Development and evaluation of novel LRRK2 inhibitors as PET ligands for in vivo imaging of LRRK2

1105 Objectives: There is an urgent need for the development of Parkinson9s disease (PD) treatments that can slow disease progression. LRRK2 (leucine-rich repeat kinase 2) has recently been identified as a causative gene for autosomal dominant Parkinson’s disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Several LRRK2 inhibitors have been developed and evaluated in vitro; however, in vivo target engagement has never been characterized. Despite research efforts invested to date, there is no radiotracer available for in vivo imaging of LRRK2 using PET. In our pilot studies, we synthesized and evaluated two tritium-labeled potent and selective kinase inhibitors, [3H]LRRK2-IN-1 (1st generation) and [3H]GNE-9605 (second-generation LRRK2 inhibitors), via in vitro (IC50, Kd, Bmax) and in vivo/ex vivo methodologies (autoradiography, bio-distribution, and blocking experiments) in rodents and human striatum tissues. Comparative studies indicated that, although LRRK2-IN-1 has a lower ΔG (lower free binding energy to the target enzyme determined via docking studies) than GNE-9605, GNE-9605 is more CNS permeable due to its higher lipophilicity than LRRK2-IN-1, suggesting more promising properties for ligands derived from second-generation LRRK2 inhibitors. We have, since then, identified candidates that are more potent and selective than current known LRRK2 inhibitors, based on in vitro assays. Radiolabeling and microPET evaluation studies are ongoing, preliminary results will be presented. Methods: We have prepared and evaluated several novel LRRK2 inhibitors and compared their in vitro properties (e.g., IC50 values,membrane permeability, and the P-glycoprotein liability) with those previously developed LRRK2 inhibitors, such as GNE compounds. We have also synthesized several corresponding precursors and carried out radiolabeling with either C-11 or F-18 to obtain the desired target molecules for further evaluation of their in vivo properties as PET ligands via microPET/CT studies, including in vivo/ex vivo bio-distribution and blocking studies. Results: Several in vitro assays were conducted to compare IC50 values (for example, in HEK293 cells with transient overexpression of LRRK2 G2019S measuring decrease of phosphoserine 935 with an antibody in a robust Meso Scale Discovery assay) for LRRK2_a and LRRK2_b (novel inhibitors) vs. GNE-7915 and GNE-9605. The results are consistently indicated that LRRK2_b (10 nM) is more potent and selective than GNE-7915 (40 nM), and GNE-9605 (90 nM). The results of the low efflux ratio (<3) from the CNS permeability measurement via MDR assay in the absence or presence of a Pgp inhibitor suggested that LRRK2_b is a promising candidate as its BBB permeability may not be a concern and it is not a good Pgp substrate. Radiolabeling of LRRK2_a and LRRK2_b with F-18 has been accomplished via one-step fluoro-for-tosyl radiosynthesis. In vivo microPET/CT evaluation studies in mice are underway. Conclusions: Potential in vivo imaging of LRRK2 with PET is an exciting, but at the same time uncharted research area, limited thus far by a lack of relevant information, resources and tools. Based on the in vitro and in vivo/ex vivo results, we have identified several candidates and will fine-tune the structure-activity relationship (SAR) to generate promising PET ligand candidates for in vivo imaging of brain LRRK2.