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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 reduced LPS-induced microglia activation and LRRK2-upregulation.G2019S-LRRK2 BAC transgenic mice (6–12 weeks) were injected with LPS (5 μg) and FX2149 (10 mg/kg) as described in the methods section. Serial coronal sections through the substantia nigra were subjected to immunohistochemistry analysis. A. Representative immunofluorescent images with anti-isolectin (green) and anti-LRRK2 (red) staining. B. Quantification of immunofluorescence of A by unbiased stereology. *p < 0.05 by ANOVA compared with vehicle group. #p < 0.05 by ANOVA compared with LPS treated group. C. Representative immunostaining with anti-phospho-LRRK2-S935 and anti-isolectin B4 (marker for microglia) antibodies by DAB detection.
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pone.0122461.g006: FX2149 reduced LPS-induced microglia activation and LRRK2-upregulation.G2019S-LRRK2 BAC transgenic mice (6–12 weeks) were injected with LPS (5 μg) and FX2149 (10 mg/kg) as described in the methods section. Serial coronal sections through the substantia nigra were subjected to immunohistochemistry analysis. A. Representative immunofluorescent images with anti-isolectin (green) and anti-LRRK2 (red) staining. B. Quantification of immunofluorescence of A by unbiased stereology. *p < 0.05 by ANOVA compared with vehicle group. #p < 0.05 by ANOVA compared with LPS treated group. C. Representative immunostaining with anti-phospho-LRRK2-S935 and anti-isolectin B4 (marker for microglia) antibodies by DAB detection.

Mentions: To further characterize the pharmacological effects of FX2149 in brains, a LPS-based mouse neuroinflammation model was used as described previously [13,16]. Consistent with previous findings [13,16], injection of LPS resulted in significant increases in LRRK2 expression, phosphorylation, and microglial activation in the substantia nigra compared with vehicle controls (Fig. 6). Treatment of mice with FX2149 significantly reduced LPS-induced LRRK2-positive immunostaining compared with vehicle controls, but it did not alter LRRK2 cytoplasmic localization. Moreover, the anti-phosphoryated-LRRK2 immunoactivity was also significantly reduced in the FX2149 treated group. As in our previous study of 68 at 20 mg/kg [13], FX 2149 at a 10 mg/kg dose significantly reduced LPS-induced isolectin B4 (microglia marker) positive immunostaining in the substantia nigra. There was a ~43% isolectin B4 immunoactivity in the FX2149 treated group compared with the LPS alone treated group. In comparison, the immunoactivity of isolectin B4 in the 68 treated group (20 mg/kg) was ~56% (Fig. 6B). Consistent with previous findings [13], anti-TH (dopaminergic neuron marker) immunostaining in the subtantia nigra did not change among various treatment groups and the control group, indicating that LPS treatment did not alter dopaminergic neuron degeneration in this acute inflammation condition.


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 reduced LPS-induced microglia activation and LRRK2-upregulation.G2019S-LRRK2 BAC transgenic mice (6–12 weeks) were injected with LPS (5 μg) and FX2149 (10 mg/kg) as described in the methods section. Serial coronal sections through the substantia nigra were subjected to immunohistochemistry analysis. A. Representative immunofluorescent images with anti-isolectin (green) and anti-LRRK2 (red) staining. B. Quantification of immunofluorescence of A by unbiased stereology. *p < 0.05 by ANOVA compared with vehicle group. #p < 0.05 by ANOVA compared with LPS treated group. C. Representative immunostaining with anti-phospho-LRRK2-S935 and anti-isolectin B4 (marker for microglia) antibodies by DAB detection.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122461.g006: FX2149 reduced LPS-induced microglia activation and LRRK2-upregulation.G2019S-LRRK2 BAC transgenic mice (6–12 weeks) were injected with LPS (5 μg) and FX2149 (10 mg/kg) as described in the methods section. Serial coronal sections through the substantia nigra were subjected to immunohistochemistry analysis. A. Representative immunofluorescent images with anti-isolectin (green) and anti-LRRK2 (red) staining. B. Quantification of immunofluorescence of A by unbiased stereology. *p < 0.05 by ANOVA compared with vehicle group. #p < 0.05 by ANOVA compared with LPS treated group. C. Representative immunostaining with anti-phospho-LRRK2-S935 and anti-isolectin B4 (marker for microglia) antibodies by DAB detection.
Mentions: To further characterize the pharmacological effects of FX2149 in brains, a LPS-based mouse neuroinflammation model was used as described previously [13,16]. Consistent with previous findings [13,16], injection of LPS resulted in significant increases in LRRK2 expression, phosphorylation, and microglial activation in the substantia nigra compared with vehicle controls (Fig. 6). Treatment of mice with FX2149 significantly reduced LPS-induced LRRK2-positive immunostaining compared with vehicle controls, but it did not alter LRRK2 cytoplasmic localization. Moreover, the anti-phosphoryated-LRRK2 immunoactivity was also significantly reduced in the FX2149 treated group. As in our previous study of 68 at 20 mg/kg [13], FX 2149 at a 10 mg/kg dose significantly reduced LPS-induced isolectin B4 (microglia marker) positive immunostaining in the substantia nigra. There was a ~43% isolectin B4 immunoactivity in the FX2149 treated group compared with the LPS alone treated group. In comparison, the immunoactivity of isolectin B4 in the 68 treated group (20 mg/kg) was ~56% (Fig. 6B). Consistent with previous findings [13], anti-TH (dopaminergic neuron marker) immunostaining in the subtantia nigra did not change among various treatment groups and the control group, indicating that LPS treatment did not alter dopaminergic neuron degeneration in this acute inflammation condition.

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