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A novel GTP-binding inhibitor, FX2149, attenuates LRRK2 toxicity in Parkinson's disease models.

Li T, He X, Thomas JM, Yang D, Zhong S, Xue F, Smith WW - PLoS ONE (2015)

Bottom Line: While most known LRRK2 inhibitors are developed to target the kinase domain, we have recently identified the first LRRK2 GTP binding inhibitor, 68, which not only inhibits LRRK2 GTP binding and kinase activities with high potency in vitro, but also reduces neurodegeneration.However, the in vivo effects of 68 are low due to its limited brain penetration.Our results highlight a novel GTP binding inhibitor with better brain efficacy, which represents a new lead compound for further understanding PD pathogenesis and therapeutic studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States of America.

ABSTRACT
Leucine-rich repeat kinase-2 (LRRK2), a cytoplasmic protein containing both GTP binding and kinase activities, has emerged as a highly promising drug target for Parkinson's disease (PD). The majority of PD-linked mutations in LRRK2 dysregulate its GTP binding and kinase activities, which may contribute to neurodegeneration. While most known LRRK2 inhibitors are developed to target the kinase domain, we have recently identified the first LRRK2 GTP binding inhibitor, 68, which not only inhibits LRRK2 GTP binding and kinase activities with high potency in vitro, but also reduces neurodegeneration. However, the in vivo effects of 68 are low due to its limited brain penetration. To address this problem, we reported herein the design and synthesis of a novel analog of 68, FX2149, aimed at increasing the in vivo efficacy. Pharmacological characterization of FX2149 exhibited inhibition of LRRK2 GTP binding activity by ~90% at a concentration of 10 nM using in vitro assays. Furthermore, FX2149 protected against mutant LRRK2-induced neurodegeneration in SH-SY5Y cells at 50-200 nM concentrations. Importantly, FX2149 at 10 mg/kg (i.p.) showed significant brain inhibition efficacy equivalent to that of 68 at 20 mg/kg (i.p.), determined by mouse brain LRRK2 GTP binding and phosphorylation assays. Furthermore, FX2149 at 10 mg/kg (i.p.) attenuated lipopolysaccharide (LPS)-induced microglia activation and LRRK2 upregulation in a mouse neuroinflammation model comparable to 68 at 20 mg/kg (i.p.). Our results highlight a novel GTP binding inhibitor with better brain efficacy, which represents a new lead compound for further understanding PD pathogenesis and therapeutic studies.

No MeSH data available.


Related in: MedlinePlus

FX2149 improved the brain penetration and inhibition of LRRK2 GTP binding and kinase activities.FX2149 (10 mg/kg) and 68 (10 and 20 mg/kg) were injected intraperitoneally into G2019S-LRRK2 BAC transgenic mice at 6–12 weeks of age for 1 hour. There were 6 mice in each experimental group. The brain homogenates were used to detect LRRK2 GTP-binding and kinase activities. A and B, LRRK2 GTP-binding assays. C and D, LRRK2 phosphorylation assays using anti-phospho-LRRK2 antibodies. E and F, FX2149 reduced G2019S-LRRK2-induced 4E-BP phosphorylation determined by anti-phospho-4E-BP western blot analysis. Ntg: non-transgenic mouse. *p < 0.05 by ANOVA compared with G2019S-LRRK2 transgenic mice treated with vehicle.
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pone.0122461.g005: FX2149 improved the brain penetration and inhibition of LRRK2 GTP binding and kinase activities.FX2149 (10 mg/kg) and 68 (10 and 20 mg/kg) were injected intraperitoneally into G2019S-LRRK2 BAC transgenic mice at 6–12 weeks of age for 1 hour. There were 6 mice in each experimental group. The brain homogenates were used to detect LRRK2 GTP-binding and kinase activities. A and B, LRRK2 GTP-binding assays. C and D, LRRK2 phosphorylation assays using anti-phospho-LRRK2 antibodies. E and F, FX2149 reduced G2019S-LRRK2-induced 4E-BP phosphorylation determined by anti-phospho-4E-BP western blot analysis. Ntg: non-transgenic mouse. *p < 0.05 by ANOVA compared with G2019S-LRRK2 transgenic mice treated with vehicle.

Mentions: To assess the effects of FX2149 on LRRK2 in brains compared with 68, both 68 and FX2149 were injected intraperitoneally into G2019S-BAC-LRRK2 transgenic mice at 10 and 20 mg/kg doses. One hour after the injection, the mouse brain homogenates were subjected to LRRK2 GTP binding and kinase assays. Both 68 (20 mg/kg) and FX2149 (10 mg/kg) reduced LRRK2 GTP binding activity in mouse brains (Fig. 5A and 5B). While FX2149 at a 10mg/kg dose had the equivalent GTP binding inhibition as 68 at a 20 mg/kg dose, compound 68 at 10 mg/kg dose did not alter brain GTP binding activity 1 hour after injection. Moreover, both 68 (20 mg/kg) and FX2149 (10 mg/kg) also significantly reduced brain LRRK2 kinase activity (Fig. 5C and 5D). To further confirm the effect of FX2149, we also assessed a LRRK2 downstream effector, 4E-BP phosphorylation. 4E-BP is a transcription factor that can be phosphorylated by LRRK2 [20]. We found that both 68 and FX2149 reduced 4E-BP phosphorylation in mouse brains (Fig. 5E and 5F). FX2149 at a 10mg/kg dose reduced 4E-BP up to 15% of the untreated control group, while 68 at a 20 mg/kg dose reduced 4E-BP up to 15% of the untreated control group. These data indicated that FX2149 was taken up into brains at a greater extent and had more potent efficacy in inhibiting GTP binding and kinase activity in mouse brains when compared with 68.


A novel GTP-binding inhibitor, FX2149, attenuates LRRK2 toxicity in Parkinson's disease models.

Li T, He X, Thomas JM, Yang D, Zhong S, Xue F, Smith WW - PLoS ONE (2015)

FX2149 improved the brain penetration and inhibition of LRRK2 GTP binding and kinase activities.FX2149 (10 mg/kg) and 68 (10 and 20 mg/kg) were injected intraperitoneally into G2019S-LRRK2 BAC transgenic mice at 6–12 weeks of age for 1 hour. There were 6 mice in each experimental group. The brain homogenates were used to detect LRRK2 GTP-binding and kinase activities. A and B, LRRK2 GTP-binding assays. C and D, LRRK2 phosphorylation assays using anti-phospho-LRRK2 antibodies. E and F, FX2149 reduced G2019S-LRRK2-induced 4E-BP phosphorylation determined by anti-phospho-4E-BP western blot analysis. Ntg: non-transgenic mouse. *p < 0.05 by ANOVA compared with G2019S-LRRK2 transgenic mice treated with vehicle.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376719&req=5

pone.0122461.g005: FX2149 improved the brain penetration and inhibition of LRRK2 GTP binding and kinase activities.FX2149 (10 mg/kg) and 68 (10 and 20 mg/kg) were injected intraperitoneally into G2019S-LRRK2 BAC transgenic mice at 6–12 weeks of age for 1 hour. There were 6 mice in each experimental group. The brain homogenates were used to detect LRRK2 GTP-binding and kinase activities. A and B, LRRK2 GTP-binding assays. C and D, LRRK2 phosphorylation assays using anti-phospho-LRRK2 antibodies. E and F, FX2149 reduced G2019S-LRRK2-induced 4E-BP phosphorylation determined by anti-phospho-4E-BP western blot analysis. Ntg: non-transgenic mouse. *p < 0.05 by ANOVA compared with G2019S-LRRK2 transgenic mice treated with vehicle.
Mentions: To assess the effects of FX2149 on LRRK2 in brains compared with 68, both 68 and FX2149 were injected intraperitoneally into G2019S-BAC-LRRK2 transgenic mice at 10 and 20 mg/kg doses. One hour after the injection, the mouse brain homogenates were subjected to LRRK2 GTP binding and kinase assays. Both 68 (20 mg/kg) and FX2149 (10 mg/kg) reduced LRRK2 GTP binding activity in mouse brains (Fig. 5A and 5B). While FX2149 at a 10mg/kg dose had the equivalent GTP binding inhibition as 68 at a 20 mg/kg dose, compound 68 at 10 mg/kg dose did not alter brain GTP binding activity 1 hour after injection. Moreover, both 68 (20 mg/kg) and FX2149 (10 mg/kg) also significantly reduced brain LRRK2 kinase activity (Fig. 5C and 5D). To further confirm the effect of FX2149, we also assessed a LRRK2 downstream effector, 4E-BP phosphorylation. 4E-BP is a transcription factor that can be phosphorylated by LRRK2 [20]. We found that both 68 and FX2149 reduced 4E-BP phosphorylation in mouse brains (Fig. 5E and 5F). FX2149 at a 10mg/kg dose reduced 4E-BP up to 15% of the untreated control group, while 68 at a 20 mg/kg dose reduced 4E-BP up to 15% of the untreated control group. These data indicated that FX2149 was taken up into brains at a greater extent and had more potent efficacy in inhibiting GTP binding and kinase activity in mouse brains when compared with 68.

Bottom Line: While most known LRRK2 inhibitors are developed to target the kinase domain, we have recently identified the first LRRK2 GTP binding inhibitor, 68, which not only inhibits LRRK2 GTP binding and kinase activities with high potency in vitro, but also reduces neurodegeneration.However, the in vivo effects of 68 are low due to its limited brain penetration.Our results highlight a novel GTP binding inhibitor with better brain efficacy, which represents a new lead compound for further understanding PD pathogenesis and therapeutic studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States of America.

ABSTRACT
Leucine-rich repeat kinase-2 (LRRK2), a cytoplasmic protein containing both GTP binding and kinase activities, has emerged as a highly promising drug target for Parkinson's disease (PD). The majority of PD-linked mutations in LRRK2 dysregulate its GTP binding and kinase activities, which may contribute to neurodegeneration. While most known LRRK2 inhibitors are developed to target the kinase domain, we have recently identified the first LRRK2 GTP binding inhibitor, 68, which not only inhibits LRRK2 GTP binding and kinase activities with high potency in vitro, but also reduces neurodegeneration. However, the in vivo effects of 68 are low due to its limited brain penetration. To address this problem, we reported herein the design and synthesis of a novel analog of 68, FX2149, aimed at increasing the in vivo efficacy. Pharmacological characterization of FX2149 exhibited inhibition of LRRK2 GTP binding activity by ~90% at a concentration of 10 nM using in vitro assays. Furthermore, FX2149 protected against mutant LRRK2-induced neurodegeneration in SH-SY5Y cells at 50-200 nM concentrations. Importantly, FX2149 at 10 mg/kg (i.p.) showed significant brain inhibition efficacy equivalent to that of 68 at 20 mg/kg (i.p.), determined by mouse brain LRRK2 GTP binding and phosphorylation assays. Furthermore, FX2149 at 10 mg/kg (i.p.) attenuated lipopolysaccharide (LPS)-induced microglia activation and LRRK2 upregulation in a mouse neuroinflammation model comparable to 68 at 20 mg/kg (i.p.). Our results highlight a novel GTP binding inhibitor with better brain efficacy, which represents a new lead compound for further understanding PD pathogenesis and therapeutic studies.

No MeSH data available.


Related in: MedlinePlus