We have developed a high-throughput and homogenous cellular TR-FRET immunoassay for the measurement of LRRK2 phosphorylation. Since acute inhibition of LRRK2 kinase activity can reduce the level of Ser935 phosphorylation, this assay can be applied to high-throughput cell-based screens for LRRK2 kinase inhibitors. Screening of a small molecule inhibitor library with this assay indeed revealed several inhibitors with previously unknown LRRK2 activity as well as provided leads to cellular pathways that could involve LRRK2.
This high-throughput assay utilizes cells expressing full-length human LRRK2 with a C-terminal GFP tag (LRRK2-GFP). We provide multiple lines of evidence suggesting that LRRK2-GFP functions and behaves similarly to the previously reported N-terminally tagged LRRK2 (GFP-LRRK2) stably expressed in HEK293 cells. First, wild-type and G2019S LRRK2-GFP displayed a diffuse cytoplasmic localization 
and upon LRRK2-IN-1 treatment, a portion of LRRK2 relocalized to more aggregate and fibrillar-like structures similar to was observed previously, where H-1152 treatment of cells induced cytoplasmic accumulation of LRRK2 that appeared to colocolize with microtubules 
. The nature of these accumulations has yet to be thoroughly investigated but has been widely observed. BacMam mediated expression of LRRK2 R1441C reproduced previous observations that many Roc and COR domain pathogenic mutants induce cytoplasmic accumulations of LRRK2 
. Interestingly, acute inhibition of R1441C mutant also induced a redistribution of LRRK2 to more filamentous aggregates similar to what was observed for the Ser910Ala/Ser935Ala mutant 
Second, LRRK2-GFP wild-type, G2019S, R1441C and D1994A showed similar Ser935 phosphorylation pattern determined by Western blot and the TR-FRET assay as reported for GFP-LRRK2 
. Third, known LRRK2 inhibitors such as LRRK2-IN-1, TAE684, sunitinib and H-1152 inhibited the phosphorylation of Ser935 on LRRK2-GFP wild-type and G2019S to a similar degree and with similar rank order potency as previously reported for GFP-LRRK2. Lastly, other phosphorylation sites such as Ser910, Ser955 and Ser973 reported for N-GFP-tagged LRRK2 can also be phosphorylated on the LRRK2-GFP and inhibited by LRRK2-IN-I (Figure S1
One of the common methods for detecting LRRK2 phosphorylation is immunoblotting, typically after immunoprecipation. The complexities of this methodology make it highly impractical for the processing and analysis of large sample numbers typically associated with screening experiments. The TR-FRET cellular assay reported here is in a fully homogenous format without washing, lysate transfer, or separation procedures. Cells transduced with BacMam LRRK2-GFP can be plated onto a 384-well assay plate, treated as desired and then incubated with 6 X lysis buffer containing Tb-labeled detection antibody added directly to the wells. To further simplify the procedure, we demonstrated that cells can be transduced, cryopreserved, later thawed and used directly to run the assay without the need for culturing (Figure S3
). This indicates that one can perform a large batch transduction and cryopreserve the cells for screening applications at a later time, which will reduce the time, labor and day-to-day variables.
We profiled 1120 compounds of the Tocriscreen™ Mini library to gain insight into the biology of Ser935 phosphorylation and demonstrate the utility of the TR-FRET cellular assay in screening. Using a maximal inhibition threshold of 50%, we observed the previously identified inhibitors of LRRK2 SP600125, indirubin-3′-monoxime and ROCK inhibitor Y-27632 induced dephosphorylation of Ser935, which increased confidence in our assays. In addition to compounds with known LRRK2 inhibitory activity, we identified several novel LRRK2 kinase inhibitors from this screen. GW441756 is a known TrkA inhibitor 
and has some degree of structure similarity to GW5074. Here we show GW441756 can inhibit the phosphorylation of LRRK2 on Ser935 in cells by both TR-FRET assays and IP-Western. We further confirmed that GW441756 is a LRRK2 kinase inhibitor in the TR-FRET LRRK2 in vitro
kinase assay with an IC50
of 320 nM. Interestingly, we did not observe in vivo
dephosphorylation of Ser935 with the PKC inhibitors GF109203X and Ro31-8220, which were present in the Tocriscreen library and identified previously to inhibit LRRK2 autophosphorylation in vitro
Interestingly, we observed that 5 out of the top 16 compounds Bay 11-7085, Bay 11-7821, IKK16, Ro106-9920 and TPCA-1 intersect with the NFκB pathway. TPCA-1 and IKK16 are IKK inhibitors, however LRRK2 is inhibited by IKK16 at an in vitro
20 times lower than the cellular IC50
, indicating that the cellular effect may be due to LRRK2 inhibition. Ro106-9920 inhibits IκB degradation and LPS induced cytokine (TNF-alpha, IL1-beta and IL-6) production 
and suppressed LRRK2 phosphorylation on Ser935 by 77% in SH-SY5Y cells and approximately 50% in HEK293 cells. Bay 11-7085 and Bay 11-7821 block NFκB activation by inhibiting the IκB complex. In 293 cells, the inhibitors Bay 11-7085 and Bay 11-7821 consistently induced dephosphorylation of both LRRK2 G2019S and G2019S/A2016T to 50% of untreated G2019S indicating an independence of direct action on LRRK2. These data indicate that IKKβ is a potential in vivo
kinase regulating LRRK2 phosphorylation. The inhibitor BX-795, which inhibits PDK1 as well as the TBK1 and IKKε kinases, decreased the phosphorylation of the cellular phosphorylation sites in HEK293 T-REx™ cells expressing GFP-LRRK2 G2019S, and the G2019S mutation showed decreased sensitivity to BX795 indicating it is indeed a direct inhibitor of LRRK2. BX795 treatment cannot then be utilized to rule in or out IKKε or TBK1 in their ability to phosphorylate LRRK2 in vivo
. Interestingly, inhibition of LRRK2 Ser935 phosphorylation occurs in the absence of receptor stimulation and may represent perturbing a basal status of modification. Together, these data indicate that the pathways that induce NFκB activation play a role in LRRK2 phosphorylation, but this regulation is likely complex.
Inhibitors of the Cdc25 phosphatase NSC-95397 and NSC-663284 or kinase inhibitors aminopurvalanol A (CDK), SB 218078 and PD407824 (CHK1) can block cell cycle progression 
and were found to also repress LRRK2 Ser935 phosphorylation. PD407824 inhibited LRRK2 in vitro
and Ser935 phosphorylation in cells indicating that indeed this is potentially another direct LRRK2 kinase inhibitor. Since the LRRK2 inhibitor resistant mutant did not show decreased sensitivity to this compound, it may well be that CHK1 could act on the cellular phosphorylation sites in a LRRK2 kinase activity independent manner. In the future, it would be interesting to investigate LRRK2 activity and phosphorylation during different stages of the cell cycle, especially since there are multiple lines of evidence linking LRRK2 to cancer 
We also observed compounds other than kinase inhibitors that affect LRRK2 phosphorylation, but do not directly target LRRK2 enzyme itself, namely GPCR ligands such as opioid-receptor like-1 (OPRL-1) antagonist JTC801, the dopamine receptor D1 antagonist BNTX maleate, dopamine D2-like receptor ligand 3′-Fluorobenzylspiperone maleate, and an agonist of the adenosine A2 receptor CV1808. Though results are complicated for some of these compounds due to their cytotoxic effects, these compounds could indicate potential signaling pathways that also alter LRRK2 function via effects on LRRK2 phosphorylation. These represent avenues for further research.
In summary, we have developed a robust high-throughput detection system for evaluating the phosphorylation of LRRK2 at Ser935 that correlates well with alternate systems. A small library was used to evaluate this screening system and hits from that screen were validated in established systems showing the reliability of the methodology. By exploiting the biology of Ser935 phosphorylation, we gained novel insights into potential pathways that intersect with LRRK2, as well as identified other possible direct LRRK2 inhibitors.