scholarly journals Allosteric inhibition of LRRK2, where are we now

2020 ◽  
Vol 48 (5) ◽  
pp. 2185-2194
Author(s):  
Ahmed Soliman ◽  
Fatma Nihan Cankara ◽  
Arjan Kortholt
Keyword(s):  
Recent Progress ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Selectivity ◽  
Gtpase Activity ◽  
Leucine Rich Repeat ◽  
Allosteric Inhibitors ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity

Parkinson's disease (PD) is the second most common neurodegenerative disease. In recent years, it has been shown that leucine-rich repeat kinase 2 (LRRK2) has a crucial function in both familial and sporadic forms of PD. LRRK2 pathogenic mutations are thought to result in an increase in LRRK2 kinase activity. Thus, inhibiting LRRK2 kinase activity has become a main therapeutic target. Many compounds capable of inhibiting LRRK2 kinase activity with high selectivity and brain availability have been described. However, the safety of long-term use of these ATP-competitive LRRK2 kinase inhibitors has been challenged by several studies. Therefore, alternative ways of targeting LRRK2 activity will have a great benefit. In this review, we discuss the recent progress in the development of allosteric inhibitors of LRRK2, mainly via interfering with GTPase activity, and propose potential new intra and interprotein interactions targets that can lead to open doors toward new therapeutics.

Phosphorylation of LRRK2: from kinase to substrate

2012 ◽  
Vol 40 (5) ◽  
pp. 1102-1110 ◽  
Author(s):  
Evy Lobbestael ◽  
Veerle Baekelandt ◽  
Jean-Marc Taymans
Keyword(s):  
Kinase Activity ◽  
Pathological Process ◽  
Rapid Decrease ◽  
Leucine Rich Repeat ◽  
Phosphorylated Protein ◽  
Pathogenic Variants ◽  
Disease Protein ◽  
Lrrk2 Kinase ◽  
Disease Modifying ◽  
Lrrk2 Kinase Activity

The PD (Parkinson's disease) protein LRRK2 (leucine-rich repeat kinase 2) occurs in cells as a highly phosphorylated protein, with the majority of phosphosites clustering in the region between the ankyrin repeat and leucine-rich repeat domains. The observation that several pathogenic variants of LRRK2 display strongly reduced cellular phosphorylation suggests that phosphorylation of LRRK2 is involved in the PD pathological process. Furthermore, treatment of cells with inhibitors of LRRK2 kinase activity, which are currently considered as potential disease-modifying therapeutics for PD, leads to a rapid decrease in the phosphorylation levels of LRRK2. For these reasons, understanding the cellular role and regulation of LRRK2 as a kinase and as a substrate has become the focus of intense investigation. In the present review, we discuss what is currently known about the cellular phosphorylation of LRRK2 and how this relates to its function and dysfunction.

scholarly journals Allosteric modulation of the GTPase activity of a bacterial LRRK2 homolog by conformation-specific Nanobodies

2020 ◽  
Vol 477 (7) ◽  
pp. 1203-1218 ◽  
Author(s):  
Margaux Leemans ◽  
Christian Galicia ◽  
Egon Deyaert ◽  
Elise Daems ◽  
Linda Krause ◽  
...  
Keyword(s):  
Turnover Rate ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Bound State ◽  
Allosteric Modulation ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Lrrk2 Kinase ◽  
Dimeric Form ◽  
Novel Strategy

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer–monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer–monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations.

scholarly journals A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson’s Disease Using RapidFire Mass Spectrometry

2015 ◽  
Vol 21 (2) ◽  
pp. 145-155 ◽  
Author(s):  
Melanie Leveridge ◽  
Lee Collier ◽  
Colin Edge ◽  
Phil Hardwicke ◽  
Bill Leavens ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
High Throughput ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Label Free ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity

LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson’s disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2- to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To date, reported screening assays for LRRK2 have been based on detection of labeled adenosine triphosphate and adenosine diphosphate or on antibody-based detection of phosphorylation events. While these assays do offer a high-throughput method of monitoring LRRK2 kinase activity, they are prone to interference from autofluorescent compounds and nonspecific events. Here we describe a label-free assay for LRRK2 kinase activity using the RapidFire mass spectrometry system. This assay format was found to be highly robust and enabled a screen of 100,000 lead-like small molecules. The assay successfully identified a number of known LRRK2 chemotypes that met stringent physicochemical criteria.

scholarly journals LRRK2 Kinase Activity Does Not Alter Cell-Autonomous Tau Pathology Development in Primary Neurons

2021 ◽  
pp. 1-10
Author(s):  
Michael X. Henderson ◽  
Lakshmi Changolkar ◽  
John Q. Trojanowski ◽  
Virginia M.Y. Lee
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Lewy Bodies ◽  
Primary Neurons ◽  
Tau Pathology ◽  
Therapeutic Development ◽  
Lrrk2 Kinase ◽  
A Minor ◽  
The Impact ◽  
Lrrk2 Kinase Activity

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD) and are also associated with genetic risk in idiopathic PD. Mutations in LRRK2, including the most common p.G2019S lead to elevated kinase activity, making LRRK2 kinase inhibitors prime targets for therapeutic development. However, the role of LRRK2 kinase activity in PD pathogenesis has remained unclear. While essentially all LRRK2-PD patients exhibit dopaminergic neuron loss, many of these patients to not have α-synuclein Lewy bodies in their brains. So, what is the neuropathological substrate of LRRK2-PD? Tau has emerged as a possible candidate due to the presence of tau pathology in the majority of LRRK2 mutation carriers and reports of hyperphosphorylated tau in LRRK2 animal models. Objective: In the current study, we aim to address whether a mutation in LRRK2 changes the cell-autonomous seeding of tau pathology in primary neurons. We also aim to assess whether LRRK2 kinase inhibitors are able to modulate tau pathology. Methods/Results: Treatment of primary neurons with LRRK2 kinase inhibitors leads to prolonged kinase inhibition but does not alter tau pathology induction. The lack of an effect of LRRK2 kinase activity was further confirmed in primary neurons expressing LRRK2G2019S and with two different forms of pathogenic tau. In no case was there more than a minor change in tau pathology induction. Conclusion: Together, our results indicate that LRRK2 kinase activity is not playing a major role in the induction of tau pathology in individual neurons. Understanding the impact of LRRK2 kinase inhibitors on pathology generation is important as kinase inhibitors move forward in clinical trials.

scholarly journals Nanobodies as allosteric modulators of Parkinson′s disease-associated LRRK2

2021 ◽  
Author(s):  
Ranjan K. Singh ◽  
Ahmed Soliman ◽  
Giambattista Guaitoli ◽  
Eliza Störmer ◽  
Felix von Zweydorf ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Common Disease ◽  
Type I ◽  
Rab Protein ◽  
Lrrk2 Kinase ◽  
Inhibitory Drug ◽  
Lrrk2 Kinase Activity

Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson′s disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.

scholarly journals G2385R and I2020T Mutations Increase LRRK2 GTPase Activity

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Dong Hwan Ho ◽  
Jihoon Jang ◽  
Eun-hye Joe ◽  
Ilhong Son ◽  
Hyemyung Seo ◽  
...  
Keyword(s):  
Risk Factor ◽  
Kinase Activity ◽  
Asian Population ◽  
Kinase Assay ◽  
Gtpase Activity ◽  
Wild Type ◽  
G2019s Mutation ◽  
Lrrk2 Kinase ◽  
Familial Parkinson’S Disease ◽  
Lrrk2 Kinase Activity

The LRRK2 mutation is a major causal mutation in familial Parkinson’s disease. Although LRRK2 contains functional GTPase and kinase domains and their activities are altered by pathogenic mutations, most studies focused on LRRK2 kinase activity because the most prevalent mutant, G2019S, enhances kinase activity. However, the G2019S mutation is extremely rare in the Asian population. Instead, the G2385R mutation was reported as a major risk factor in the Asian population. Similar to other LRRK2 studies, G2385R studies have also focused on kinase activity. Here, we investigated GTPase activities of G2385R with other LRRK2 mutants, such as G2019S, R1441C, and I2020T, as well as wild type (WT). Our results suggest that both I2020T and G2385R contain GTPase activities stronger than that of WT. A kinase assay using the commercial recombinant proteins showed that I2020T harbored stronger activity, whereas G2385R had weaker activity than that of WT, as reported previously. This is the first report of LRRK2 I2020T and G2385R GTPase activities and shows that most of the LRRK2 mutations that are pathogenic or a risk factor altered either kinase or GTPase activity, suggesting that their physiological consequences are caused by altered enzyme activities.

scholarly journals P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity

2018 ◽  
Vol 475 (7) ◽  
pp. 1271-1293 ◽  
Author(s):  
Alexia F. Kalogeropulou ◽  
Jing Zhao ◽  
Marc F. Bolliger ◽  
Anna Memou ◽  
Shreya Narasimha ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Rab Proteins ◽  
Missense Mutations ◽  
Leucine Rich Repeat ◽  
Lrrk2 Gene ◽  
Ubiquitin Binding Domain ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity ◽  
In Cells

Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson's disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.

scholarly journals Mitochondrial DNA damage as a potential biomarker of LRRK2 kinase activity in LRRK2 Parkinson’s disease

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
C. P. Gonzalez-Hunt ◽  
E. A. Thacker ◽  
C. M. Toste ◽  
S. Boularand ◽  
S. Deprets ◽  
...  
Keyword(s):  
Dna Damage ◽  
Clinical Trials ◽  
Mitochondrial Dna ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Mtdna Damage ◽  
Mitochondrial Dna Damage ◽  
Lrrk2 G2019s ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity

Abstract Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson’s disease (PD) and LRRK2 kinase inhibitors are currently being tested in early phase clinical trials. In order to ensure the highest chance of success, a biomarker-guided entry into clinical trials is key. LRRK2 phosphorylation, and phosphorylation of the LRRK2 substrate Rab10, have been proposed as target engagement biomarkers for LRRK2 kinase inhibition. However, a pharmacodynamic biomarker to demonstrate that a biological response has occurred is lacking. We previously discovered that the LRRK2 G2019S mutation causes mitochondrial DNA (mtDNA) damage and is LRRK2 kinase activity-dependent. Here, we have explored the possibility that measurement of mtDNA damage is a “surrogate” for LRRK2 kinase activity and consequently of kinase inhibitor activity. Mitochondrial DNA damage was robustly increased in PD patient-derived immune cells with LRRK2 G2019S mutations as compared with controls. Following treatment with multiple classes of LRRK2 kinase inhibitors, a full reversal of mtDNA damage to healthy control levels was observed and correlated with measures of LRRK2 dephosphorylation. Taken together, assessment of mtDNA damage levels may be a sensitive measure of altered kinase activity and provide an extended profile of LRRK2 kinase modulation in clinical studies.

Pharmacological inhibition of LRRK2 cellular phosphorylation sites provides insight into LRRK2 biology

2012 ◽  
Vol 40 (5) ◽  
pp. 1158-1162 ◽  
Author(s):  
Jing Zhao ◽  
Spencer B. Hermanson ◽  
Coby B. Carlson ◽  
Steven M. Riddle ◽  
Kurt W. Vogel ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Kinase Activity ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Neuroprotective Agents ◽  
Leucine Rich Repeat ◽  
Lrrk2 Kinase ◽  
Activity Dependent ◽  
Lrrk2 Kinase Activity ◽  
Insight Into

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinson's disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.e. kinase activity) has been facilitated by the development of both biochemical and cell-based assays for LRRK2. LRRK2 is phosphorylated on Ser910, Ser935, Ser955 and Ser973 in the N-terminal domain of the enzyme, and these sites of phosphorylation are likely to be regulated by upstream enzymes in an LRRK2 kinase-activity-dependent manner. Knowledge of these phosphorylation sites and their regulation can be adapted to high-throughput-screening-amenable platforms. The present review describes the utilization of LRRK2 phosphorylation as indicators of enzyme inhibition, as well as how such assays can be used to deconvolute the pathways in which LRRK2 plays a role.

scholarly journals Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization

2009 ◽  
Vol 284 (52) ◽  
pp. 36346-36356 ◽  
Author(s):  
Saurabh Sen ◽  
Philip J. Webber ◽  
Andrew B. West
Keyword(s):  
Kinase Activity ◽  
Leucine Rich Repeat ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity
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