GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor

Abstract

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinson's disease. Here we report the discovery and characterization of 2-arylmethyloxy-5-subtitutent-N-arylbenzamides with potent LRRK2 activities exemplified by GSK2578215A which exhibits biochemical IC(50)s of around 10 nM against both wild-type LRRK2 and the G2019S mutant. GSK2578215A exhibits exceptionally high selectivity for LRRK2 across the kinome, substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.3-1.0 μM in cells and in mouse spleen and kidney, but not in brain, following intraperitoneal injection of 100mg/kg.

Figure 1. GSK2578215A inhibits LRRK2 in vitro.

(a) Chemical structure of GSK2578215A 1. (b) Enzyme activity of GSK2578215A. GST-LRRK2(1326–2517), GST-LRRK2[G2019S](1326–2517), GST-LRRK2[A2016T](1326–2517) and GST-LRRK2[G2019S + A2016T](1326–2517) were assayed using 20 μM Nictide in the presence of 100 μM ATP. Results are average of duplicate experiments.

Figure 2. LRRK2 homology model.

GSK2578215A (magenta solvent-accessible surface) binds in the same way to normal and A2016T mutant without a steric clash.

Figure 3. GSK2578215A inhibits LRRK2 in cells.

(a) HEK 293 cells stably expressing wild-type GFP-LRRK2, GFP-LRRK2[G2019S], GFP-LRRK2[G2019S + A2016T], and GFP-LRRK2[A2016T] were treated with DMSO or increasing concentrations of GSK2578215A for 90 min. Cell lysates were subjected to immunoblotting for detection of LRRK2 phosphorylated at Ser910 and Ser935 and for total LRRK2. (b) As in (a) except LRRK2-IN-1 was used.

Figure 4. GSK2578215A inhibits endogenously expressed LRRK2.

(a) Endogenous LRRK2 from EBV immortalized human lymphoblastoid cells from a control subject and a Parkinson’s disease patient homozygous for the LRRK2[G2019S] mutation. After treatment of the cells with DMSO or the indicated concentration of GSK2578215A (or LRRK2-IN-1) for 90 min, cell lysates were subjected to immunoblot analysis with the purified indicated antibody for western analysis. Immunoblots were performed in duplicate, and results were representative of at least two independent experiments. (b) As in (a) except mouse Swiss 3T3 cells were used.

Figure 5. Pharmacodynamic analysis for GSK2578215A.

Pharmacodynamic study of GSK2578215A from brain, spleen and kidney following intraperitoneal injection at dose of 100 mg/kg. Tissues were collected and endogenous LRRK2 was resolved by SDS–PAGE and blotted with a phospho-specific antibody directed against Ser910, Ser935 and total LRRK2.