Development of LRRK2 Inhibitors for the Treatment of Parkinson's Disease

CHAPTER TWO

Development of LRRK2 Inhibitors for the Treatment of Parkinson’s Disease K.V. Christensen*, G.P. Smith*, D.S. Williamson† *Neuroscience Drug Discovery, H. Lundbeck A/S, Valby, Denmark † Vernalis (R&D) Ltd, Cambridge, United Kingdom

Contents 1. Introduction 2. LRRK2 Biology 2.1 Genetic Evidence for the Possible Role of LRRK2 in PD 2.2 Localisation and Function of LRRK2 3. Structural Biology of LRRK2 4. Overview of Selective Inhibitors of LRRK2 4.1 Diaminopyrimidines 4.2 Arylbenzamides 4.3 Indolinones 4.4 Indazoles 4.5 Cinnolines/Quinolines 4.6 Pyrrolopyrimidines 4.7 Thiophenes 4.8 Triazolopyridazines 5. Conclusion References

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Keywords: Rab GTPase, α-Synuclein, Type II pneumocytes, Homology model, Crystallographic surrogates, Diaminopyrimidines, Indazoles, Pyrrolopyrimidines

1. INTRODUCTION Parkinson’s disease (PD), as first described by James Parkinson in 1817, is a neurodegenerative brain disorder characterised by four cardinal motor symptoms: bradykinesia, postural instability, resting tremor and rigidity [1,2]. A number of nonmotor symptoms are, however, increasingly Progress in Medicinal Chemistry, Volume 56 ISSN 0079-6468 http://dx.doi.org/10.1016/bs.pmch.2016.11.002

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2017 Elsevier B.V. All rights reserved.

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acknowledged as being part of the disease manifestation. These include hyposmia, constipation, problems with speech and swallowing, orthostatic hypertension, mood disorders, cognitive impairment and sleep disorders [3,4]. Historically, the pathological hallmarks of PD are neurodegeneration of the dopamine-producing neurons in the substantia nigra pars compacta (SNc) as well as the presence in various brain regions of intracellular inclusions of aggregated proteins, also denoted as Lewy bodies and neurites. A major constituent of Lewy bodies and neurites is misfolded α-synuclein, a protein encoded by the SNCA gene [5]. Recent data also suggest that PD is not only a disease of the basal ganglia system, but rather a progressing synucleopathy affecting both the peripheral and central nervous system (CNS) [6]. In support, individuals with genomic duplications and triplications of the SNCA gene, thus giving rise to increased levels of α-synuclein, have an increased risk of developing PD [7]. Presently, PD is estimated to affect between 7 and 10 million people worldwide and prevalence varies with gender and geographic region; approximately 1 million people in the United States are diagnosed with PD. The majority of PD cases manifest after the age of 50 (late-onset PD) and disease risk increases with age in Western countries; the prevalence of PD between 60 and 69 years of age is approximately 500 in 100,000 people rising to, at the age of 80, a prevalence of 3000 in 100,000 people [8]. Together, this suggests that the overall prevalence will increase with a growing elderly population [9,10]. Currently, all approved therapies for PD are symptomatic [11,12]. Thus, disease modification and delay of disease progression are two major unmet need areas in PD where no therapy has yet been approved. For developing such therapies, an increased molecular understanding of the biological mechanisms underlying the disease is crucial. Importantly, recent evidence supports a strong genetic contribution to PD that points towards such biological mechanisms.

2. LRRK2 BIOLOGY 2.1 Genetic Evidence for the Possible Role of LRRK2 in PD Linkage and genome-wide association studies (GWASs) have identified a number of rare and common genetic risk loci that are associated with an increased risk of late-onset PD [13–25]. At chromosome 12, one genetic risk locus (originally identified as PARK8) has gained a lot of interest [26]. The causative gene was identified as leucine-rich repeat kinase 2 (LRRK2) [27]. Several rare genetic mutations in the coding region of LRRK2 have been

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Fig. 1 LRRK2 sequence and domains with common mutations associated with Parkinson’s disease annotated.

associated with late-onset autosomal dominant PD. Besides the MAP kinase (MAPK) domain LRRK2-Gly2019Ser (G2019S) mutation (Fig. 1), which is the most common genetic cause of late-onset LRRK2-associated PD, at least seven other mutations in the coding region of LRRK2 (N1437H, R1441C/G/H, Y1699C, S1761R and I2020T) have been shown to cause late-onset familial PD [26–36]. All eight mutations are inherited in an autosomal dominant fashion, suggesting a gain-of-function protein. Interestingly, all the established pathogenic mutations are also situated in one of the three functional domains that form the enzymatic core of LRRK2, suggesting that modulation of enzymatic activity impacts disease risk. Common variation at the LRRK2 locus has also been associated with increased risk of PD [22,23]. GWASs have identified a number of common single-nucleotide polymorphisms in the coding region of the LRRK2 gene as risk variants, for example, carriers of LRRK2 variants A419V, R1628P, M1646T and G2385R all have an increased risk of developing PD [37–44]. Collectively, genomic evidence suggests that at least 8%–10% of individuals in both the Caucasian and Asian populations have an increased risk of PD that is highly associated with LRRK2 exonic variation [41]. A common protective haplotype variant, N551K/R1398H/K1423K, that decreases the risk of PD in several populations, has gained particular interest [41,45–47]. Biochemical studies suggest that the GTPase domain of the presumed R1398H variant underlying this protective effect has an increased affinity for GTP, when compared to both disease-associated LRRK2 variants and wild-type LRRK2 [48]. The R1398H variant is also situated in the Roc domain, further substantiating the notion that LRRK2 enzymatic activity is a modulator of disease risk. Clinical symptoms of PD associated with LRRK2 pathogenic variants are similar to sporadic PD, suggesting that causal disease mechanisms are similar [41–53]; however, compared to patients with sporadic PD patients with G2019S PD tend to have a more uniform rate of disease progression regardless of onset age [54]. In terms of disease risk, polygenetic risk profiling indicates that multiple additional PD-associated genetic factors can modify both the disease risk and age of onset in PD patients carrying the

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Asian LRRK2 risk variants G2385R and R1628P [55]. Similarly, a common single-nucleotide polymorphism in the SNCA gene has been shown to decrease the age of onset of G2019S-associated PD [56].

2.2 Localisation and Function of LRRK2 In mammals, LRRK2 mRNA is highly enriched in brain, lung, kidney, and blood [57–63]. Some species variation in LRRK2 protein expression and localisation has been observed. In rats and mice, LRRK2 is highly expressed in the cortex and striatum, most particularly in pyramidal neurons of layer V and in striatal medium spiny neurons [64]. Overall, rats have a more restricted distribution of LRRK2 when compared with mice. Mice, but not rats, show high levels of LRRK2 expression in the SNc [65]. In postmortem human brain, LRRK2 mRNA expression and localisation was found to be restricted to the cerebral cortex, caudate–putamen and SNc, whereas immuno-labelling studies showed prominent localisation of LRRK2 to neurons in the caudate–putamen [66,67]. In contrast to the expression pattern, the function of the LRRK2 protein has proven difficult to unravel (Fig. 2). LRRK2 genetically interacts with the PARK16 locus, which encodes the small Rab GTPase Rab7L1/ Rab29. More specifically, two common SNPs in the promoter region of

Fig. 2 LRRK2 sequence, domains, phosphorylation sites, interaction partners and cellular substrates.

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Rab7L1 are significantly associated with protection against LRRK2-associated PD [68], suggesting cross talk between the Rab GTPase and LRRK2 biologies, and lately Rab7L1 has been proposed as an in vivo LRRK2 substrate [69]. Rab7L1 has also been identified as an LRRK2 interactor that functionally interacts with several LRRK2 disease variants to promote relocalisation to, and clearance of, trans Golgi-derived vesicles [70]. LRRK2 interacts with 14-3-3 proteins via the LRR domain. This interaction is dependent upon phosphorylation at a cluster of amino acid residues at positions serine 910 (Ser910), serine 935 (Ser935), serine 955 (Ser955) and serine 973 (Ser973) in LRRK2 [71–73]. This phosphorylation cluster precedes the LRR domain in the LRRK2 protein. Phosphorylation at the cluster and the subsequent binding of 14-3-3 proteins are likely to play an important role in the cellular regulation, localisation and function of LRRK2. These phosphorylations are observed both in vitro and in vivo [72,74], and recent reports suggest that, in immune cells, at least eight different kinases, and at least one phosphatase, could be involved in phosphorylating and dephosphorylating LRRK2 at these four positions [75,76]. Upon full LRRK2 inhibition, in vivo and in vitro dephosphorylation occurs, and the interaction with 14-3-3 proteins is lost, suggesting that LRRK2 phosphorylation and the concomitant interaction are both dependent on an active ATP-bound LRRK2 conformation. Thus, the phosphorylation sites at Ser910, Ser935, Ser955 and Ser973 can be used as pharmacodynamic markers for LRRK2 target engagement in vitro and in vivo. No validated preclinical in vivo model exists that can predict the therapeutic potential of a drug in terms of either a delay in disease progression or disease modification in PD. Also, rodent animals that carry G2019S or any other LRRK2 pathogenic variants do not present with PD symptomatology. Links to other PD disease biologies have been extensively explored since the discovery of LRRK2. So far, no replicated findings have been reported showing interaction between LRRK2 and α-synuclein in transgenic mouse models coexpressing wild-type or disease variants of human LRRK2 and human α-synuclein [77–79]. Encouragingly, recent observations showing a rescue effect by both LRRK2 ablation and LRRK2 kinase inhibition in the rat adeno-associated virus α-synuclein overexpression model might pave the way for testing LRRK2 kinase inhibitors on disease-related mechanisms [80,81]. Safety aspects of modulating LRRK2 kinase activity have also been assessed in the literature. Of particular interest are the in vivo observations

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that support a functional role for LRRK2 in the regulation of microvesicular turnover and protein degradation pathways. In homozygous LRRK2 knockout rats and mice, type II pneumocytes in the lung are enlarged and vacuolated due to intracellular accumulation of small vesicular structures called lamellar bodies [82–85]. The lamellar bodies contain surfactant that, under normal conditions, is secreted into the alveolar space of the lung. Parallel to the observations in the rat lung, an increased number of secondary lysosomes in proximal tubular epithelium have been observed in the kidneys of homozygous LRRK2 knockout rats and mice, suggesting LRRK2 involvement in protein degradation pathways via the lysosomal system [84–86]. Studies using transgenic knock-in mice carrying a kinase-dead version of the human LRRK2 gene suggest that the phenotypes are associated with a lack of LRRK2 kinase function, and further support for this hypothesis comes from in vivo pharmacology studies in rodents and nonhuman primates using LRRK2-selective inhibitors [82,87]. The most common pathogenic form of LRRK2-associated PD results in an amino acid substitution from glycine to serine at amino acid position 2019 (G2019S) in the kinase domain of the LRRK2 protein. Biochemical studies have shown that this glycine-to-serine substitution leads to an increased kinase activity of LRRK2 that increases LRRK2 autophosphorylation [74]. Upon dimerisation, LRRK2 phosphorylates itself in cis at serine 1292 (Ser1292). The endogenous level of phosphorylation at Ser1292 is very low and therefore not easy to detect in vivo. In in vitro systems, increased levels of Ser1292 phosphorylation have been observed for all rare disease-causing genetic variants of LRRK2, thus confirming a strong correlation between phospho-Ser1292 (pSer1292) levels and disease risk [88]. Since autophosphorylation at Ser1292 is dependent on, and correlates with, LRRK2 kinase activity, the Ser1292 phosphorylation level might be useful as a disease-relevant marker for LRRK2 inhibition in vitro. As indicated earlier, recently a number of small Rab GTPases have been identified as LRRK2 substrates [69]. In particular, Rab8A, Rab10, Rab12 and Rab7L1 have been shown to be phosphorylated by LRRK2 in the Rab GTPase switch region, and Rab8A has also been shown to have increased interaction with a pathological species of α-synuclein [89]. Upon phosphorylation, it is hypothesised that the Rab GTPase is activated, which in turn leads to increased membrane tethering, but exactly how the interplay between α-synuclein, LRRK2 and Rab GTPase biologies modulates risk of disease is still unknown.

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3. STRUCTURAL BIOLOGY OF LRRK2 The human LRRK2 gene consists of 51 coding exons and encodes a large 2527 amino acid multidomain protein including an N-terminal armadillo domain, an ankyrin-like (ANK) domain, several leucine-rich repeats (LRR), a Ras-like GTPase domain (ROC) along with its C-terminal domain (COR), a MAP kinase domain and a C-terminal WD40 domain (Fig. 1). Based on overall sequence identity, it belongs to the ROCO family sharing the highest overall sequence identity to the mammalian paralogue LRRK1. However, when focusing on the kinase domain, LRRK2 and LRRK1 are not close homologues. Here, LRRK2 is closer to the superfamily of MAP kinases. LRRK2 is predicted to consist of multiple domains through sequence analysis, in the absence of published crystal structures of either the full-length protein or its individual domains [90,91]. The consensus is that recombinantly expressed full-length LRRK2 protein, or any fragment thereof, is typically unstable, insoluble or permanently bound to chaperones, thus presenting difficulties for crystallography or other biophysical or biochemical studies. Recently, an LRRK2 structural model has been proposed. The 3D model is based on a combination of domain-based homology models, cross-linking studies coupled with mass spectrometric analysis, small-angle X-ray scattering, negative stain EM and computational approaches. The authors suggest that LRRK2 is a functional dimer positioned in a head-to-tail orientation, thus allowing N- and C-terminal domains to interact with the central enzymatic core in order to regulate LRRK2 activity [92]. The enzymatic core of the protein bears both a kinase and a GTPase domain; the latter is comprised of Ras complex proteins (Roc, a Ras-like GTPase) and C-terminal of Roc (COR) [93]. This Roc-COR domain is conserved across all members of the ROCO protein family, of which LRRK2 is a member; further understanding of LRRK2 structure has arisen by analysis of related ROCO proteins from bacteria and amoebae [94–96]. The core of the LRRK2 sequence is surrounded by protein–protein interaction domains, known as ANK, LRR and WD40, a sequence of 40 amino acids terminating in a tryptophan (W)–aspartate (D) dipeptide. LRRK2 normally exists in a dimeric complex, which is disrupted by removal of the WD40 domain [97]. The pathogenic exonic variants of LRRK2 are predominantly located in the kinase or GTPase domains, and this suggests that

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the changes that relate to increased risk of PD could change the catalytic properties of LRRK2. Since most pathogenic variants of LRRK2 increase kinase activity or decrease GTPase activity, it is possible that LRRK2 kinase function could therefore be altered by affecting ATP binding in the kinase domain, binding of GTP in the Roc-COR domain or by impacting on LRRK2 dimerisation through changes in the protein–protein interaction domains nearer the protein surface. Although the role of each LRRK2 domain is becoming better understood, and many parts of the protein provide potential drug interaction sites which could modulate kinase activity, all drug discovery efforts disclosed to date have focussed on the kinase domain. This is not surprising, given the success of kinase inhibition as a means of therapeutic intervention in other disease areas. However, balancing the exquisite selectivity required to give a molecule with the desired safety profile together with typical CNS-penetrant drug-like properties is particularly challenging to the medicinal chemist. Homology modelling studies based on B-Raf (a kinase with 33% sequence identity to LRRK2 and a high degree of conservation around the ATP binding site) have been undertaken [98]. Liu and coworkers proposed that the LRRK2 G2019S mutation, most commonly linked to PD, occurs in the DYG motif of the activation loop of the ATP binding site. The position of the DYG (or DFG) motif in kinases plays an important role in switching the protein from an active (DYG-in) to an inactive (DYG-out) form. Fig. 3 illustrates the LRRK2 Asp residue (shown as D2107 of the Hinge

Activation loop

G2019S D2017

D1994

Fig. 3 Overview of ATP binding site of LRRK2.

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Development of LRRK2 Inhibitors for PD

DYG motif ) forming an interaction with the beta phosphate of ATP (probably Mg2+ ion mediated). The glycine residue (G) in the DYG loop enables a more flexible conformation, so that the kinase can switch between the active and inactive forms. It is likely that mutation of the glycine of the DYG loop to a serine in the LRRK2 G2019S mutant stabilises the kinase in its active form via hydrogen bond interactions with other residues in the ATP binding site, such as the backbone of Asp1194 in the catalytic loop. B-Raf has subsequently been used as the basis for homology modelling of other inhibitor chemotypes, such as indolinones [99].

4. OVERVIEW OF SELECTIVE INHIBITORS OF LRRK2 The combined genetic and biochemical evidence supports a hypothesis in which the LRRK2 kinase function is causally involved in the pathogenesis of sporadic and familial forms of PD, and therefore that LRRK2 kinase inhibitors could be useful for treatment. In the last 5 years, efforts to identify selective and brain-penetrant LRRK2 inhibitors have made significant advances. Prior to 2011, reported LRRK2 inhibitors were typically legacy compounds from previous kinase inhibitor programmes, with significant off-target kinase activities. These included the natural product staurosporine 1a and its derivative K-252a 1b [100], which inhibit wild-type LRRK2 (IC50 ¼ 1–40 and 3.6–25 nM, respectively) as determined by radioactive, time-resolved fluorescence resonance energy transfer (TF-FRET) and AlphaScreen® in vitro assays, as well as the ROCK2 inhibitor H-1152 (2) [101] (wild-type LRRK2 IC50 ¼ 244 nM and G2019S LRRK2 IC50 ¼ 150 nM). H N

H N

O

O

HN N

N

N

O

N O H

H O

O N 1a

O O

Me

N O S O

Me Me

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Me 1b

2

The kinase inhibitor field has focussed on developing therapeutics for oncology, although marketed kinase inhibitors are now available for idiopathic pulmonary fibrosis and arthritis [102]. Medicinal chemists have been

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adapting the properties of these molecules so that they are more aligned with the properties required for a CNS agent, with lower molecular weight, fewer hydrogen bonds and a cLogP optimally around 3 [103]. Nearly all compounds that have been reported are type I binders that compete with ATP. A study of the effect of type II kinase inhibitors with LRRK2 inhibitory activity concluded that it would be challenging to develop type II inhibitors for the G2019S mutation, due to stabilisation of the active kinase conformation by the Ser2019 [104]. The field has now matured so that tool compounds have become available for understanding LRRK2 biology in vitro and in vivo, and some of these have been progressed into advanced preclinical toxicology assessment. One of the principal challenges in their in vivo profiling is the absence of any preclinical models of PD that are modelled by LRRK2 dysfunction. Evidence of in vivo activity has been typically measured by indirect measurements of LRRK2 activity using LRRK2 Ser910 and Ser935 phosphorylation or directly using the LRRK2 Ser1292 autophosphorylation site. In this review, these efforts towards the discovery of selective LRRK2 inhibitors will be discussed, grouped by chemotype. The reader is also directed to previous reviews of LRRK2 inhibitors [105–110].

4.1 Diaminopyrimidines The diaminopyrimidine scaffold has proved a particularly fruitful chemotype for the development of LRRK2 inhibitors and has contributed significantly to advancing our understanding of LRRK2 biology. Some of the first reported selective LRRK2 inhibitors, such as CZC-25146 (3) [111] and LRRK2-IN-1 (4) [112], were diaminopyrimidine based, and this chemotype has subsequently been optimised towards clinical candidates. Me N

O

N N

HN

O N

F

N

O

N H

N

N H

Me

Me

Me S O O

N

O

O

N Me

N N N

CZC-25146 (3)

LRRK2-IN-1 (4)

Me

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In the case of 3, an analogue of the nonselective kinase inhibitor Sunitinib was immobilised on a solid-phase matrix. The ability of compounds to block the binding of LRRK2 from mouse kidney lysate was then used to identify diaminopyrimidine 3 as a potent and selective LRRK2 inhibitor (IC50 ¼ 10–30 nM). This was confirmed in a TR-FRET assay with wild-type LRRK2 and G2019S IC50s reported as 4.76 and 6.87 nM, respectively. Selectivity for LRRK2 was good, with off-target activity against seen in just 5 out of 184 kinases with biochemical IC50s less than 300 nM using KinoBeads™. No cytotoxicity was observed below 5 μM and 3 attenuated G2019S LRRK2-induced neuronal injury and death in a concentration-dependent manner, with an EC50 of approximately 100 nM. In addition, in a neurite morphology assay using human cortical neurons, measuring neurite length and branch points, 3 exhibited an EC50 of approximately 4 nM. Although it was reported to have negligible brain levels, probably due to suboptimal physicochemical properties for blood– brain barrier penetration, this compound was one of the first selective LRRK2 tool compounds that could be used to interrogate LRRK2 biology in vitro. LRRK2-IN-1 (4) is another selective and potent diaminopyrimidinebased LRRK2 inhibitor (LRRK2 wild-type and G2019S IC50s ¼ 13 and 6 nM, respectively), which resulted from an optimisation of hits arising from the screening of a 300-member compound library against a panel of 442 diverse kinases using an in vitro ATP-site competition binding assay. Kinase selectivity was assessed using three different methods; the Ambit/ DiscoveRx KINOMEscan®, Dundee profiling and KiNativ™ technology. In the KINOMEscan®, 12 kinases showed inhibition below 10% of the control out of 440 kinases at a 10 μM concentration. In the Dundee panel, an IC50 of 45 nM was reported for DCLK2 and IC50s greater than 1 μM for seven other kinases. The KiNativ™ profiling in human peripheral blood mononuclear cells confirmed cross-reactivity against DCLK1 and MAPK7, with an EC50 measured for MAPK7 of 160 nM. Interestingly, 4 was effective in reducing TNF release at nanomolar concentrations, suggesting that this compound has off-target effects that complicate interpretation of the activity of this compound [113]. Compound 4 inhibited LRRK2 Ser910 and Ser935 phosphorylation in HEK293 cells at doses of between 1 and 3 μM for wild-type LRRK2, and at slightly lower doses for the G2019S variant. No effect on Ser910 and Ser935 phosphorylation was observed in the drug-resistant LRRK2 A2016T and LRRK2 (double A2019T+G2019S) variants. This confirmed that the effect

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of 4 was LRRK2 mediated and not caused by off-target effects. Similar effects were observed on endogenous levels of LRRK2 in human lymphoblastoid cells from a control individual, PD patient homozygous for the LRRK2 G2019S mutation, human-derived neuroblastoma SHSY5Y cells and in mouse Swiss 3T3 cells. Compound 4 was next assessed in vivo. After i.p. dosing at 100 mg/kg in mice, complete inhibition of the serine biomarkers was observed in kidney at 1 and 2 h. No effect on brain LRRK2 Ser910 or 935 phosphorylation was observed, due to the poor brain penetration properties of 4. Cl

N HN Me

O

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Cl

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N

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O Me

Me

NH Me

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Me N

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N O

N TAE684 (5)

HG-10-102-01 (6)

N Me

TAE684 (5) [114] is another diaminopyrimidine with LRRK2 activity, originally identified as an inhibitor of anaplastic lymphoma kinase (ALK), but subsequently discovered to have LRRK2 inhibitory activity (IC50s ¼ 7.8 and 6.1 nM against WT LRRK2 and G2019S LRRK2, respectively). Compound 5 displays a different profile to the previously reported compound LRRK2-IN-1. It inhibits the LRRK2 A2016T mutant (IC50 ¼ 93.3 nM), in contrast to LRRK2-IN-1 which is much less potent against the A2016T mutant (IC50 ¼ 2450 nM), the G2019S mutant (IC50 ¼ 6 nM) or WT (IC50 ¼ 13 nM). Using ALK as a crystallographic surrogate for LRRK2, it was hypothesised that the isopropyl sulphone moiety of 5 avoided a steric clash with the A2016T residue that is likely with the anthranilic acid ring of 4. The kinase selectivity was assessed in the Dundee panel (124 kinases) and KINOMEscan® binding was assessed against a panel of 442 kinases. In the Dundee panel at a concentration of 1 μM, six other kinases were inhibited at greater than 90% inhibition. In the KINOMEscan® panel, binding was reported for six other kinases with Kds below 100 nM, illustrating that 5 is less selective than other compounds such as 4.

Development of LRRK2 Inhibitors for PD

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Compound 5 showed significant inhibition of wild-type LRRK2 pSer935 and pSer910 in the concentration interval of 0.1–0.3 μM in stably transfected HEK293 cells. This is an order of magnitude more potent than reported for LRRK2-IN-1 (4). Slightly higher concentrations were required for LRRK2 G2019S. Consistent with the reported inhibition of the drug-resistant mutant LRRK2 A2016T in the biochemical assay, it was observed that inhibition in HEK 293 cells transfected with LRRK2 A2016T and LRRK2 A2016T+G2019S could be achieved at concentrations of 1–3 μM. Oral bioavailability for 5 was excellent (F ¼ 84%), with a long half-life of 11.3 h and excellent plasma exposure of 6374 h ng/mL after 10 mg/kg p.o. dosing. A brain-to-plasma ratio of around 2 was estimated based on AUC ratios. At doses of 10 and 50 mg/kg p.o., however, no inhibition of phosphorylation of Ser910 and Ser935 was observed, despite the significant brain exposure measured. HG-10-102-01 (6) [115] is a diaminopyrimidine-based compound which was one of the first examples reported to be brain penetrant, and to demonstrate inhibition of Ser910 and 935 phosphorylation in mouse brain. It was slightly less potent against wild-type LRRK2 than 4 and 5 with an LRRK2 IC50 of 20.3 nM, but was slightly more potent against LRRK2 G2019S with a reported IC50 of 3.2 nM. Like TAE684, HG-10-102-01 also showed inhibition of some drug-resistant mutants LRRK2 (A2016T IC50 ¼ 153.7 nM) and LRRK2 G2019S+A2016T (IC50 ¼ 95.9 nM). Docking of 6 in a homology model based on ALK suggested that there was a lower possibility for steric clash with the A2016T mutation compared to 4. Removal of the 4-anilino substitution thus maintained LRRK2 potency but improved the CNS-penetrant properties of the molecule. Compound 6 was active in inhibiting the phosphorylation of LRRK2 Ser910 and Ser935 in HEK293 cells (both stably expressing wild-type and G2019S LRRK2) at a concentration of 1 μM for wild-type LRRK2 and 0.3 μM for G2019S LRRK2. Inhibition in the 1–3 μM range was observed in cells expressing the LRRK2 A2016T and LRRK2 G2019S +A2016T drug-resistant mutants. Similar cellular inhibition of the serine markers was observed in endogenously expressed LRRK2 in human lymphoblastoid cells derived from a control and from a PD patient homozygous for G2019S LRRK2, as well as in mouse Swiss 3T3 cells and in mouse embryonic fibroblast cells. Compound 6 displayed good oral bioavailability (F ¼ 67%) but displayed high clearance in vitro (T½ in mouse liver microsomes ¼ 13 min) and a short

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half-life in vivo (0.13 h), indicating high first-pass metabolism. Dosing at 100 mg/kg i.p. in mouse gave complete inhibition of phosphorylation of Ser910 and 935 in spleen, kidney and brain. At 50 mg/kg, near complete inhibition was observed in brain with only partial inhibition at 30 and 10 mg/kg. The same chemotype was also optimised by Genentech/Biofocus. An LRRK2 homology model based on JAK2 [116] was used to guide the optimisation of the chemistry. This was validated by docking (Glide, Schr€ odinger, Inc.) using known selective LRRK2 inhibitors from the literature prior to analysis of HTS hits. With a view to obtaining kinase selectivity, the authors analysed the locations, accessibility and properties of those residues in the LRRK2 ATP binding site least conserved in the kinome, and highlighted Phe1883, Leu1949, Ser1954 and Arg1957. Leu1949 was considered to be the most attractive selectivity handle, given that it is two residues away from the hinge binding motif in the adenine pocket of the ATP binding site. Leucine, present in 25.3% of kinases at this position near the hinge, has a shorter side chain than phenylalanine or tyrosine, present in 18.1% and 40.5% of other kinases, respectively. Leu1949 therefore provides a small cavity which can be exploited to help impart selectivity on inhibitors for LRRK2. Using this model, the team rationalised that the 2,4-diaminopyrimidine moiety of the HTS hit 7 binds to the ATP adenine site through a pair of hydrogen bonds to the backbone amide NH and the carbonyl oxygen of Ala1950. The amide carbonyl forms a weak hydrogen bond with the guanidinium side chain of Arg1957, the aniline ring binds in a flat hydrophobic cleft along the hinge and the 4-morpholinoamide group points towards the side chain of Phe1883. A key strategy in optimisation of the HTS hit was optimisation of selectivity for LRRK2 by accessing the pocket formed by Leu1949. Indeed, selectivity against JAK2 was improved by small substituents in the ortho-position of the aniline ring with greatest selectivity observed with methoxy substitution (optimised hit). This was confirmed by only wild-type and G2019S LRRK2 being inhibited by more than 50% in a panel of 63 kinases at Invitrogen. The physicochemical properties of the optimised hit (8) were within the typical parameters for CNS penetration and the compound was assessed for brain penetration in wild-type and P-gp/BCRP knockout mice. At a dose of 1 mg/kg i.v., total and free brain-to-plasma AUC ratios were determined in wild-type mice to be 1.4 and 0.61, respectively, and in the P-gp/BCRP knockout mice total and free AUC ratios were determined to be 2.9 and 1.3, showing the compound was most likely

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Development of LRRK2 Inhibitors for PD

a weak P-gp and/or BCRP substrate. The brain exposures in wild-type mice at 30 mg/kg i.p. were determined after 30 min to be 6.2 and 0.37 μM, illustrating the value in using this compound as an in vivo tool compound. Me H N

H N

N Cl

Me

Me O

O

8

7 Me

Cl NH

N

NH

N

N N

O

N

O

O

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H N N

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N CF3

H N

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CF3

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N Me

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F

NH Me

GNE-7915 (10)

Further optimisation of this series [117] quickly identified the corresponding trifluoromethyl analogue 9 as a promising lead, with improved permeability and no efflux liability, as well as an improved free brain-to-plasma AUC ratio of 0.5, compared to 0.17 for the chlorine analogue. This was ascribed to intramolecular hydrogen bonds between a fluorine atom and the aminomethyl NH, as well as between the aniline NH and the methoxy group. Kinase selectivity profiling of 9 at 0.1 μM against 178 kinases showed excellent selectivity, with 60-fold selectivity for LRRK2, while at 1 μM against 63 kinases, it showed 600-fold selectivity for LRRK2. Further kinase profiling identified TTK, a kinase with potential toxicity concerns with 55% and 98% inhibition at 0.1 and 1 μM, respectively. A strategy for optimisation of selectivity against TTK by C-5 substitution of the aniline ring was employed. This was based on a difference between Ser1954 in LRRK2 and Asp608 in TTK, equivalent residues in the enzymes. This was exploited by the authors, leading to a hypothesis that small C-5 substituents would be expected to impart selectivity for LRRK2. GNE7915 (LRRK2 Ki ¼ 1 nM) (10) was identified as part of this optimisation where C-5 substitution with fluorine imparted selectivity for LRRK2 against TTK (53-fold) and JAK2 (3200-fold), while the aminoethyl

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substitution in this series imparted reduced clearance in vivo, compared with the aminomethyl example. In the case of 10 it is a methoxy group which is predicted to occupy the space near Leu1949, and this also forms a key structural feature of the earlier aminopyridine-derived inhibitors of LRRK2, compounds 3–6 thus providing strong support for this hypothesis. Invitrogen profiling of 10 against a panel of 187 kinases at 0.1 μM only picked up TTK inhibition at greater than 50% inhibition. In the larger DiscoveRx panel of 392 unique kinases, only TTK and ALK were identified with greater than 65% probe displacement at 0.1 μM. CEREP receptor profiling of GNE-7915 only picked up 5HT2B inhibition (>70% at 10 μM), as confirmed by in vitro functional assays. Compound 10 displayed good permeability (MDR1-MDCK Papp ¼ 10.4  106 cm/s), with no measurable efflux. Brain penetration in rat was good, with an unbound brain/plasma AUC ratio of 0.5. In vivo PK studies in rats showed a favourable profile with a low clearance of 8.3 mL/min/kg, a half-life of 3.1 h and oral bioavailability of 40%. A pharmacodynamic assessment of LRRK2 inhibition showed 10 to have an IC50 of 9 nM in vivo based on unbound brain concentration for inhibition of phosphorylation of Ser1292 in bacterial artificial chromosome (BAC) transgenic mice expressing the LRRK2 G2019S mutation. Further profiling in cynomolgus monkey showed a low plasma clearance of 11 mL/min/kg, a long half-life of 7.7 h and oral bioavailability of 24% at an oral dose of 20 mg/kg. Additional optimisation of this series [118] was focussed on reduction of size, improvement of aqueous solubility and replacement of the aniline moiety to avoid the potential for ortho-quinone-imine reactive metabolite formation. An important feature of this work was to maintain the selectivity handle afforded by the methoxy group in 10 identified in the JAK2-derived homology model. In addition, optimisation of the physicochemical and ADME properties, by using the vector that extends into the solvent-exposed region of the active site occupied by 10, was a key strategy in the evolution of this series. Replacement of the aniline with a variety of pyrazole isomers identified the 1,4-dimethyl-pyrazole 11a as the most potent example (LRRK2 Ki ¼ 2 nM) and also that possessing the greatest JAK2/LRRK2 selectivity (560-fold). This showed the crucial selectivity imparted by the methyl substitution, compared to the nonsubstituted 1-methylpyrazole, 11b, which showed virtually no selectivity for LRRK2, with a JAK2/ LRRK2 selectivity ratio of 2.7. Although the pyrazole replacements were not susceptible to P-gp efflux, they were reversible and/or time-dependent inhibitors of cytochrome P450 (CYP) 1A2. The strategy for avoiding this

53

Development of LRRK2 Inhibitors for PD

was to increase the bulk of substitution on the pyrazole nitrogen with sterically demanding groups which would cause steric clashes with the CYP1A2 active site. Importantly, it was established that geminal demethylation on cyanoethyl substitution of the pyrazole N was successful in avoiding both reversible inhibition and time-dependent inhibition of CYP1A2 in 12. Replacement of the 5-methyl of 12 with 5-chloro and the 2,2-dimethyl-cyanoethyl with 2,2-dimethyl-hydroxyethyl delivered molecule 13, with no reversible or time-dependent inhibition of CYP1A2. Compound 13 had an LRRK2 Ki of 6 nM in the biochemical assay, and a cellular IC50 of 28 nM. Selectivity was excellent against a panel of 185 kinases at Invitrogen, with only one other kinase inhibited at greater than 76% (TSSK1) at a concentration of 1 μM. In vivo, compound 13 showed an acceptable clearance in rat (Clp ¼ 21 mL/min/kg) and dog (Clp ¼ 22 mL/min/kg) and acceptable bioavailability in dog (F ¼ 31%). Brain penetration in rat was modest, with an unbound brain/unbound plasma AUC ratio of 0.37. The pharmacodynamic effect of compound 13 was assessed after i.p. dosing in BAC transgenic mice expressing the G2019S LRRK2. The unbound brain IC50 based on LRRK2 pSer1292 inhibition was determined to be 12 nM. CF3

N HN

N

N H

R

Me HN

N

N H

HN

Me

N N

Me 11a R = Me 11b R = H

12

N H

Me

N N

N N CN Me

N

Cl

Me

Me

CF3

N

CF3

N

Me

OH Me 13

Compound 13 was subsequently reported to have elevated turnover in human hepatocytes due to high levels of glucuronidation (>60%) after 3 h. Additionally, oral bioavailability was poor in cynomolgus monkey (oral bioavailability ¼ 5% at 1 and 20 mg/kg) attributed to high intestinal metabolism, and this compound was not progressed further due to uncertainty in the human PK prediction. Further optimisation of this series [119] then focussed on removing the alcohol group in 13, the most likely source of the glucuronidation and a factor in the modest brain penetration. The first approach was to attempt to cyclise the tertiary alcohol, as illustrated by compound 14. Although potent against LRRK2, with a Ki of 3 nM and a cellular

54

K.V. Christensen et al.

IC50 of 42 nM, this was not pursued due to a low ratio of unbound drug between brain tissue and plasma (Bu/Pu) at 0.2. A series of gem-disubstituted cyanopyrazoles was then identified. This is illustrated by GNE-0877 (15), which showed improved cellular potency (LRRK2 IC50 ¼ 3 nM), low intrinsic clearance in vitro and in vivo and no evidence of the glucuronidation seen with 13. The enhanced potency was reasoned to be due to a better fit afforded by the gem-dimethyl cyano group. The dimethyl groups should form improved van der Waals interactions with hydrophobic residues in the protein. The cyano group forms an electrostatic interaction with the side chain of Arg1957. Compound 15 displayed good selectivity for LRRK2 at a screening concentration at 0.1 μM in the Invitrogen panel of 188 kinases, with 4 other kinases inhibited at a >50% level (Aurora B, RSK2, RSK4 and RSK3). Selectivity against TTK was 212-fold (LRRK2 Ki ¼ 150 nM). In vivo, 15 displayed a good free brain-to-plasma ratio of 0.6, good oral bioavailability of 88% and a rat in vivo clearance (44 mL/min/kg) which was comparable to in vitro rat hepatocyte clearance (25 mL/min/kg). Compound 15 was found to be a reversible CYP1A2 inhibitor (IC50 ¼ 0.7 μM), but did show time-dependent inhibition of the enzyme. Additional profiling of 15 showed that the compound was a pan-inducer of CYP enzymes in human hepatocytes. It was then hypothesised that incorporating more polarity distal to the pyrazole may mitigate the risk of induction for other human CYP enzymes, while maintaining the branched pyrazole N-alkyl substitution necessary for avoiding CYP1A2 inhibition. Initially, 3-oxetyl and 4-tetrahydropyranyl substitution on the pyrazole nitrogen was investigated, but could not be progressed due to incurring a short half-life, an undesirable in vitro–in vivo disconnection and limited brain penetration in rats. In order to address the concerns surrounding the suboptimal PK parameters for these compounds, it was decided to incorporate a weakly basic nitrogen into the solvent-exposed region of the molecule to increase the volume of distribution and thus the in vivo half-life. Incorporation of a piperidine-N-oxetyl substituent on the pyrazole did indeed increase the volume of distribution and extend the half-life; however, compound 16 was shown to be a P-gp substrate in MDR1-MDCK cells (efflux ratio ¼ 5.1). The low unbound rat clearance for compound 16 (45 mL/min/kg) prompted further optimisation, in particular, minimisation of the P-gp efflux. Moderation of the basicity by introduction of a fluorine atom in the 3-position of the piperidine ring did reduce the efflux ratio to 2.2 but not enough to facilitate good brain penetration

55

Development of LRRK2 Inhibitors for PD

(Bu/Bp ¼ 0.08). Replacement of the methyl group on the pyrazole with chlorine had the effect of removing efflux in both the MDCK-MDR1 and MDCK-BCRP paradigms (ER ¼ 0.8 and 0.9, respectively). The lead compound GNE-9605 (17) displayed an LRRK2 Ki in the biochemical assay of 2 nM as well as a cellular IC50 of 19 nM, with off-target kinase activity greater than 50% observed only for TAK1-TAB1 at a screening concentration of 1 μM in the Invitrogen 178 kinase panel. No other off-target activity was observed in a representative panel of receptors and ion channels. Compound 17 showed low clearance in both human liver microsomes and hepatocytes (5 and 1 mL/min/kg, respectively). No time-dependent or reversible inhibition of CYP isoforms was observed, justifying the strategy to place polarity distal to the pyrazole ring. In the in vivo pSer1292 assay in BAC transgenic mice, 15 and 17 displayed unbound brain IC50s of 3 and 20 nM, respectively. Compound 15 was selected in favour of 17, partly because of the greater in vivo potency, for further evaluation in preclinical toxicology and genotoxicology studies. CF3

N Me

N

HN

N H

CF3

N

Me

HN

N

N H

Me

Me O

N N Me Me

N N

NC GNE-0877 (15)

14

CF3

N HN Me

N

N H

CF3

N

Me HN

N

N H

Me

Cl

N N

N N N

F N

O O 16

GNE-9605 (17)

Both 10 and 15 have undergone repeat dosing safety assessments to study the consequences of LRRK2 inhibition. Dosing of 10 and 15 in male C57BL/6 mice for 15 days at doses of 200 or 300 mg for 10, or 30 or

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K.V. Christensen et al.

65 mg for 17 BID, did not induce any lung or kidney pathology. In male and female Sprague–Dawley rats, no effects were observed on lung or kidney at once daily doses for 7 days up to 100 mg for 10 or up to 200 mg for 15. Dosing of both compounds in nonhuman primates [82], however, induced a lung phenotype characterised by formation of lamellar bodies in type II pneumocytes. Cynomolgus monkeys were dosed for 7 days with 10, 25 or 65 mg/kg QD of 10 via oral gavage for 7 days. At the two higher doses vacuolation of type II pneumocytes in the lung was observed in both sexes. A follow-up repeat dose 29-day study was performed in cynomolgus monkeys with both 10 and 15 confirming this initial finding. These findings in lung tissue were morphologically the same as seen in LRRK2 KO mice. No kidney phenotype was observed, with no effect observed on renal function.

4.2 Arylbenzamides In 2012, a novel arylbenzamide GSK2578215A (18) [120] was identified by GSK as a potent LRRK2 inhibitor. Hits for this series were identified in a screen of the GSK KCS (a kinase-focussed set of compounds for lead discovery) using a homogenous time-resolved fluorescence (HTRF) assay that monitored inhibition of phosphorylation of the peptide-substrated LRRKtide by baculoviral-derived recombinant 6His-Tev-LRRK2 (1326–2527). The inhibitory potency for 18 was assessed as having an IC50 of 8.9 nM against G2019S LRRK2 and 10.9 nM against WT LRRK2. The potency against the A2016T mutant (IC50 ¼ 81.1 nM) was reduced eightfold, a much smaller reduction than has been observed for LRRK2-IN-1. Kinase selectivity was assessed against a panel of 131 kinases in the Dundee panel and a panel of 329 kinases in the KINOMEScan®. At a concentration of 10 μM, excellent selectivity was observed in the Dundee panel and only one kinase with >50% inhibition was observed. Only two kinases (ALK and FLT3) had an ambit score less than 10 in the KINOMEscan® assay. A homology model was used to rationalise its LRRK2 binding mode and kinase selectivity. This model had been previously developed from ligand-bound X-ray crystal structures of rho-associated protein kinase 2 (ROCK2) [101]. Although LRRK2 and ROCK2 are not closely related kinases, it was discovered via biochemical screening that ROCK inhibitors, including H-1152 (2), also inhibit LRRK2. This work highlighted another key residue in the LRRK2 ATP binding site of importance for kinase selectivity, namely Ala2016, which is close to the DYG motif.

57

Development of LRRK2 Inhibitors for PD

In particular, mutation of Ala2016 to Thr reduced inhibition by 2 significantly. In contrast, 18 was able to inhibit A2016T mutant and wild-type LRRK2 to the same extent. The docked pose of 18 showed that this compound was able to avoid a steric clash with Ala2016, in contrast to H-1152 and other inhibitors such as LRRK2-IN-1 [112]. Cellular potency assessed by monitoring pSer935 and pSer910 inhibition showed GSK2578215A had comparable cellular potency to LRRK2-IN-1 in HEK293 cells stably transfected with WT or G2019S, where significant dephosphorylation at 0.3–1.0 μM was observed. Consistent with the biochemical results, lower cellular potency was observed in the inhibitorresistant mutations (A2016T+G2019S, A2016T) at concentrations between 1 and 3 μM. Similar cellular potency was observed in human lymphoblastoid cells derived from a control and from a PD patient homozygous for the LRRK2 G2019S mutation. Similar dose-dependent inhibition of Ser910 and Ser935 phosphorylation of endogenous LRRK2 was observed in mouse Swiss 3T3 cells. Pharmacokinetic profiling of 18 in mice showed low oral bioavailability (F ¼ 12.2%), a half-life of 1.14 h and plasma exposure 635.3 h/ng/mL. Brain penetration was assessed to be good with a brain/plasma ratio greater than >1. At a dose of 100 mg/kg i.p. in mice complete pSer910 and pSer935 inhibition was observed in kidney and spleen, but no inhibition was observed in brain which might be explained by low free drug levels in the brain. Me N N

F N H N

H N O

O

N GSK2578215A (18)

N

O

O

BMPPB-32 (19)

Another arylbenzamide from this series, BMPPB-32 (19) (LRRK2 WT Ki ¼ 1.5 nM and G2019S Ki ¼ 6.2 nM), was profiled in Drosophila flies expressing hLRRK-G2019S, which have an activity-dependent loss phenotype, and 19 normalises this loss of reduction in visual gain [121].

4.3 Indolinones The first compounds reported based on the indolinone scaffold included the Raf-1 kinase inhibitor GW5074 (20) and indirubin-3-monoxime (21)

58

K.V. Christensen et al.

[122]. Compound 20 was reported to be more potent against the LRRK2G2019S mutant (IC50 ¼ 880 nM) than LRRK2 WT (IC50 ¼ 3150 nM). Me O

Br HO N

OH I

N

Me

N

O

N H O

F

Br N H Indirubin-3⬘monoxime (21)

GW5074 (20)

N H Me

N H

O

N H

Me

Sunitinib (22)

A group at Novartis using Sunitinib, 22, as their starting point [123] discovered that replacement of the 5-fluoro substituent with a methoxy group (23) maintained LRRK2 potency with an IC50 of 46 nM, but improved kinase selectivity inhibiting only 5 other kinases out of 36 with an IC50 of less than 1 μM, compared to 22’s inhibition of 20 other kinases out of 54 with an IC50 of less than 1 μM. Docking studies using a homology model based on IRAK4 using the 5-methoxy derivative led these scientists to form a new fused ring, via the 2-methyl pyrrole and inversion of the amine, to yield the 4,5,6,7-tetrahydro-1H-pyrropyridine derivative 24, which maintained potency on LRRK2. The Novartis group highlighted the importance of interaction with Arg1957 in their IRAK4-derived homology model in rationalising their indoline-derived series, as initially proposed by Genentech and further supported by the Pfizer group. IRAK4 has 26% sequence identity to LRRK2 in the kinase domain and 46% similar residues. It was observed that examples from the indolinone series had particularly high affinity for RET kinase. This was rationalised by a cocrystal structure with RET kinase, in which the role of Ala2016 in LRRK2 (Ser891 in RET) was highlighted. Me O

N

Me

O

N H Me

N H O

O N H 23

N

Me

N H O

O N H 24

NH2

59

Development of LRRK2 Inhibitors for PD

The tetrahydropyridine ring was amenable to a variety of substitutions both in terms of LRRK2 potency and kinase selectivity and was thus a convenient handle to adjust physicochemical and PK properties. The most prominent compound from this series was compound 25, which inhibited LRRK2 with an IC50 of 9 nM, but also displayed equipotent activity against KDR (IC50 ¼ 6 nM) and RET (IC50 ¼ 6 nM). O

O N Me

N H O

O

N

N

N Me Me

N H

N O

N H O

O N H

25

26

N-Methylation of the morpholine analogue compound 26 reduced potency by 50-fold from an LRRK2 IC50 of 3 nM for compound 26 to an IC50 of 1.5 μM for the N-methylated indolinone derivative. Further replacement of the 5-methoxy with CF3, N-methylpiperazine or N-acetylamino did not affect LRRK2 potency or kinase selectivity. Compound 25 had promising properties for in vivo investigation with acceptable aqueous solubility and favourable PK properties in mice. It was assessed using in vitro competition pull-down experiments. The indolinone derivative starting point was cross-linked to a sepharose solid support that be used to assess the binding of the test compound to LRRK2 in vitro. Mouse brain extracts were mixed with this compound or a negative control which had poor cell and poor brain penetration. Both compounds showed a high degree of LRRK2 binding at 1 μM or greater. Compound 25 was dosed via p.o. administration to C57BL/6 mice. A clear dose-dependent effect of in vivo brain binding to LRRK2 was observed for compound 25. Another group also explored indolinones [124] using the FLT-3 inhibitor 27 as their starting point. Potency was reported with an LRRK2 IC50 of 265 nM. An LRRK2 homology model based on B-Raf was used to guide compound design. The close analogue of GW5074 (28) was the most potent compound prepared. This displayed a 53-fold improvement in activity (LRRK2 IC50 ¼ 15 nM) compared to GW5074 (IC50 ¼ 880 nM). The remarkable effect of a 3,5-dibromo substituent was illustrated by the much poorer activity witnessed with the phenyl and 4-hydroxy-phenyl analogues. Replacement of the Cl with phenyl led to a drop in potency (LRRK2 IC50 ¼ 204 nM), while methoxy was only slightly less potent (LRRK2 IC50 ¼ 15 nM). The potency of compound 28 was explained from the

60

K.V. Christensen et al.

homology model with Glu1948 of LRRK2 forming a strong hydrogen bond to the N–H motif of the indole. The 3,4-dibromo-4-phenol moiety binds to the rear of the back pocket with hydrophobic interaction observed between the phenol ring and the gatekeeper residue M1947. Me

N Me Br

O

OH

Cl

Cl

Br

O

O

N H

N H

27

28

4.4 Indazoles Merck reported indazole MLi-2 (29) [87] as a useful tool for exploring LRRK2 biology in vivo. Compound 29 exhibits an in vitro IC50 of 0.76 nM with an LRRK2 selectivity of >100-fold for other kinases. Five other kinases were reported with an IC50 of <1 μM. These were CLK4 (IC50 ¼ 225 nM), MAP3K14 (IC50 ¼ 244 nM), MAP3K5 (IC50 ¼ 428 nM), CLK2 (IC50 ¼ 605 nM) and TTK (IC50 ¼ 935 nM). In tet-inducible LRRK2 SH-SY5Y cells, 29 displayed excellent cellular activity with an IC50 of 1.4 nM, measuring inhibition of LRRK2 pSer935 phosphorylation. As a marker of inhibitory activity, LRRK2 serine 935 phosphorylation is an indirect measure of cellular activity; a close analogue, 35S-MLi-A (30), was developed as a radioligand. Using tet-inducible SH-SY5Y cells, 30 exhibited high levels of displaceable and saturable binding with a Kd of 0.2 nM. Comparable binding was observed in cells expressing G2019S LRRK2 with a Kd of 0.52 nM measured for the radioligand. Compound 29 exhibited a Ki of 3.4 nM in this radioligand binding assay. Me

H N

Me

N

H N N

O

O Me N N

N

O Me

MLi-2 (29)

N N

N

Me N S O O

[35S]MLi-A (30)

PK studies in mice showed an oral bioavailability of 45% at a p.o. dose of 10 mg/kg. The mean plasma concentration was obtained at 0.75 h postdose.

Development of LRRK2 Inhibitors for PD

61

A mean residence time of 2.7 h after i.v. dosing and 11 h after p.o. dosing was measured. This was ascribed to a long absorption time. After 1 h, the unbound brain concentration had increased to 273 nM in a dose-dependent fashion. Doses above 10 mg/kg gave full LRRK2 pSer935 inhibition. Compound 29 was then evaluated in an 11-day in-diet dosing regimen at between 3 and 120 mg/kg/day. A dose of 30 mg/kg/day was identified as giving full LRRK2 pSer935 inhibition at 4, 8 and 24 h time points. No reduction was observed on total LRRK2 levels in mouse brain; however, a significant and dose-dependent reduction was observed in mouse kidney after 11 days dosing of 29. The free brain pSer935 IC50 was determined to be 0.8 nM. Having established 30 mg/kg/day as the optimal dose, a 15-week in-diet dosing regime was undertaken in MitoPark mice. The MitoPark mouse is a PD model [125] in which a PD phenotype develops as a result of DAT-Credependent knockout of the mitochondrial transcription factor (TFAM). Asymptomatic 5-week-old MitoPark mice were treated for 15 weeks at a dose of 30 mg/kg/day which is sufficient to sustain more than 90% pSer935 LRRK2 inhibition over a 24-h period in both wild-type and MitoPark mice. Total LRRK2 protein levels were unchanged in the 29-treated MitoPark mice. Although 29 successfully demonstrated a pharmacodynamic effect based on pSer935 inhibition, no effect was observed in the trajectory of the progressive motor phenotype observed in MitoPark mice. In this model, drug treatment starts at week 5 and a significant decline in motor and rearing behaviour is measured by week 11 in vehicle-treated MitoPark mice. No difference was observed in the compound 29-treated group. Neurochemical analysis also showed that 29 had no effect on the decline of striatal dopamine, DOPAC or tyrosine hydroxylase levels observed in MitoPark mice at 8 weeks of age. Histopathology and morphologic assessments were made of the kidney and lungs of the MitoPark mice that were treated with 29. MitoPark mice receiving 30 mg/kg/day of in-diet dosing did not exhibit darkened kidneys, which have been reported for LRRK2 KO mice.

4.5 Cinnolines/Quinolines A group at Elan Pharmaceuticals employed a kinase inhibitor-focussed screen using an HTRF assay. A series of 4-aminoquinoline3-carboxamides including 31 (LRRK2 IC50 ¼ 781 nM) and 32 (LRRK2 IC50 ¼ 35 nM) was identified [126]. Initial drawbacks identified for these starting points were potential off-target activity for colony-stimulating factor

62

K.V. Christensen et al.

1 receptor (CSF1R) and poor CNS penetration due to these compounds being P-gp substrates. R

NH O NH2 N

Me S O O

31 R = Ph 32 R = iPr

Introduction of a nitrogen atom at the C2 position was postulated to form an internal hydrogen bond with the carboxamide and attenuate P-gp liability. The direct analogue of 32 containing the cinnoline core, 33 [127], showed balanced potency for WT and G2019S with IC50s of 15 and 27 nM, respectively. The cyclobutyl analogue 34 had a similar potency (LRRK2 WT IC50 ¼ 10 nM and LRRK2 G2019S ¼ 19 nM) and also showed a brain-to-plasma ratio of 0.76 with no P-gp efflux observed in in vitro MDR1-MDCK permeability experiments. Optimisation of this series was ultimately abandoned due to poor kinase selectivity for most of the compounds screened. R

NH O NH2 N

Me S O O

N 33 R = iPr 34 R = Cyclobutyl

An alternative strategy undertaken by the team was replacement of the primary amide in 32 with a nitrile to give 35, a compound of similar potency (LRRK2 WT IC50 ¼ 31 nM and LRRK2 G2019S IC50 ¼ 28 nM). Replacement of the 4-(methylsulphonyl)phenyl moiety of 35 with 1-methyl-1H-pyrazol-4-yl improved potency to single-digit nanomolar with an LRRK2 WT IC50 of 7 nM and G2019S IC50 of 5 nM. Cellular activity in HEK293 cells based on pSer-935 inhibition indicated an IC50 of 435 nM for 35 and for 36 an IC50 of 140 nM. Me Me

NH CN

R

35 R: 4-(Methylsulphonyl)-phenyl 36 R:1-Methyl-1H-pyrazol-4-yl

N

Both compounds showed selectivity towards LRRK2 in a panel of 40 kinases with no other kinases inhibited above 80% at 1 μM. CNS

Development of LRRK2 Inhibitors for PD

63

penetration was assessed to be good, with good brain/plasma ratios in mice and no P-gp efflux observed in in vitro permeability assessment in MDR1-MDCK transfected cells. The lead compound 36 was further evaluated in BAC transgenic mice expressing mouse LRRK2 with the G2019S mutation. At oral doses of 30 and 100 mg/kg, after 3 h 36 showed relatively moderate effects with 30% and 44% reduction, respectively, in pSer935 inhibition, reflecting the moderate CNS exposure of the compounds.

4.6 Pyrrolopyrimidines The pyrrolopyrimidine scaffold has proved relatively fruitful in providing a chemotype that could be optimised within CNS physicochemical space and deliver compounds with in vivo activity. For example, Pfizer reported the discovery of PF-06447475, 39 [128], a potent and selective pyrrolopyrimidine-based brain-penetrant, selective and potent LRRK2 inhibitor. The starting point for this optimisation was the attractive imidazopyrimidine hit 37, identified in an HTS from the Wyeth compound collection. This compound was included in a focus screen by the Pfizer group, who employed a variety of approaches including internal kinasetargeted library and virtual screening based on similarity using atom pairs and feature trees. The pyrrolopyrimidine 38 was subsequently identified, being a compound that had arisen from a previous programme targeting the Janus kinase (JAK) family. JAK crystallographic data were available which suggested that pyrrolopyrimidine was probably a two-point hinge binder, with the aryl group pointing towards the DFG motif and the amine occupying the ATP-ribose pocket. Optimisation of the pyrrolopyrimidine 38 showed that the 3-methyl substitution on the piperidine was not required for activity. The resulting unsubstituted piperidine was slightly more potent (IC50 ¼ 6 nM). Attempts to drive down the lipophilicity with a 3- or 4-methoxy or a 3-methoxymethyl group did not improve potency or stability. The morpholine analogue, PF-06447475 (39), displayed both improved potency and microsomal stability. Activity was also good in the LRRK2 whole-cell assay and further profiling using the ActivX Kinativ assay technology was undertaken in human PBMC lysates. This approach uses biotinylated acyl phosphates of ATP that irreversibly react with protein kinases on conserved lysine residues in the ATP binding pocket, and gives both a measure of potency and kinase selectivity.

64

K.V. Christensen et al.

Me

O

Me

N

37

N H

O

N

CN

N

N

N

N

CN

CN

N

N

O

O

Me

N

N

N N H

N 38

N N

N H

PF-06447475 (39)

N

N H

39b

Pfizer deployed a homology modelling-based approach in their design of 39. LRRK2 homology models were constructed based on TAK1, which was also used by the Genentech group and others in the optimisation of other LRRK2 inhibitor chemotypes [100,129,130]. A crystallographic surrogate-based approach was then used to assist with further fine-tuning of kinase selectivity. This utilised mammalian STE-20-like protein kinase 3 (MST3), since this was a major off-target hit for the series of interest, and its ATP binding site has 73% similarity to that of LRRK2. In a ligand-bound crystal structure of 39 in complex with MST3, the nitrile group formed a charge-assisted hydrogen bond with the conserved Lys38 (Lys1906 in LRRK2), which maintained its salt bridge with Glu55 (Glu1920 in LRRK2) within the αC-helix. Despite this interaction being conserved across the kinome, it appeared to be contributing to compound selectivity. This was consistent from previous observations with other kinases that it is possible to modulate selectivity by varying steric or electrostatic interactions in this area. Interaction with Arg1957 was also targeted by the Pfizer group as a means of obtaining selectivity for LRRK2. Although this residue is probably solvent exposed and flexible, there is only a 3% incidence of arginine at this position in the kinome. The Pfizer authors conducted molecular dynamics simulations, and concluded that Arg1957 may be involved in interactions with residues up to 68% of the time, thereby restricting its motion and orienting it in a position for interaction with the morpholine moiety of 39. Additionally, it was hypothesised that Arg1957 could interact with other LRRK2 domains, which would also restrict its motion. X-Ray crystallography suggested that the nitrile was responsible for enhancing both potency and selectivity via a charge-assisted hydrogen bond with Lys38. Attempts to optimise the 3-cyanophenyl with further substitution or heterocyclic replacements failed to improve potency. Further work was then undertaken exploring substitution and manipulation of the morpholine ring in an attempt to improve off-target activity towards MST

65

Development of LRRK2 Inhibitors for PD

kinases, for example, introduction of an oxadiazole substitution (39b) improved selectivity towards MST2 and MST4 but at the cost of a reduction in LRRK2 potency (LRRK2 IC50 ¼ 8 nM). Compound 39 was then assessed to have good brain penetration with good permeability with no P-gp efflux. In vivo PK studies demonstrated good oral bioavailability in rat (F ¼ 41%) but not in dog (F ¼ 0.6%) or nonhuman primate (F ¼ 3.5%) at a dose of 5 mg/kg. Compound 39 demonstrated an in vivo brain-free IC50 of 8 nM. Me

O

H N

N

H N

N

O N

Me

NH

Cl

O JH-II-127 (40)

Another pyrrolopyrimidine, JH-II-127 (40), was reported in 2015 [131]. Using compound 10 as their starting point, a series of 5,6-ring closed analogues was prepared. Their hypothesis was that an additional hydrogen bond donor could be made with M1949, in addition to an improved hydrophobic binding afforded by a fused bicyclic. Compound 40 was reported with an LRRK2 WT IC50 of 6.5 nM and an LRRK2 G2019S IC50 of 2.2 nM. A molecular docking study based on the crystal structure of ROCO kinase revealed the expected binding between the hinge region and the pyrrolopyrimidine, and an additional halogen interaction is postulated between M1947 and the 5-Cl. Compound 40 dose dependently inhibited LRRK2 Ser935 and Ser910 phosphorylation in HEK293 cells stably transferred with LRRK2 with substantial inhibition achieved at 0.3 μM. The effect of 40 was studied in human lymphoblastoid cells derived from a control and from a PD patient homozygous for the LRRK2 G2019S mutation. A similar effect was observed on inhibition of LRRK2 pSer935 and pSer910 with an apparent potency greater than that observed in HEK293 cells. Similar effects were also observed on inhibition of LRRK2 pSer935 in mouse Swiss 3T3 cells. Good oral bioavailability (F ¼ 116%) was observed after 10 mg/kg p.o. in mouse with a short half-life of 0.66 h and good plasma exposure. A total brain/plasma ratio of 0.45 was measured after 2 mg/kg i.v. dosing. At 100 mg and 30 mg/kg i.p. doses, full pSer935 inhibition was observed in kidney, spleen and brain. No effects on total LRRK2 expression were

66

K.V. Christensen et al.

observed. Kinase selectivity was assessed against a panel of 138 kinases (Dundee profiling at a concentration of 1 μM). Only two other kinases were inhibited at a more than 90% level, these being SmMLCK and CHK2. A follow-up IC50 determination gave an IC50 of 81.3 nM for SmMLCK and an IC50 of 27.6 nM for CHK2. In the DiscoveRx KINOMEscan, excellent selectivity was observed against 451 kinases with significant binding observed only to TTK and RPS6KA4.

4.7 Thiophenes A group from Merck reported in 2016 a thiophene-based series of LRRK2 inhibitors [130]. The novel thiophene-based compound 41 was identified from a screen of the Merck sample collection, despite having been assessed in 274 HTS campaigns with only limited activity in any of these screens. The LRRK2 Ki was reported as 377 nM against wild type and 234 nM against the G2019S enzyme. O

O

S Me S

Me Me

Me Me

N

S N NH

N NH 41

O

S

42

S nPr S

Me

N NH

43

Initial SAR studies identified the cyclopentyl analogue 42 (LRRK2 WT Ki ¼ 389 nM, LRRK2 G2019S Ki ¼ 94 nM) which was selective for LRRK2 when assessed at a concentration of 10 μM against a panel of 192 kinases. The high cLogP of 5.5 of the cyclopentyl analogue resulted in a not unexpected high in vivo clearance in rat (Cl ¼ 96 mL/min/kg) and poor cerebrospinal fluid exposure of 10 nM after dosing at 5 mg/kg i.p., ascribed to high plasma protein (99.3%) rather than P-gp efflux. Initial SAR indicated that the oxidation state of the sulphur was important for activity with the sulphone much less active. Alkyl substitutions were well tolerated, whereas aromatic substituents were less well tolerated and the geminal methyl groups were not important for activity. The sulphur could be replaced with oxygen or nitrogen but not carbon. Interestingly the NH(cyclopentyl) analogue showed a larger difference in inhibitory values between LRRK2 WT and G2019S variants. Methylation of the pyrazole or replacement with 2-thiazole was not tolerated. The potency afforded by the pyrazole was hypothesised to be due to formation of two hydrogen

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bonds to hinge amino acids Glu-1948 and Ala-1950. Introduction of a nitrogen atom into the cyclohexanone ring of the series to give a lactam series improved cLogP with a reduction of 1 unit, and increased CNS MPO scores by approx. 0.5 for matched pairs. These effects could be seen in a reduction of in vivo intrinsic clearances and higher observed oral bioavailability values. For example, the compound 43 (LRRK2 wild-type Ki ¼ 84 nM, LRRK2 G2019S Ki ¼ 39 nM) showed 98% oral bioavailability after 10 mg/kg p.o. dosing and an i.v. clearance of 11.9 mL/min/kg compared to an oral bioavailability of 33% and a clearance of 56.0 mL/min/kg. The kinase TrkA was used as a surrogate for LRRK2 in crystallography studies as compounds from this series showed weak activity in a TrkA Caliper assay and an LRRK2 homology model was created based on the related TAK1 crystal structure. The gatekeeper+2 residue in Trk A (Tyr-591) was observed to be close to the SMe for a compound in this series, where the corresponding G+2 residue is Leu1949. This position in other kinases has a larger residue and this could explain why larger substituents such as the cyclopentyl can be accommodated and impart selectivity for LRRK2 in relation to Aurora A and B, both of which have a tyrosine at the G+2 residue.

4.8 Triazolopyridazines Triazolopyridazines have been identified by several teams and this scaffold has delivered molecules with high kinase selectivity, but ultimately this chemotype has proved challenging to optimise towards compounds with real value as in vivo tools. A group from Elan identified a series of triazolopyridazines [113] from an HTS campaign using an HTRF assay which measured inhibition of phosphorylation of LRRKtide. N N

N

N N N

S

CF3

Me N

S 44

45

N

N OMe

S O

N

N

N

S

CF3

S 46

Interestingly, compound 44 was confirmed subsequently in a TR-FRET assay as showing enhanced inhibitory activity against G2019S vs wild-type LRRK2 by almost a factor of 5, with IC50s of 50 and 242 nM, respectively. Kinase selectivity at 1 μM against 40 other kinases showed no significant off-target activity. The drawbacks to this starting point were the poor aqueous solubility (9 μM) and low permeability (21 nM/s). Some of these

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properties were improved with the analogous hit 45 which showed improved permeability (280 nM/s) and no P-gp liability. The C-3 phenyl of 44 was replaceable with 2-thienyl group with no loss of potency in compound 46 and an exploration of removing the N from the central heterocyclic core was only successful in compound 47 which showed an LRRK2 WT IC50 of 101 nM and an LRRK2 G2019S IC50 of 47 nM.

N

N

S

N N

CF3

N N

Me

N

N

O

S O

S

S

47

3

N

6

N

S

Cl

S N

48

49

The 6-alkylthio analogue 48 showed remarkable selectivity for G2019S (7.6:1) with an LRRK2 WT IC50 of 76 nM and an LRRK2 G2019 IC50 of 10 nM, although this compound showed loss of kinase selectivity and was not pursued further. A homology model of LRRK2 was developed based on MLK1. However, the P-loop was remodelled using homologous structures of kinases with ATP analogues. The DYG sequence was built to resemble PKC-1. Docking of compound 44 showed that the H-bond in the triazole ring accepts an H-bond from hinge residue A1950. Attempts to replace the 2-thienyl substituent of 46 indicated that the five-membered heterocycles were more potent with LRRK2 G2019S IC50s in the range 35–13 nM, with the 1H-pyrazol-5-yl the most potent replacement. The six-membered rings were less potent, displaying LRRK2 IC50s in the range 1023–1045 nM, and maintained the selectivity in favour of G2019S (ratios 5.7–3.2). Cycloalkyl substituents in place of the 2-thienyl were disfavoured. Optimisation of the arylthio substitution showed that single-digit nanomolar inhibition was achievable where C-3 was 5-thiazolyl and C-6 was 3-halophenyl thio group. The chloro analogue 49 displayed IC50s of 31 and 8 nM against WT and G2019S, respectively. The cellular activity based on inhibition of pSer935 gave an IC50 of 1.397 μM for 49. The 1000-fold shift in the cellular activity was ascribed to the poor permeability of these compounds. Attempts to replace the sulphur with O, NH, CO or C(CN) were unsuccessful and this was rationalised on the basis that the wider dihedral

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angles were less optimal than the thioether which had a dihedral angle around 90 degrees. Ultimately, this series was abandoned due to the metabolic liability of the sulphur. Incubation of the start point 44 in NADPH-supplemented rat liver microsomes at 30°C showed complete consumption of the parent molecule and formation of the weakly active sulphone and sulphoxide. This oxidation was less pronounced in human liver microsomes. A group from Pfizer reported in 2014 the same chemotype from an HTS campaign [132]. Optimisation of the C-3 position of the triazolopyridazine scaffold identified the pyrazole analogue 50 as a promising compound with high kinome selectivity (0/39 hits observed at 1 μM) in internal kinase profiling and in the DiscoveRx KINOMEscan® panel at 1 μM, high selectivity was observed. Using TyK2 as a crystallographic surrogate for LRRK2 the C1-N atom of the triazole binds to the hinge with the methylpyrazole occupying the ribose pocket pointing towards solvent. In vivo profiling of 50 in the rat at 10 mg/kg showed relatively poor brain penetration with a free brain-to-plasma ratio of 0.14 attributed to efflux by the breast cancer-related transporter. Following an in vivo study of this compound in mice at doses up to 300 mg/kg/day for 14 days, no effect was observed either on brain or kidney pSer935 levels. Dosing the compound in G2019S transgenic mice at doses up to 300 mg/kg (free brain concentration ¼ 1809 nM) did induce modest effects, with a 20% reduction in brain pSer935 and pSer1292 phosphorylation. Kidney pSer935 levels were reduced by 20% and pSer1292 levels by 40%. OMe N N

N

N

S OMe

N N Me 50

The same chemotype was picked up by the Southern Research Institute [133] using a high-throughput screen tracking ADP formation assay with ADP-Hunter technology and their optimisation work resulted in the identification of the tool compounds SRI-29132 (51) and SRI-29451 (52).

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N

4N

N

S

N

N

N N N

S Me 51

O

N

O

5N

S Me

S 52

Biochemical IC50s reported were 312 nM for 51 and 27 nM for 52 in a G2019S LRRK2 trans-peptide phosphorylation assay and 570 nM for 51 and 2 nM for 52 in a cis-autophosphorylation assay using Alphascreen® technology utilising an antibody that recognised LRRK2 Thr(P)-1503. An IC50 of 2 μM for 51 was measured for inhibition of Ser935 phosphorylation in primary macrophages incubated with the compound for 72 h and then stimulated with lipopolysaccharide (LPS) for 6 h. Compound 51 inhibited TNF release in LPS-stimulated WT and G2019S LRRK2 primary macrophage cultures at a concentration of 5 μM. Confirmation that this was an LRRK2-mediated effect was confirmed by a study with compound 51. In this case, no inhibition was observed in macrophage cultures from KO-LRRK2 treated with LPS. Interestingly, LRRK2-IN-1 (4) was effective in reducing TNF release at nanomolar concentrations, suggesting that this compound has off-target effects that complicate interpretation of its effects. Excellent kinase selectivity was observed for 51 in a screen that involved over 600 kinases at 1 and 10 μM. Only at 10 μM was significant inhibition of glycogen synthase kinase 3, aurora kinase A and phosphatidylinositol 3 kinase γ observed. The authors proposed a homology docking model of SRI-29132 (51) based on an amoeba-derived LRRK2 crystal structure. The polarisable sulphur atom of the thioether is thought to interact with the positively charged γ-amine of Lys 1906 and N-4, while N-5 binds to amide NH of Glu-1948, Ala-1950 in the hinge and the morpholine ring binds in a hydrophobic pocket formed in the P-loop. Compounds 51 and 52 demonstrated both moderate permeability in the MDR1-MDCK assay and good permeability in the parallel artificial membrane permeation assay. Both compounds showed rapid oxidation in microsomes in the presence of NADPH and this high clearance meant that that inhibition of endogenous Ser935 phosphorylation in mouse brain was only significant from 5 min post i.v. injection.

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5. CONCLUSION In summary, despite the absence of LRRK2 kinase domain X-ray crystal structures, extensive in silico modelling based on kinases with similar ATP binding sites, and off-target kinase hits of particular LRRK2 inhibitor chemotypes, has provided useful guidance for inhibitor optimisation. This has allowed a consensus to be reached on the ligand–protein interactions in the kinase domain which gives rise to LRRK2 potency and/or kinase selectivity, and thus selective LRRK2 compounds have become available which can be used to interrogate the biology of LRRK2. Given that the LRRK2 kinase domain inhibitors reported thus far have given rise to kidney and lung abnormalities in in vivo toxicological studies, more targeted approaches on other domains of LRRK2 may be necessary to give rise to improved therapeutic benefits. To fully explore these potential targets, high-resolution structural insights into LRRK2, crystallographically or otherwise, are still required. Such studies have been further supported by efforts using other crystallographic surrogates with increased similarity to LRRK2. Most studies have been undertaken with Roco proteins from lower organisms [95,96]. Gilsbach and coworkers studied the amoeba-derived Roco4 kinase domain from Dictyostelium discoideum. These authors solved the X-ray structure of Roco4 kinase wild-type, as well as the PD-related mutants G1179S and L1180T (G2019S and I2020T) in LRRK2, and the structure of Roco4 in complex with the LRRK2 inhibitor H1152. Two mutations in the ATP binding site of Roco4 enabled ligand-bound X-ray crystal structures of selective LRRK2 inhibitors including LRRK2-IN-1 to be obtained. These crystal structures, along with ligand-observed NMR experiments, further supported the binding modes of compounds previously hypothesised in docking models. This showed that suitable surrogates for human LRRK2 can provide valuable insights into the optimisation of LRRK2 inhibitors, and it is hoped that similar approaches will provide further insight into drugging this attractive therapeutic target. It appears that the field has made large strides in the last 5 years in developing selective and brain-penetrant LRRK2 inhibitors which have progressed to advanced preclinical safety studies. The three chemotypes which have so far been progressed furthest in enlightening the field on LRRK2 biology are the diaminopyrimidines, as exemplified by GNE-7915 (10), pyrrolopyrimidines such as PF-06447475 (39) and the indazole MLi-2 (29) (Fig. 4). All are selective for LRRK2, brain penetrant and have been

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A

D

D

D

A N

H N

O

N

A

H N

CF3 N

H N N

NH

O

N Me O

F

CN

N

N

Me N

N

O O GNE-7915 (10)

PF-06447475 (39)

O Me

N MLi-2 (29)

Fig. 4 Summary of LRRK2 inhibitors progressed furthest in preclinical studies. Key donor (D)/acceptor (A) kinase hinge binding motifs indicated.

used in chronic safety experiments in rodent and monkeys to determine both the effect of peripheral and centrally acting LRRK2 inhibition. Experience so far has shown that numerous diverse starting points for lead optimisation are possible for LRRK2. Although LRRK2 has so far not been crystallised, the use of homology modelling and crystallographic surrogates have allowed the optimisation of chemical structures such that compounds of high selectivity with good brain penetration and appropriate pharmacokinetic properties are now available for understanding the biology of LRRK2. Although direct inhibition of the ATP binding pocket has been shown to be a relatively successful approach in identifying LRRK2 inhibitors, other approaches for modulating LRRK2 are potentially possible [134]. It would seem likely that, in the near future, compounds will progress further to clinical trials. One of the major challenges remaining to be overcome will be the level of target engagement required for therapeutic effect which, in the absence of any reliable preclinical disease model of LRRK2 dysfunction, is unknown, leading to uncertainty on the therapeutic index. Advanced preclinical safety studies have revealed concerns that chronic full LRRK2 inhibition may have effects on lung and kidney. The lung in particular is difficult to monitor in a clinical situation and so caution will be needed in further chronic studies to explore the effect of LRRK2 inhibition. An impedance, however, to answering the question on whether LRRK2 inhibitors are useful in the management and treatment of PD is just as likely to be the availability of tools to assist in clinical trials. Confirmation of central target engagement and dose estimation will be challenging as a validated PET ligand has not been disclosed for LRRK2 and instead indirect methods such as estimating central occupancy from CSF measurements of compound concentration or mechanistic effect will probably be needed. Biomarkers of

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LRRK2 activity will also be needed to be able to confirm target engagement and, in this respect, the identification of proteins from the Rab GTPase family as the endogenous substrates for LRRK2 will allow the development of new assays. Such studies will give more confidence on the level of target engagement and the mechanistic effect required for advancing the first clinical candidate into human clinical trials. In addition, further understanding is required on which patient populations would be best targeted by an LRRK2 inhibitor. Although proof of concept is most likely to be obtained in G2019S-associated PD, it still remains to be proven which other patient populations with other LRRK2 mutations, or indeed idiopathic PD, will gain benefit from an LRRK2 inhibitor. The first compound to be advanced into clinical trials is now eagerly awaited, to ascertain whether LRRK2 inhibition represents a breakthrough therapy in the management and treatment of PD.

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