ATP-competitive LRRK2 inhibitors interfere with monoclonal antibody binding to the kinase domain of LRRK2 under native conditions. A method to directly monitor the active conformation of LRRK2?

Frank Gillardon; Elisabeth Kremmer; Thomas Froehlich; Marius Ueffing; Bastian Hengerer; Christian J Gloeckner

doi:10.1016/j.jneumeth.2012.12.015

ATP-competitive LRRK2 inhibitors interfere with monoclonal antibody binding to the kinase domain of LRRK2 under native conditions. A method to directly monitor the active conformation of LRRK2?

J Neurosci Methods. 2013 Mar 30;214(1):62-8. doi: 10.1016/j.jneumeth.2012.12.015. Epub 2013 Jan 12.

Authors

Frank Gillardon¹, Elisabeth Kremmer, Thomas Froehlich, Marius Ueffing, Bastian Hengerer, Christian J Gloeckner

Affiliation

¹ CNS Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany. Frank.Gillardon@boehringer-ingelheim.com

PMID: 23318290
DOI: 10.1016/j.jneumeth.2012.12.015

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. LRRK2 kinase activity is required for toxicity in neuronal cell cultures suggesting that selective kinase inhibitors may prevent neurodegeneration in patients. Directly monitoring LRRK2 activity in cells would be advantageous for the development of small molecule LRRK2 inhibitors. Here, we demonstrate that a monoclonal anti-LRRK2 antibody directed against the activation segment binds less efficiently to native LRRK2 protein in the presence of ATP-competitive LRRK2 inhibitors. Since kinase inhibitors prevent autophosphorylation and refolding of the activation segment, we hypothesize that the antibody preferentially binds to the active conformation of LRRK2 under native conditions.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / analogs & derivatives
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / pharmacology
Adenosine Triphosphate / metabolism
Amino Acid Sequence
Amino Acid Substitution
Animals
Antibodies, Monoclonal / metabolism*
Antigen-Antibody Reactions
Benzodiazepinones / pharmacology
Binding, Competitive
Enzyme Activation
Epitopes / chemistry
Epitopes / metabolism*
HEK293 Cells
Humans
Immunoprecipitation
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
Mice
Mice, Inbred C57BL
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Parkinson Disease / enzymology
Parkinson Disease / genetics
Phosphorylation
Protein Binding
Protein Conformation
Protein Kinase Inhibitors / pharmacology*
Protein Processing, Post-Translational
Protein Serine-Threonine Kinases / antagonists & inhibitors*
Protein Serine-Threonine Kinases / chemistry
Protein Serine-Threonine Kinases / metabolism
Protein Structure, Tertiary
Pyrimidines / pharmacology
Recombinant Proteins / antagonists & inhibitors
Swiss 3T3 Cells

Substances

2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine
Antibodies, Monoclonal
Benzodiazepinones
Epitopes
LRRK2-IN1
Protein Kinase Inhibitors
Pyrimidines
Recombinant Proteins
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
Adenosine Triphosphate
LRRK2 protein, human
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
Lrrk2 protein, mouse
Protein Serine-Threonine Kinases