Spencer B. Hermanson 1 , Coby B. Carlson 1 , Steven M. Riddle 1 , Jing Zhao 2 , Kun Bi 1 and R. Jeremy Nichols 2 1 Life Technologies Corporation, Madison, WI U.S.A; 2 The Parkinson’s Institute, Sunnyvale, CA U.S.A. High throughput screening of small molecules that inhibit LRRK2 phosphorylation on Serine 935 reveals novel inhibitors and cellular pathways that affect LRRK2 Abstract LRRK2 is phosphorylated on Serines 910, 935, 955 and 973, which has been shown to be likely regulated b ll l ki h i difi d i f db k h i h ki Figure 4. Tocris Mini Library Screen Using LRRK2 pSer935 Cellular Assay Tocris Library Hit Profile in LRRK2 Cellular Assay by cellular kinases. These sites are modified in an apparent feedback mechanism where LRRK2 kinase activity potentiates the kinase activity against these sites or perhaps negatively regulates the phosphatase of these sites (Figure 1). Acute inhibition of LRRK2 leads to rapid dephosphorylation of the cellular phosphosites. Inhibition of Serine 935 may indicate perturbation of the signaling pathway upstream or direct inhibition of LRRK2. Immunological detection of Serine 935 phosphorylation has been adapted to a homogenous TR‐FRET based high‐throughput cellular assay for LRRK2. This assay was applied to a small compound library to assess applicability of the assay to screening against a larger compound library. We identified ~20 compounds that led to the cellular dephosphorylation of LRRK2 Serine 935 in SH‐SY5Y cells. We counter screened these hits against LRRK2 in vitro and indeed some of these compounds had direct inhibitory effects on LRRK2 and interestingly, others did not. We confirmed a selection of the cellular pathways indicated by these hits with further pharmacological and reverse genetic approaches. We provide evidence that cell based screening for phosphorylation of Serine 935 (and potentially other phosphosites) can yield novel therapeutic targets for Parkinson’s disease as well as new potential direct -50 -25 0 25 50 75 100 0 10 20 30 40 50 Percent Inhibition Frequency -100 -75 -50 -25 0 25 50 75 100 125 -100 -75 -50 -25 0 25 50 75 100 125 Percent Inhibition Replicate 1 Percent Inhibition Replicate 2 To demonstrate the utility of the LRRK2 cellular assay in HTS applications a compound screen was performed using the Tocris mini library – a collection of 1120 biologically active compounds. Table 2. Percent inhibition of some top hits from the Cytotoxicity Assay In vitro Biochemical LRRK2 assay Max % Inhibition IC50 (µM) IC50 (µM) IC50 (µM) 97.5 Bay 11-7085 Irreversible inhibitor of TNFa-induced IkB phosphorylation 96.6 8.3 >20 >100 85.5 Bay 11-7821 Irreversible inhibitor of TNFa-induced IkB phosphorylation 94.2 5.2 20 >100 81.7 IKK 16 Inhibitor of IKK 82.5 2.1 >20 0.78 Compound Name Known Compound Activity (Tocris description) In vivo Cellular TR-FRET assay LRRK2 G2019S % Inhibition (at 20 M) Figure 2. BacMam Enabled Expression of LRRK2 in multiple cell lines Figure 1. Schematic of LRRK2 regulation at Serines 910/935/955& 973 LRRK2 is found to be phosphorylated on Serines 910/935/955/973. This model shows that LRRK2 kinase activity potentiates a kinase activity towards these cellular phosphosites. Conversely, LRRK2 may negatively regulate a phosphatase that dephosphorylates LRRK2. inhibitors of LRRK2. Percent inhibition of some top hits from the screen with their biological activity shown and the maximal inhibition observed. Titrations of these compounds were evaluated to derive cellular IC50 values. Since 935 inhibition can be indicitive of direct inhibition of LRRK2, hese hits were tested in vitro against recombinant LRRK2 for inhibition (potent hits in Red). 69.8 Ro 106-9920 Inhibitor of NFkB activation 76.4 7.6 20 >100 54.8 TPCA-1 Inhibitor of IKK-2 46.2 9.2 >20 ND 76.1 NSC 95397 Selective Cdc25 dual specificity phosphatase inhibitor 95.9 2.2 7.8 1.9 67.3 PD 407824 Inhibitor of Chk1 and Wee1 65.3 0.6 15 0.68 59.1 NSC 663284 Cdc25 phosphatase inhibitor 45.6 4.9 2 0.13 55.3 SB 218078 Inhibitor of checkpoint kinase 1 (Chk1) 58.9 0.1 >20 6 88.3 SP 600125 JNK and other kinase inhibitor 103.1 0.9 >20 0.4 69.7 GW 441756 TrkA inhibitor 72.2 2.3 >20 0.65 67.3 Y-27632 p160ROCK inhibitor 68.8 14.5 >20 0.88 100 LRRK2 IN-1 Positive Control Compound 100.2 0.1 >20 0.008 α-pS935 α-LRRK2 910/GFP Veh. Cont. p910 0.4 0.6 0.8 1.0 1.2 pS910 0.4 0.6 0.8 1.0 1.2 p910/GFP Veh. Cont. Figure 5. Testing Library hits against LRRK2 and the Inhibitor desensitized mutant A2016T in HEK293 cells and against endogenous LRRK2 U‐2 OS SH‐SY5Y Human Astrocytes A. B. C. 1 DMSO 2 LRRK2IN1 3 GW 441756 4 BNTX maleate 5 NSC 95397 6 Y-27632 7 PD 407824 8 JTC 801 9 IKK 16 10 Bay 11-7085 11 SB 218078 12 Bay 11-7821 13 Ro 106-9920 14 SP 600125 15 BX795 α LRRK2 α-Tubulin α-pErk1/2 Figure 5. A. Inhibitors revealed to affect LRRK2 phosphorylation at Ser935 in the LanthaScreen assay were tested in the HEK‐293 T‐Rex cells stably, inducibly expressing Nterm GFP LRRK2. The indicated compounds were assayed at 20uM in GFP‐LRRK2 expressing cells. Phosphorylation of GFP‐LRRK2 on Serines 910/935/955 & 973 was assessed. Graphs are ratios of GFP/phosphosites set to DMSO control for each antibody. B. The efficacy of selected inhibitors that directly inhibited LRRK2 were also tested against the i hibit it t t t (IRM) A2016T f LRRK2 C Th DMSO LRRK2IN1 GW 441756 BNTX maleate NSC 95397 Y-27632 PD 407824 JTC 801 IKK 16 Bay 11-7085 SB 218078 Bay 11-7821 Ro 106-9920 SP 600125 p %V α-pS910 α-pS935 α-pS955 α-pS973 α-GFP 0.0 0.2 0.4 p935 0.0 0.2 0.4 0.6 0.8 1.0 1.2 p955 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1 2 p973 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1 2 p935/GFP % Veh. Cont. p955/GFP % Veh. Cont. p973/GFP % Veh. Cont. 0.0 0.2 pS935 0.0 0.2 0.4 0.6 0.8 1.0 1.2 pS955 0.0 0.2 0.4 0.6 0.8 1.0 1.2 GS IRM GS IRM GS IRM GS IRM GS IRM GS IRM GS IRM GS IRM GS IRM GS IRM pS973 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1 DMSO 2 RRK2IN1 3 W441756 7 D407824 9 IKK 16 10 y 11-7085 11 B218078 12 y 11-7821 14 P 600125 15 BX795 α-pS910 α-pS935 α-pS955 α-pS973 α-GFP p %V p935/GFP % Veh. Cont. p955/GFP % Veh. Cont. p973/GFP % Veh. Cont. A. U‐2 OS cells, SH‐SY5Y Cells and human astrocytes were transduced with BacMam viruses of GFP tagged LRRK2 and the indicated mutants. Cells were exposed to 20% virus for 24 hours, media was exchanged and cells were allowed to incubate an additional 24 hours before imaging by fluorescence microscopy. B. Cellular phosphorylation of LRRK2 GFP tagged at the carboxy and amino termini is compared. C. Cellular phosphorylation of LRRK2 and the indicated mutants were evaluated in the context ype LRRK2 GFP DMSO LRRK2‐IN1 D. inhibitor resistant mutant (IRM) A2016T of LRRK2. C. The compounds were evaluated against endogenous LRRK2 in the mouse Raw Macrophages. GS=G2019S, IRM=G2019S/A2016T LR GW PD Bay SB Bay SP Figure 6. In Vitro Evaluation of Kinase Activity against LRRK2 Ser910/935/973 LanthaScreen® LRRK2 [pSer935] Cellular Assay -4 -3 -2 -1 0 1 0.00 0.05 0.10 0.15 0.20 0.25 log [LRRK2-IN-1] (M) TR-FRETratio B. of LRRK2‐IN1 treatement in U2OS cells and SH‐ SY5Y cells. D. Bacmam delivered LRRK2‐GFP relocalizes after acute inhibition with LRRK2‐IN1 in U2OS cells. Wild‐ty Figure 3. HTS Amenable Cellular Assay for Monitoring the Phosphorylation of LRRK2 Ser935 BacMam-enabled LanthaScreen® pLRRK2 [Ser935] Assay in U-2 OS cells 0.06 0.08 0.10 0.12 WT G2019S D1994A R1441C 520nm/495nm Figure 6. GST-LRRK2 (aa882-1300) (A) or MBP (B) were subjected to in vitro kinase assay with the indicated kinases in the presence of 32P- ATP. Reaction products were visualized by autoradiography and colloidal blue staining (A) and immunoblot with anti-GST (Green) and anti- phosphoserine 910, 935 & 973 antibodies (Red) (C). Similar units of kinase activity were used as assessed against the non specific substrate myelin basic protein (MBP) shown in B. AutoP=autophospohrylation Figure 7. In Vivo Evaluation of Kinase Activity against LRRK2 Ser910/935/973 LRRK2 siRNA profiling: CHEK1 LRRK2 siRNA profiling: CDC25c C. D. 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 MOCK Neg1 GFP LRRK2 LRRK2 LRRK2 Emission Ratio A. IC50 (µM) U-2 OS SHSY5Y HEK293T Compound Name WT G2019S WT G2019S WT G2019S LRRK2-IN-I 0.08 0.06 0.22 0.094 0.03 0.01 JAK3 Inh. VI 0.24 0.18 1.2 0.73 1.17 0.31 Indirubin-3-oxine 5.12 2.54 >10 3.8* 5.96 3.55 Sunitinib 0.27 0.15 0.23 0.11 0.60 0.48 GW5074 >20 0.03* >20 >20 >20 0.28* H-1152 2.80 0.85 1.7 0.62 3.88 1.43 H-89 >20 >20 >20 >20 nd nd Staurosporine 0.001 0.001 nd nd 0.001 0.002 Using Time‐Resolved Förster Resonance Energy Transfer (TR‐FRET) technology, we developed a high‐ throughput compatible homogenous cellular assay for monitoring LRRK2 phosphorylation at Ser935 0.0001 0.001 0.01 0.1 1 10 0.02 0.04 [LRRK2-IN-1] M 5 BacMam-enabled LanthaScreen pLRRK2 [Ser935] Assay in SH-SY5Y cells 0.0001 0.001 0.01 0.1 1 10 0.02 0.03 0.04 0.05 0.06 0.07 0.08 WT G2019S R1441C [LRRK2-In-I], M 520nm/495nm Figure 7. In vivo evaluation of pathway hits on LRRK2 phosphorylation: LanthaScreen assay combined with reverse genetics and pharmacological interrogation. A. p935 Lantha cellular assays were performed in 384 well dishes with LRRK2 specific siRNAs. B. Control screen with LRRK2‐IN1. C &D. CHK1 and CDC25 siRNA delivery suppresses LRRK2 phosphorylation compared to control and mock siRNAs while LRRK2 and GFP siRNAs served as positive controls. B. Analysis of NFkB pathway signaling on LRRK2 pSer935. Raw macrophages were treated with the indicated inhibitors and concentrations for 90min. Where indicated, 30min into treatment, cells were co‐ treated with LPS treatment for 1hr. DMSO 2IN1 LRRK2 inhibitor TAE684 LRRK2 inhibitor CLI‐095 (TAK‐242) inhibitor of TLR4 intracellular domain OxPAPC inhibitor of TLR2/4 via blocking LPS receptor (5Z)-7-Oxozeaenol TAK1 inhibitor BX795 IKK/TBK1, MARK, PDK1 inhibitor IKK16 IKKb inhibitor Bay7085 inhibitro of IKK LRRK2 siRNA profiling: CHEK1 0.01 0.1 1 10 100 0.00 0.04 0.08 0.12 0.16 0.20 Neg LRRK2 GFP oligo 1 MOCK oligo 2 oligo 3 [siRNA oligo] (nM) Emission Ratio LRRK2 siRNA profiling: CDC25c 0.01 0.1 1 10 100 0.00 0.04 0.08 0.12 0.16 0.20 Neg LRRK2 GFP oligo 1 MOCK oligo 2 oligo 3 [siRNA oligo] (nM) Emission Ratio This work was supported by LifeTechnologies TM , and grants from the Michael J Fox Foundation and the benevolence of the Brin/Wojicki Foundation to RJN. throughput compatible homogenous cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2‐GFP fusion proteins were transiently expressed in a variety of cell backgrounds via BacMam gene delivery system, and were then plated in 384‐well assay plates (Schematic in A). The phosphorylation at Ser935 in these cells was detected using a terbium labeled anti‐Ser935 phosphorylation specific antibody that generates TR‐FRET signals between terbium and GFP. B. A variety of LRRK2 expression constructs were evaluated in U2OS and SH‐SY5Y cells against a titration of the selective LRRK2 inhibitor LRRK2‐IN1. Consistent with previous reports and our western blot results, wild type and G2019S LRRK2 are constitutively phosphorylated at Ser935 in cells measured by TR‐FRET. The phosphorylation level was reduced for R1441C mutant and little could be detected for the kinase activity‐dead mutant D1994A*. TABLE 1. The TR‐FRET cellular assay was further validated with reported LRRK2 inhibitors including LRRK2‐ IN1 in U2Os, SH‐SY5Y cells and HEK293 cells demonstrating an applicability to multiple systems. Cellular IC50 values are reported for each inhibitor. nd=not determined. •Conclusions: •BacMam gene delivery systems efficiently express LRRK2 in multiple cell lines. •TR‐FRET based detection of phospho935 LRRK2 with a Terbium labeled antibody‐GFP pair was successfully adapted to multiwell assays to create a LanthaScreen cellular assay for LRRK2 •The LanthaScreen cellular assay is amenable to high throughput screening for direct inhibitors as well as for biological inputs. •Factors that activate NFB also induce LRRK2 phosphorylation at Ser935, revealing that LRRK2 is not fully phosphorylated in these cells. •Repression of the CHK1 & CDC25 expression results in a decrease in LRRK2 phosphorylation.