Can the increasing number of newly developed leucine-rich repeat kinase 2 inhibitors validate or invalidate a potential disease-modifying therapeutic approach for Parkinson's disease?

Abstract

Leucine-rich repeat kinase 2 (LRRK2) is considered an attractive therapeutic target for potential disease-modifying treatment of Parkinson's disease (PD). Both genetic and cell biological evidence have contributed to the hypothesis that LRRK2 kinase inhibition may have therapeutic potential in PD. This hypothesis was widely translated in drug discovery programs as illustrated by the growing number of patents covering newly discovered LRRK2 kinase inhibitors and emanating from at least 20 different public and private research organizations. With work still under way, this research shows the feasibility of developing potent, selective and brain permeable LRRK2 kinase inhibitors. The growing availability of these pharmacological tools should contribute to filling in the gaps in our knowledge on the safety and efficacy of LRRK2 kinase inhibition and validate/invalidate this therapeutic strategy for further development. Validation criteria should include a lack of toxic effects following long-term treatment with inhibitors as well as confirmation of target engagement in cells and animal models leading to improvement of pathological features in phenotypic assays. The state of advancement of the field of LRRK2 is such that existing biological tools and expertise can be combined with the increasing number of available LRRK2 kinase inhibitors to address these key issues.

Keywords::

• kinase inhibitor
• leucine-rich repeat kinase 2
• Parkinson's disease
• phenotypic assay

Clinical treatment of Parkinson's disease (PD) today is based on alleviating symptoms resulting from the death of midbrain dopaminergic neurons. These neurons are key regulators in neuronal circuits of voluntary movement (the so-called basal-ganglia-thalamocortical circuits). The available symptomatic treatments for PD are designed to counteract the imbalances in these circuits caused by dopaminergic neuron death and brain dopamine deficiency through pharmacological, neurosurgical or neurostimulatory means. PD symptomatic treatment strategies, such as treatment with the dopamine precursor L-DOPA, have been in clinical practice for more than five decades now and do not halt the neurodegenerative process; therefore, there is a general consensus in the clinical and biomedical research community that disease-modifying therapy for PD is a major unmet medical need.

The identification of genes involved in PD and the study of PD gene functions has provided researchers with valuable insight into potential therapeutic targets [1]. Of these, leucine-rich repeat kinase 2 (LRRK2) has emerged in recent years as a very attractive therapeutic target for potential disease-modifying treatment of PD. LRRK2 was first genetically linked to dominantly inherited familial PD in 2004 [2,3] and geneticists have since concluded that LRRK2 is the most common cause of genetic Parkinsonism [4]. The importance of LRRK2 as a causative agent of PD was further confirmed in 2011 with the publication of genome-wide association studies pointing to genomic variation at the LRRK2 locus as a risk factor for sporadic PD [5,6]. The dominant mode of inheritance points to a toxic gain of function mechanism for LRRK2 PD. Biological evidence supporting this hypothesis has been: i) the observation that the most prevalent disease mutation located in LRRK2's kinase activation loop (G2019S) leads to increased LRRK2 activity; ii) LRRK2 mutant overexpression is toxic in certain conditions in cells and in vivo; and iii) kinase inactive LRRK2 variants annul LRRK2 mutant toxicity (for reviews that summarize these studies, see Refs. [7,8]). Although much remains to be elucidated in our understanding of LRRK2 (patho)biology, the above arguments were sufficient to justify drug discovery projects in multiple institutions around the world.

In this issue of EOTP, Kethiri and Bakthavatchalam inventorize the most recent patents on LRRK2 kinase inhibitors, which illustrate the extent and success of multiple drug discovery programs to discover novel LRRK2 kinase inhibiting compounds [9]. Consistent with research on LRRK2 kinase inhibitors commencing shortly after the first publications proposing LRRK2 inhibition as a potential PD therapy (2004 – 2006), the first patents on LRRK2 kinase inhibitors were submitted in 2006 – 2007 and the number of submitted patents has risen ever since. A previous overview of patents on LRRK2 kinase inhibitors authored by Deng et al., covering the period of 2006 – 2011 showed that 15 patents had been filed up to 2011 [10]. Two years later, that number has more than doubled with 31 patents on LRRK2 kinase inhibitors published up to the end of 2013. The broad impact of this area is also exemplified by the > 20 different research organizations authoring LRRK2 kinase inhibitor patents.

Kinase inhibitors which have been approved for clinical use are primarily therapies for cancers. Along the discovery and development path of the now clinically approved kinase inhibitors, much know-how and expertise have been built up which are facilitating programs targeting new kinases, including for non-oncology indications. One of the first hurdles to clear in developing kinase inhibitors is that of obtaining potent and selective inhibitors for the targeted kinase. This challenge is being met for LRRK2 as a number of kinase inhibitors are reported at the low nanomolar range in potency, whereas the most favorable inhibitors tested for selectivity inhibit only a handful of non-LRRK2 at up to 100-fold higher concentrations than concentrations effective for LRRK2 kinase inhibition (see Kethiri and Bakthavatchalam (2014) and references therein). However, developing kinase inhibitors for the treatment of a brain disorder brings other challenges, including designing compounds which will cross the blood–brain barrier. Reports of brain permeable LRRK2 kinase inhibitors (see Kethiri and Bakthavatchalam (2014) and references therein) suggest that it is feasible to overcome this hurdle as well. Besides expression in brain, LRRK2 is also expressed in peripheral tissues such as kidney or lung. It remains to be seen whether inhibition of LRRK2 in peripheral organs will be beneficial or detrimental to clinical outcomes of treatment in order to determine optimal CNS-periphery biodistribution of compounds. In all, reports of LRRK2 inhibitors since 2006 show that it will be feasible to develop potent, selective and brain permeable LRRK2 kinase inhibitors for use in clinical trials.

However, does the growing number of developed and patented LRRK2 kinase inhibitors mean that we are closer to a PD disease-modifying therapy targeting LRRK2? The answer to this question lies in validating LRRK2's activity as a therapeutic target for PD. A first key issue to assess is safety of the LRRK2 kinase inhibitors. Knockout of LRRK2 leads to abnormalities in peripheral organs in rodents [11,12], and it will, therefore, be necessary to test for such abnormalities in animals after long-term treatment with LRRK2 kinase inhibitors.

Besides safety, the therapeutic efficacy of LRRK2 kinase inhibition needs to be confirmed. Two key issues in LRRK2 inhibitor efficacy are understanding the cellular mechanism of action of LRRK2 kinase inhibitors and delineating the cellular events by which LRRK2 mediates pathology. One of the striking molecular consequences of LRRK2 kinase inhibition in cells is its dephosphorylation at a cluster of phosphorylation sites, including at S910 and S935, attributed to protein phosphatase 1 [13,14]. This inhibitor-induced dephosphorylation of LRRK2 is widely accepted as a measure of cellular activity for LRRK2 kinase inhibitors. One subtle but important consideration in interpreting this test is that the phosphosites measured are not autophosphorylation sites; therefore, cellular dephosphorylation may be a result of inhibitor binding to LRRK2 rather than LRRK2 activity inhibition. It will not be possible to rule out that potent LRRK2 kinase inhibitors may engage LRRK2 in such a way that it does not induce LRRK2 dephosphorylation in cells. Further work is, therefore, required to elucidate the specific molecular events caused by LRRK2 kinase inhibitors in cells. In this regard, LRRK2 kinase interactors and substrates have begun to be reported and characterized, including LRRK2 cellular partners mediating cell death, which may in follow-up studies demonstrate molecular events such as phosphorylations or protein–protein interactions regulated by LRRK2 kinase inhibitors [7,8,15]. Pursuing this work as well as identifying and confirming the as-yet-unknown physiological substrate(s) of LRRK2 will undoubtedly be instrumental in designing alternative cellular activity assays for LRRK2 kinase inhibition.

A second issue in assessing the efficacy of LRRK2 inhibition is that currently there are no widely accepted phenotypic models for LRRK2-mediated pathology. In cells, a straightforward choice would be to attempt to counteract the apoptotic cell death using LRRK2 kinase inhibitors, which is reported to occur after acute LRRK2 mutant overexpression in cell lines and primary neurons; however, reports with such experiments are lacking as of today, perhaps owing to technical difficulties with this approach or the variability in the toxicity observed [15]. In vivo, several LRRK2 transgenic animals have been reported; however, few display key pathological features such as dopaminergic neuron death or Lewy-body like proteinaceous deposits. Those LRRK2 transgenic animals that do display pathology have a late onset and moderate phenotype making them difficult to implement in phenotypic testing of kinase inhibitors [16,17]. In contrast, models of viral vector-mediated LRRK2 overexpression in brain report toxicity in dopaminergic neurons and may, therefore, be useful in efficacy studies, on condition that toxic phenotypes are reproducible from study to study [8,15,18,19]. Other options for phenotypic assays lie in exploiting the observation that LRRK2 mutants are linked to negative effects on neurite morphology [7], in line with hypotheses that axon degeneration is an early step in the neurodegenerative process (reviewed by Burke and O’Malley [20]).

Filling in the gaps in our knowledge on the safety and efficacy of LRRK2 kinase inhibition will determine whether this therapeutic approach can be validated for further development. Validation criteria include a lack of toxic effects following long-term treatment with inhibitors and confirmation of target engagement in cells and animal models leading to improvement of pathological features in phenotypic assays. The state of advancement of the field of LRRK2 is such that existing biological tools and expertise can be combined with the growing number of available LRRK2 kinase inhibitors to address these key issues.

Declaration of interest

This specific work was not funded by any specific body, however funding for research on this topic in the recent past has come from Research Fund - Flanders (FWO), KU Leuven, King Baudouin Foundation, Agency for Innovation for Science and Technology (IWT) and the Michael J Fox Foundation. The author has previously performed contract research for Oncodesign and Ipsen. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.