Mutations in the LRRK2 gene are the leading cause of familial Parkinson's disease (PD), often resulting in increased kinase activity and phosphorylation (modification) of several serine amino acids (S910 and S935). Inhibition of LRRK2 kinase activity is a potential therapeutic target for treating PD, which reduces phosphorylation of these serine residues. Additionally, the phosphorylation state of the LRRK2 protein may serve as an indicator of disease progression. This project aims to identify binding reagents that are able to discriminate between different phosphorylation states of LRRK2, as no such reagents currently exist.
The primary objective of this study is to identify one or more Slow Off-rate Modified Aptamers (SOMAmer) reagents that bind with high affinity to phosphorylated LRRK2 but not non-phosphorylated LRRK2. The secondary objective is to identify SOMAmer reagents that bind to both phosphorylated and non-phosphorylated LRRK2 (total LRRK2).
This study will utilize wild type (WT) LRRK2 protein, which can exist in multiple phosphorylation states, and LRRK2 protein that cannot be phosphorylated at four specific sites (910, 935, 955 and 973) due to the deliberate introduction of mutations (4xSA-LRRK2). We will screen 81 potential LRRK2 SOMAmer reagents to determine if they show differences in binding between the two proteins. Reagents that able to discriminate between WT LRRK2 and 4xSA-LRRK2 will be tested for their ability to bind the LRRK2 protein in a complex matrix such as cell lysate (breakdown of cells). Lastly, we will use an analytical technique capable of determining which phosphorylation state of LRRK2-specific SOMAmer reagents recognize the cell lysate.
Impact on Diagnosis/Treatment of Parkinson's disease:
No molecular recognition reagents are available that can discriminate between the different phosphorylation states of LRRK2. If validated, these reagents may have immediate utility in the research setting as well as in tracking PD progression.
Next Steps for Development:
The SOMAmer reagents identified in this project may ultimately be incorporated into a quantitative diagnostic assay to measure phosphorylated and total LRRK2 concentrations in biological samples, such as human cerebrospinal fluid (clear fluid found in the brain and spinal cord) and blood.