Development of Novel Radioligands for Imaging LRRK2 in Parkinson’s Disease

Objectives: LRRK2 (Leucine-rich repeat kinase 2) has recently been proven to be a promising drug target for Parkinson’s disease (PD) due to its apparent enhanced activity caused by PD-associated mutations; for example, G2019S mutation in LRRK2 can cause up to 30% of sporadic PD in some populations. To date, there have been no publications in which a LRRK2 inhibitor has been radiolabeled and used for in vitro or in vivo studies of LRRK2. In the present study, we radiolabeled the LRRK2 ligand, LRRK-IN-1, for the purposes of performing in vitro (IC50, Kd, Bmax, autoradiography) and in vivo (biodistribution and blocking experiments) evaluations in rodents and human striatum tissues.

Methods: Both [3H]-labeled ligands (LRRK2-IN-1 and GNE-9605) were prepared with high radiochemical purity (~99%) and a specific molar radioactivity of 23-41Ci/nmol via tritium/hydrogen (T/H) exchange using Crabtree’s catalyst. For IC50, Kd and Bmax determination, LRRK2-IN-1 was used as a competing drug for nonspecific binding assessment. The specific binding of the tracer was further evaluated via an in vivo blocking study in mice with a potent LRRK2 inhibitor, Pf-06447475 (0, 1, 3, 10 mg/kg). [³H]GNE-9605, one of the most promising second generation LRRK2 inhibitors, was also synthesized and evaluated. Comparative in vivo bio-distribution and blocking studies and in vitro ARG studies were carried out.

Results: In the radioligand binding assays, specific binding of [³H]LRRK2-IN-1 was observed both in the rat tissues (kidney and brain) and in the post-mortem human tissues (brain). The binding of [³H]LRRK2-IN-1 was displaced by unlabeled LRRK2-IN-1 with IC50 values of 40±4 in rat kidney, 65±3 in rat brain striatum and 73±6 nM in human brain striatum tissues. In vitro binding studies demonstrated a saturable binding site for [3H]LRRK2-IN-1 in rat kidney, rat brain striatum and human brain striatum with Kd values of 26±3 and 43±8, 48±2 nM, respectively. In rat, the density of LRRK2 binding sites (Bmax) was higher in kidney (6.4±0.04 pmol/mg) than in brain (2.5±0.03 pmol/mg); however, in human brain striatum, the Bmax was 0.73±0.01 pmol/mg protein. Autoradiography imaging in striatum of rat and human brain tissues gave results consistent with binding studies. In in vivo biodistribution and blocking studies in mice, co-administration with Pf-06447475 dose-dependently reduced the uptake of [³H]LRRK2-IN-1 (%ID/g) with 50-60% reduction at the highest dose (10 mg/kg) in the kidney or brain. Comparative studies of [³H]GNE-9605 vs. [³H]LRRK2-IN-1 indicated that, although LRRK2-IN-1 has a lower ΔG (lower free binding energy determined via docking studies) than GNE-9605, GNE-9605 is more CNS permeable due to its higher lipophilicity than LRRK2-IN-1. Conclusions: Our studies demonstrated that LRRK2 binding sites are present at high levels in both periphery (kidney) and brain, suggesting that this enzyme can serve as a suitable target for PET studies. Our studies also indicated that [³H]LRRK2-IN-1 is able to specifically label LRRK2 binding sites in vitro and thus can be used as a research tool for radioligand binding assays. However, the low level of brain uptake limits its use as a suitable PET ligand for brain imaging of LRRK2. [³H]GNE-9605 showed more promising properties as a ligand, as it has a higher uptake and higher specific binding in the brain as compared to [³H]LRRK2-IN-1.