Type II kinase inhibitors that target Parkinson's disease-associated LRRK2

Abstract

Increased kinase activity of leucine-rich repeat kinase 2 (LRRK2) is associated with Parkinson's disease (PD). Numerous LRRK2-selective type I kinase inhibitors have been developed, and some have entered clinical trials. Here, to our knowledge, we present the first type II kinase inhibitors that target LRRK2. Targeting the inactive conformation of LRRK2 is functionally distinct from targeting the active-like conformation using type I inhibitors. We designed these inhibitors with a combinatorial chemistry approach fusing selective LRRK2 type I and promiscuous type II inhibitors using iterative cycles of synthesis supported by structural biology and activity testing. Our lead compounds are selective and potent toward both LRRK2 and LRRK1, a close relative of LRRK2. Through cellular assays, cryo-electron microscopy structural analysis, and in vitro motility assays, we show that our inhibitors stabilize the open, inactive LRRK2 kinase conformation. These new conformation-specific compounds will be invaluable as tools to study LRRK2's function and regulation and expand the potential therapeutic options for PD.

Fig. 1.. LRRK2 type II inhibitor design strategy.

(A) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2^RCKW, and LRRK2^KW. (B and C) Close-up of the inhibitor binding pocket from cryo–electron microscopy (cryo-EM) maps and models of LRRK2^RCKW bound to the type I inhibitor MLi-2 [Protein Data Bank (PDB): 8TXZ] (B) and type II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange, C-lobe; light orange, N-lobe; black, DYG motif; gray, G-loop; green, activation loop. (D) Scheme depicting our hybrid design strategy to develop potent type II inhibitors targeting LRRK2.

Fig. 2.. Co-crystal structure of RN129 bound to CLK3 and cryo-EM structure of RN277 bound to LRRK2^RCKW.

(A) The co-crystal structure of RN129 (28) with CLK3 highlighting the type II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3). (B) Ribbon diagram of the atomic model of LRRK2^RCKW:RN277:E11 DARPin complex (PDB: 9DMI) built into the cryo-EM map. (C and D) Close-ups of the active sites of the cryo-EM structures of LRRK2^RCKW:RN277 (C) and LRRK2^RCKW:GZD824 (PDB: 8TZE) (D). (E) Superposition of the atomic model of LRRK2^RCKW:RN277:E11 DARPin complex (in lighter shades) and our previously published structure of a LRRK2^RCKW:MLi-2:E11 DARPin complex (PDB: 8TXZ) (in darker shades). Only the kinase domains, which were aligned on their C-lobes, are shown. Major features of the kinase, including those that are indicators of type I and type II inhibitor binding, are shown.

Fig. 3.. Synthesis of RN277 and RN341.

The convergent synthesis route of compounds 30 (RN277) and 32 (RN341). The detailed procedures and analytics are shown in the supplementary information. rt, room temperature; NIS, N-iodosuccinimide; DCM, dichloromethane; TEA, triethylamine; DMF, dimethylformamide; XPhos, dicyclohexyl[2′,4′,6′-tris(propan-2-yl)[1,1′-biphenyl]-2-yl]phosphane; EA, ethyl acetate; DIPEA, N,N-diisopropylethylamine; TFA, trifluoracetic acid.

Fig. 4.. Kinome selectivity of RN341.

(A) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue or light blue. The 18.5 K ∆T_m shift of LRRK2^KW is highlighted in red. For all screened kinases, the bubble size and color correlates with the degree of ∆T_m shift, as indicated in the legend. (B) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20-K ∆T_m shift of LRRK2^KW is highlighted in red. The bubble size or color for each kinase correlates with the ∆T_m shifts, as indicated in the legend (as in A). Kinases with ∆T_m > 6 K are labeled. (C) Waterfall plots of the ReactionBiology ³³PanQinase screen of RN341 at 1 and 10 μM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to <22% of the control value are labeled. (D) Off-target validation from both screens via in cellulo nanoBRET assay in two biological replicates, error bars ± SD, EC₅₀ (JNK2) = 2.7 μM, EC₅₀ (STK10) = 1.5 μM, EC₅₀ (MAPK14) = 1.7 μM, EC₅₀ (TTK) = 3.2 μM, EC₅₀ (CDKL1) = 17 μM, EC₅₀ (CLK1) = 6.0 μM, EC₅₀ (JNK3) = 15 μM, EC₅₀ (DYRK2) ≥ 20 μM, EC₅₀ (SLK) > 20 μM, EC₅₀ (DDR2) > 20 μM, and EC₅₀ (STK17B) ≥ 20 μM. (E) Representative immunoblot from 293T cells transiently co-transfected with LRRK1 and its substrate GFP-Rab7 before treatment with a dilution series of RN277 and RN341. Lysed cells were immunoblotted for LRRK1, GFP-Rab7, phospho-Rab7 (pS72), and GAPDH. (F) Quantification of the GFP-pRab7/GFP-Rab7/LRRK1 ratio from three independent Western blots. Statistical analysis performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons of means. P < 0.0001 for all inhibitor concentrations versus DMSO; error bars ± SEM.

Fig. 5.. Inhibition of LRRK2’s phosphorylation of Rab8a in vitro and in cellulo.

(A and B) Dose-response curve of RN277 (A) and RN341 (B) inhibiting LRRK2^RCKW-mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a versus non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341. (C) Representative immunoblot from 293T cells transiently co-transfected with LRRK2 and GFP-Rab8a, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. (D) Sample from (C) run separately under identical conditions and immunoblotted for phospho-S935 LRRK2 and GAPDH. (E) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from three independent immunoblots (C). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **P = 0.0049, DMSO versus MLi-2; ***P = 0.0004, DMSO versus Ponatinib; ***P = 0.0006, DMSO versus 5 μM RN277; ***P = 0.0003, DMSO versus 10 μM RN277; *P = 0.0406, DMSO versus 5 μM RN341; **P = 0.0065, DMSO versus 10 μM RN341; error bars ± SEM. (F) Quantification of the pS935 LRRK2/LRRK2 ratio (run under identical conditions on separate blots) from three independent immunoblots (D). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ****P < 0.0001 for all conditions versus MLi-2; error bars ± SEM. (G) Representative immunoblot from 293T cells transiently co-transfected with GFP-Rab8a and either GFP-11 tagged wild-type (WT) or GFP-11 tagged G2019S LRRK2, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. (H) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from four independent immunoblots (G). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **P = 0.0077, WT LRRK2 DMSO versus MLi-2; *P = 0.0324, WT LRRK2 DMSO versus 5 μM RN277; *P = 0.0461, WT LRRK2 DMSO versus 5 μM RN341; ****P < 0.0001 for all inhibitor treatments versus G2019S LRRK2 DMSO; error bars ± SEM.

Fig. 6.. LRRK2-specific type II inhibitors RN277 and RN341 rescue kinesin motility in the presence of LRRK2^RCKW.

(A) Schematic of the single-molecule in vitro motility assay. (B) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type I inhibitor MLi-2 (5 μM) in the presence or absence of LRRK2^RCKW. Scale bars, 5 μm (x) and 30 s (y). (C) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2^RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; ***P = 0.0007, DMSO condition; ***P = 0.0003, MLi-2 condition, one-way ANOVA with Šidák’s multiple comparisons test within drug only. (D) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type II inhibitors Ponatinib, RN277, and RN341 (10 μM) in the presence or absence of LRRK2^RCKW. Scale bars, 5 μm (x) and 30 s (y). (E) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2^RCKW concentration in the absence (DMSO) or presence of type II inhibitors Ponatinib, RN277, and RN341 (10 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; ***P = 0.0003, one-way ANOVA with Šidák’s multiple comparisons test within drug only.