capable of protecting Drosophila against PQ-induced neurodegeneration [30,45].These observations suggest that switching off hLRRK2 and dLrrk2 by specific PF-06447475 might be an achievable therapeutic approach in sporadic as well as familial PD.
PF-475 is a second-generation LRRK2 inhibitor with high potency, selectivity, and good blood-brain barrier (BBB) permeability properties in mice and rats [11].

Discussion
Parkinson's disease is a complex disease mainly characterized by movement disorder associated with a selective degeneration of DAergic neurons [1] believed to deteriorate by the interaction of genes (e.g., LRRK2) and environmental factors (e.g., exposure to neurotoxin PQ) [43].However, the gene-environmental interplay in PD is not yet fully understood.Remarkably, D. melanogaster has become a simple and powerful system to model PD and understand the pathophysiology of the disorder [44].Here, we confirmed that Drosophila flies chronically exposed to PQ induce a shortage of life span, promote significant high levels of MDA as an indication of LPO and OS, and provoke locomotor impairment as an indication of functional degeneration of DAergic neurons [26,30,45].Taken together, these observations suggest that PQ might be a potential neurotoxin to  Previous studies have shown that reduced expression of dLrrk2 in the DAergic neurons of transgenic flies conferred PQ resistance and the absence of neurodegeneration [28][29][30].Specifically, it has been shown that transgenic flies dramatically increased locomotor activity, reduced the lipid peroxidation (LPO) index alone or in the presence of PQ, presented an extended life span, and showed DAergic neuron integrity and/or functionality.Our present investigation shows similar observations when w[1118] flies were treated with PF-475 and PQ.As expected, PF-475 was inoperant in knockdown flies.Indeed, TH > Lrrk2-RNAi/+ flies exposed to PF-475 and PQ behave similarly as knockdown flies treated with PQ alone.Taken together, these observations suggest that both knockdown and pharmacologic models are biologically comparable and that reduced or suppressed LRRK2 expression by pharmacologic or gene LRRK2 blockage might delay or prevent motor symptoms in persons at risk of suffering Parkinsonism by impeding structural or functional impairment (= neurodegeneration) in the DAergic neurons.However, the pharmacology approach might be ideal over gene therapy for clinical trials.

Conclusion
LRRK2 is a serine-threonine kinase involved in multiple cellular processes and signaling pathways in PD [61].Therefore, inhibition of LRRK2 kinase function is a promising therapeutic strategy for PD treatment [62].Here, we demonstrate that the small-molecule kinase inhibitor PF-06447475 can protect Drosophila against OS-induced neurodegeneration.Since Drosophila might be biologically homologous to mammalian model organisms (e.g., mice, rats), the fly is an important resource to test and discover new therapeutic compounds.The present Drosophila-PQ model might serve as a reference assay, which, together with in silico data results from chemical libraries and/or natural compounds, offers a unique opportunity to speed up the development of novel LRRK2 inhibitors.
Recent cryo-electron microscopy (cryoEM) analysis of the hLRRK2 protein (resolution 3.10 Armstrong) has provided the means to delve into the structure-function interaction between hLRRK2, its natural ligand ATP, and the inhibitor PF-475 [8].By using in silico molecular docking analysis, we found that PF-475 binds to hLRRK2 (PDB: 7LI4) with a higher bonding affinity than ATP (1.189-fold increased), involving 16/21 (76%) identical aa residues in the binding pocket of the kinase (Table 1).Interestingly, by using mammalian STE20-like protein kinase 3 (MST3, residues 1-303; NP_001027467; PDB ID: 4U8Z; ATP-binding site residue shows 73% similarity to human LRRK2) as a surrogate crystallographic system for hLRRK2, Henderson and coworkers [11] determined ASP1887, VAL1893, LYS1906, GLU1948, and ALA1950 as important aa residues in the interaction with PF-475.Similarly, we found identical aa interacting in the pocket of hLRRK2 and the inhibitor (Table 1, Suppl.Fig. s2).Interestingly, a BLAST analysis revealed a 30% identity and 48% similarity between the full length in aa residues of protein dLrrk2 (Uniprot Q9VDJ9|LRRK2_DROME, length 2,445) and hLRRK2 (Uniprot Q5S007|LRRK2_HUMAN, length 2,527).Moreover, a similar BLAST analysis showed a 34% identity and 51% similarity between the kinase domain aa residues of dLrrk2 (Uniprot Q9VDJ9, location 1,728-2,034) and hLRRK2 (Uniprot Q5S007, location 1,879-2,138), including an identical matched sequence at the ATP binding site (dLrrk2: 1911 KIADYGI 1917 ; hLRRK2: 2015 KIADYGI 2021 ) according to software tool FIMO (Find Individual Motif Occurrences [54].Since PF-475 binds to the ATP pocket in the kinase domain through a DXG motif [55,56], it is reasonable to think that PF-475 operates as an effective blocker of dLrrk2 through a similar molecular interaction with the human DXG motif.Not surprisingly, PF-475 bound to the binding pocket of dLrrk2 with a higher affinity than ATP (-5.7 vs. -8.6 fitDock score).Taken together, our findings suggest that both hLRRK2 and dLrrk2 are biologically and functionally equivalent.Therefore, the use of Drosophila as an invaluable surrogate in vivo PD model to investigate other potential LRRK2 inhibitors either from natural products (e.g., [57]) or chemical libraries (e.g., [58]) in a simple, fast, reliable, and inexpensive manner.Provided that PF-475 (and other kinase inhibitors) interact with mammalian (hLRRK2) or invertebrate (dLrrk2) kinase in a similar molecular mechanism, we anticipate that pharmacological inhibitors might effectively inhibit LRRK2 in PD patients [59,60].Therefore, we anticipate that the use of flies with LRRK2 mutations associated with familial PD (e.g., G2019S, BDSC#602459) might increase the success of finding an effective inhibitor of hLRRK2 for the treatment of PD.

Fig. 2
Fig. 2 PF-06447475 prolongs life span and locomotor activity in Drosophila flies chronically exposed to PQ. Female flies (n = 60 per treatment) were left untreated or treated with PQ, PF-06447475, or PQ and PF-06447475 as is described in Materials and Methods.The graphs show (A) the survival and (B) climbing analysis of flies.The PF-06447475 increases the survival index and percentage of climbing

Fig. 4
Fig. 4 PF06447475 reduces LPO index in w[1118] flies exposed to PQ. Female flies were treated as is described in Materials and Methods.The graph shows MDA (Malondialdehyde) concentration as a measure of LPO index.The molar absorptivity of MDA (1.56 × 10 5 M − 1 cm − 1 ) was used to express lipid peroxidation levels as nMol of MDA per mg of head flies.The letter n represents the number of fly heads examined per treatment (n = 30).**p < 0.05, significant differences between transgenic and control line in each treatment.Error bars indicate ± SD

Table 2 ,
track 13) showed a comparable survival and climbing activity to untreated flies (Table 2, track 11), whereas PF-475 co-administered with PQ (Table 2, track 14) showed comparable survival Differences were considered statistically significant at p < 0.05.

Table 2
Drosophila flies treated with PF06447475 are resistant to PQ-induced life span decrease and locomotor impairment