Design of Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Crystallographic Surrogate Derived from Checkpoint Kinase 1 (CHK1)

Williamson, D.S.; Smith, Garrick; Acheson-Dossang, P.; Bedford, Simon T.; Chell, Victoria; Chen, I‐Jen; Daechsel, Justus C.A.; Daniels, Zoe; David, Laurent; Dokurno, P.; Hentzer, Morten; Herzig, Martin C.; Hubbard, Roderick E.; Moore, Jonathan D.; Murray, James B.; Newland, Samantha; Ray, Stuart C.; Shaw, T.; Surgenor, A.E.; Terry, Lindsey; Thirstrup, Kenneth; Wang, Yikang; Christensen, Kenneth Vielsted

doi:10.1021/acs.jmedchem.7b01186

Cited by 46 publications

(61 citation statements)

References 86 publications

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“…The protection of the ATP-binding site and the hinge region by MLi-2 are consistent with other inhibitor bound homolog kinase structures (57,58) although our data also re ects the dynamic change that the binding of MLi-2 has not only on the kinase domain but also on LRRK2 RCKW overall. Essentially any region in LRRK2 RCKW that interfaces with the kinase domain will sense binding of nucleotide or an inhibitor.…”

Section: Mapping the Conformational Changes Induced By Mli-2 Using Hysupporting

confidence: 90%

“…In contrast to MLi-2 and LRRK2-IN-1, which are type I kinase inhibitors, we show that in the presence of a type II kinase inhibitor, rebastinib, G2019S LRRK2 remained cytosolic con rming the predictions of Deniston and coworkers (2020) (13), that docking to MTs is extremely sensitive to the conformational state of the kinase domain. Although the MLi-2 complex is catalytically inactive, MLi-2, which is a competitive inhibitor of ATP, nevertheless locks the kinase into an active-like conformation (58). In contrast, rebastinib is likely to stabilize a DYG/DFG-out/open and inactive conformation of the kinase domain.…”

Section: Discussionmentioning

confidence: 99%

“…Looking more carefully at the protected regions, we saw that the binding of MLi-2 reduces the H-D exchange in the ATP-binding site, the activation loop, the αC helix and the hinge region ( Figure 5). These regions that would be predicted to contact the inhibitor (57,58) all show signi cantly reduced deuterium uptake. Peptides, for example, in the hinge region (aa 1948-1958), including the aD helix, experienced a large increase in protection upon MLi-2 binding (50% vs. 20%).…”

Section: Mapping the Conformational Changes Induced By Mli-2 Using Hymentioning

confidence: 99%

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Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

Schmidt

Weng

Aoto

et al. 2020

Preprint

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Background: Leucine-Rich Repeat Kinase 2 (LRRK2) is a complex multi-domain protein where LRRK2-mutations are associated with Parkinson´s Disease (PD). To explore how pathogenic PD-mutations hijack the finely tuned activation process of LRRK2, we here used a multi-tiered approach. Methods: First, the spatial and temporal distribution of full-length LRRK2 was investigated by a real-time cell-based assay in the presence and absence of LRRK2-kinase inhibitors. In a 2nd layer we explored the consequences of PD mutations as well as removal of the N-terminal domains employing a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We focused on the biochemical characterization of LRRK2RCKW variants based on kinase assays using Rab8a or LRRKtide as substrates. Next, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the solvent accessible regions of LRRK2RCKW in the presence and absence of the LRRK2 inhibitor MLi-2. Finally, Molecular Dynamics simulations on the kinase domain were applied to elucidate differences in breathing dynamics between wild type and mutants of the DYGψ motif. Results: Our cellular approaches revealed that the kinase inhibitors MLi-2 and rebastinib both freeze the kinase domain in a stable conformation, however, only MLi-2 resembles an active conformation and induces filament formation. LRRK2RCKW showed, regardless of the mutation it was combined with, filament formation, indicating a shielding function of the N-terminal domains. This shielding function is impaired for pathogenic mutations in full length LRRK2. LRRK2RCKW retained kinase activity similar to full-length LRRK2. HDX-MS provided a comprehensive allosteric portrait of the kinase domain and revealed how MLi-2 binding is sensed by the entire protein. Molecular Dynamics simulations suggest that, while all domains contribute to regulating kinase activity and spatial distribution, it is the highly dynamic kinase domain, driven by the DYGψ motif, that coordinates the overall domain crosstalk and serves as a regulatory hub for the intrinsic regulation of LRRK2.Conclusion: These studies confirm our hypothesis that the N-terminal scaffolding domains shield the catalytic domains in an inactive state. PD mutations, MLi-2, or Rab GTPases can all unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules.

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Section: Mapping the Conformational Changes Induced By Mli-2 Using Hysupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Mapping the Conformational Changes Induced By Mli-2 Using Hymentioning

confidence: 99%

See 1 more Smart Citation

Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

Schmidt

Weng

Aoto

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…In contrast to MLi-2 and LRRK2-IN-1, which are type I kinase inhibitors, we show that in the presence of a type II kinase inhibitor, rebastinib, G2019S LRRK2 remained cytosolic confirming the predictions of Deniston and coworkers (2020) (13), that docking to MTs is extremely sensitive to the conformational state of the kinase domain. Although the MLi-2 complex is catalytically inactive, MLi-2, which is a competitive inhibitor of ATP, nevertheless locks the kinase into an active-like conformation (58). In contrast, rebastinib is likely to stabilize a DYG/DFGout/open and inactive conformation of the kinase domain.…”

Section: Filament Formation Is Dependent On Unleashing the Catalytic mentioning

confidence: 99%

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

Schmidt

Weng²,

Aoto³

et al. 2020

Preprint

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In a multi-tiered approach, we explored how Parkinson's Disease-related mutations hijack the finely tuned activation process of Leucine-Rich Repeat Kinase 2 (LRRK2) using a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We hypothesized that the N-terminal domains shield the catalytic domains in an inactive state. PD mutations, type-I LRRK2 inhibitors, or physiological Rab GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules. Mapping solvent accessible regions of LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how inhibitor binding is sensed by the entire protein. Molecular Dynamics simulations of the kinase domain elucidated differences in conformational dynamics between wt and mutants of the DYGψ motif. While all domains contribute to regulating kinase activity and spatial distribution, the kinase domain, driven by the DYGψ motif, coordinates domain crosstalk and serves as an intrinsic hub for LRRK2 regulation.

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“…3C). Since inhibitors that capture the kinase in an ATP-bound active-like state (25,26) are known to induce filament formation (13,14) and all other filament forming LRRK2 mutants (N1437H, R1441G/C and Y1699C) also results in hyper-kinase activity in cells (11)(12)(13), we expect that our structure captures an active kinase conformation, which may be facilitated through oligomerization. Taken together, our data suggests that MTbinding plays a scaffolding role in the oligomerization and activation of LRRK2.…”

mentioning

confidence: 99%

Thein situstructure of Parkinson’s disease-linked LRRK2

Watanabe

Buschauer

Böhning

et al. 2019

Preprint

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Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using in situ cryo-electron tomography and subtomogram averaging, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double-helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase points towards the microtubule, while the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to obtain structures of previously unsolved proteins in their cellular environment and provides insights into LRRK2 function and pathogenicity.

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Design of Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Crystallographic Surrogate Derived from Checkpoint Kinase 1 (CHK1)

Cited by 46 publications

References 86 publications

Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

Thein situstructure of Parkinson’s disease-linked LRRK2

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