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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 attenuated G2019S-LRRK2-induced neuronal degeneration in SH-SY5Y cells.SH-SY5Y cells were co-transfected with GFP and various pcDNA3.1-LRRK2 plasmids at a 1:15 ratio as described in the method section. After 4-h transfection, cells were treated with FX2149 for 48 hours. A. Cell viability was measured by counting the healthy viable GFP positive cells that contained at least one smooth extension (neurite) that was twice the length of the cell body. *p< 0.05 by ANOVA compared to wild type LRRK2. #p < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle. B. TUNEL assays. The experiments were repeated three times. *p < 0.05 by ANOVA compared to vector control. #p < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle.
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pone.0122461.g004: FX2149 attenuated G2019S-LRRK2-induced neuronal degeneration in SH-SY5Y cells.SH-SY5Y cells were co-transfected with GFP and various pcDNA3.1-LRRK2 plasmids at a 1:15 ratio as described in the method section. After 4-h transfection, cells were treated with FX2149 for 48 hours. A. Cell viability was measured by counting the healthy viable GFP positive cells that contained at least one smooth extension (neurite) that was twice the length of the cell body. *p< 0.05 by ANOVA compared to wild type LRRK2. #p < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle. B. TUNEL assays. The experiments were repeated three times. *p < 0.05 by ANOVA compared to vector control. #p < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle.

Mentions: GTP binding activity and elevated kinase activities have been implicated in PD-linked mutant, G2019S-LRRK2, resulting in neurodegeneration [6,13]. SH-SY5Y cells contain dopamine and are often used as a PD cell model [6]. To assess whether FX2149 alters mutant LRRK2-induced neuronal degeneration, G2019S-LRRK2 construct transiently transfected into SH-SY5Y cells was used as a toxicity model as previously described [6]. Treatment of FX2149 significantly increased the viability of cells expressing G2019S-LRRK2 compared with vehicle treated cells (Fig. 4A). Moreover, FX2149 at 100 nM significantly reduced the TUNEL-positive cells expressing mutant G2019S-LRRK2 and had effects equivalent to that of 68 at 10 nM (Fig. 4B).


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 attenuated G2019S-LRRK2-induced neuronal degeneration in SH-SY5Y cells.SH-SY5Y cells were co-transfected with GFP and various pcDNA3.1-LRRK2 plasmids at a 1:15 ratio as described in the method section. After 4-h transfection, cells were treated with FX2149 for 48 hours. A. Cell viability was measured by counting the healthy viable GFP positive cells that contained at least one smooth extension (neurite) that was twice the length of the cell body. *p< 0.05 by ANOVA compared to wild type LRRK2. #p < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle. B. TUNEL assays. The experiments were repeated three times. *p < 0.05 by ANOVA compared to vector control. #p < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4376719&req=5

pone.0122461.g004: FX2149 attenuated G2019S-LRRK2-induced neuronal degeneration in SH-SY5Y cells.SH-SY5Y cells were co-transfected with GFP and various pcDNA3.1-LRRK2 plasmids at a 1:15 ratio as described in the method section. After 4-h transfection, cells were treated with FX2149 for 48 hours. A. Cell viability was measured by counting the healthy viable GFP positive cells that contained at least one smooth extension (neurite) that was twice the length of the cell body. *p< 0.05 by ANOVA compared to wild type LRRK2. #p < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle. B. TUNEL assays. The experiments were repeated three times. *p < 0.05 by ANOVA compared to vector control. #p < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle.
Mentions: GTP binding activity and elevated kinase activities have been implicated in PD-linked mutant, G2019S-LRRK2, resulting in neurodegeneration [6,13]. SH-SY5Y cells contain dopamine and are often used as a PD cell model [6]. To assess whether FX2149 alters mutant LRRK2-induced neuronal degeneration, G2019S-LRRK2 construct transiently transfected into SH-SY5Y cells was used as a toxicity model as previously described [6]. Treatment of FX2149 significantly increased the viability of cells expressing G2019S-LRRK2 compared with vehicle treated cells (Fig. 4A). Moreover, FX2149 at 100 nM significantly reduced the TUNEL-positive cells expressing mutant G2019S-LRRK2 and had effects equivalent to that of 68 at 10 nM (Fig. 4B).

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