Dual Leucine Zipper Kinase Inhibitors for the Treatment of

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Cite This: J. Med. Chem. 2018, 61, 8078−8087

Dual Leucine Zipper Kinase Inhibitors for the Treatment of Neurodegeneration Miniperspective Michael Siu,*,† Arundhati Sengupta Ghosh,† and Joseph W. Lewcock*,‡ †

Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States Denali Therapeutics, 151 Oyster Point Boulevard, South San Francisco, California 94080, United States

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ABSTRACT: Dual leucine zipper kinase (DLK, MAP3K12) is an essential driver of the neuronal stress response that regulates neurodegeneration in models of acute neuronal injury and chronic neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and ALS. In this review, we provide an overview of DLK signaling mechanisms and describe selected small molecules that have been utilized to inhibit DLK kinase activity in vivo. These compounds represent valuable tools for understanding the role of DLK signaling and evaluating the potential for DLK inhibition as a therapeutic strategy to prevent neuronal degeneration.

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INTRODUCTION Neurodegeneration resulting from acute brain injury or chronic neurodegenerative disease represents a significant unmet medical need with no effective treatments that are able to impact the resulting functional deficits. A major challenge in developing therapeutics for these indications lies in the diverse mechanisms that are thought to underlie neuronal loss in these settings.1,2 Although human genetic studies conducted over the past decade have identified a number of risk factors, in many cases the exact mechanisms underlying degeneration remain poorly defined.3 As a result of this heterogeneity, multiple therapeutic strategies have been proposed based on the hypothesis that core pathways exist that regulate degeneration resulting from a neuronal injury or disease.4,5 The neuronally enriched kinase DLK (dual leucine zipper kinase, also known as mitogen activated protein 3 kinase 12, MAP3K12) may represent a druggable node within such a conserved pathway.6 Pharmacological inhibition or genetic deletion of DLK is sufficient to attenuate the neuronal injury response and can result in potent protection of neurons from degeneration in response to a range of neuronal insults,7−12 suggesting that inhibition of DLK may represent an attractive therapeutic strategy. DLK is a member of the mixed lineage kinase (MLK) family that contains an N-terminal kinase domain followed by two © 2018 American Chemical Society

leucine zipper domains and a glycine/serine/proline rich Cterminal domain whose function is not well understood (Figure 1A).13,14 Palmitoylation of DLK is required for proper function in neurons, suggesting that it is likely associated with intracellular vesicles.15 Activation of DLK in neurons induces stress-specific JNK signaling via MKK4/7 and increases PERK signaling through an unknown mechanism (Figure 1B).8,16,17 The induction of these pathways generates a broad transcriptional injury response in neurons through the regulation of transcription factors including c-Jun and ATF4,7,8,18 which leads to apoptosis and axon degeneration in multiple neuron types including retinal ganglion cells and motor neurons.7,9,10 Notably, the physiological JNK1 signaling present in neurons does not appear to be affected by DLK, suggesting that this pathway specifically regulates JNK2/3-dependent stress signaling.8,11 Levels of DLK protein within neurons are tightly controlled via ubiquitination, and JNK activation in neurons results in hyperphosphorylation and stabilization of DLK, creating a feed forward loop to both amplify and spatially propagate this signaling event.19 Recent studies have identified additional factors that either phosphorylate DLK or contribute to DLK activation following insult.16,20−22 However, none of Received: March 8, 2018 Published: June 4, 2018 8078

DOI: 10.1021/acs.jmedchem.8b00370 J. Med. Chem. 2018, 61, 8078−8087

Journal of Medicinal Chemistry

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The function of DLK is highly conserved through evolution and was first identified using genetic screens in invertebrate species examining neuromuscular junction formation during development.23,24 Soon after, screens to identify factors regulating the response to axonal injury in C. elegans identified the worm homolog of DLK (DLK-1) as a central regulator of this process.25 Interestingly, contrary to what is observed in the mammalian central nervous system, loss of DLK-1 in C. elegans attenuated axonal regeneration rather than neuronal degeneration following injury. Similar DLK dependent axon regeneration phenotypes have been identified in other systems including peripheral neurons in vertebrates,14−18 and the regulation of these two disparate outcomes by the same kinase has resulted in some debate regarding whether DLK inhibition following neuronal injury would be desirable.26 However, the fundamental role DLK is highly conserved across experimental systems where it acts to convey neuronal injury signals to the nucleus,14,18,19 with the outcome of this response being determined by the experimental setting examined. In the adult mammalian central nervous system, which is not a permissive context for regeneration, activation of DLKdependent neuronal stress signaling appears to uniformly result in neuronal cell death and axon degeneration as is the case in chronic neurodegenerative disease.7,9,10 Therefore, pharmacological inhibition of DLK would be expected to be therapeutically beneficial for these indications.

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DLK AS A DRUG TARGET Targeting the JNK pathway downstream of DLK has long been of therapeutic interest based on the essential role of this pathway in neuronal degeneration.27 Of the three JNK genes present in humans, JNK3 has been of particular interest based on its functional role in neuronal degeneration and selective expression in the CNS.28−32 However, a number of challenges have impeded the successful development of CNS-penetrant JNK kinase inhibitors including the high level of sequence homology and overlapping function of different JNK isoforms.33 Cephalon pioneered an alternative approach through targeting upstream kinases rather than JNK directly. This led to the development of pan-MLK inhibitors such as CEP-1347 (1) and CEP-5214 (2) (Figure 2), which were based on the natural product K-252a.34−38 Pan-MLK inhibitor 1 demonstrated potent neuroprotection of neurons from degeneration in vitro as well as in multiple animal models including the MPTP model of Parkinson’s disease and the R6/2 model of Huntington’s disease.39−42 Compound 1 displayed relatively poor kinase selectivity and inhibited multiple kinases including GCK and CDC42 in addition to all members of the MLK family tested.37 The DLK IC50 for compound 1 was found to be 0.114 μM,43 which was less potent than inhibition of other MLKs including MLK3. These and other results led to the hypothesis that MLK3 inhibition was responsible for a substantial portion of the neuroprotective effects.44 However, a range of subsequent studies suggest that inhibition of DLK was likely to be a major contributor to these phenotypes.8,12,45 On the basis of these encouraging preclinical results, this molecule (1) was progressed to clinical trials for Parkinson’s disease but failed to demonstrate benefit.46 A retrospective analysis suggests that this compound was unlikely to reach sufficient concentration in the brain to achieve inhibition of the target MLKs, including DLK, at the doses used in the clinic.38 The discovery of DLK as a regulator of axon degeneration that occurred following the completion of this clinical trial12 led

Figure 1. Schematic of DLK domain structure and signaling pathway. (A) DLK domain structure. DLK comprises an N-terminal serine/ threonine kinase domain, two leucine zipper domains, and a glycine/ proline rich C-terminal domain. DLK is palmitoylated at cysteine 127, a modification that is required for function. DLK can be phosphorylated on serines 272 and 302 in the activation loop as well as additional sites outside the activation loop including threonine 43 and serine 533. Phosphorylation of the sites outside the activation loop regulates DLK stability. (B) Overview of DLK signaling in neurons. A variety of cellular stresses result in DLK activation, which in turn activates the JNK signaling pathway resulting in the phosphorylation of a number of substrates including the transcription factor c-Jun. This requires the scaffolding protein JIP3. DLK/JNK pathway activation also induces PERK signaling, resulting in an upregulation of the transcription factor ATF4. These transcription factors induce a transcriptional stress response within neurons that includes PUMA, Bcl-2, DDIT3 (CHOP), and many additional genes. DLK protein levels are tightly controlled by the opposing functions of the ubiquitin ligase Phr1 and the deubiquitinating enzyme USP9x. DLK can interact with the closely related kinase LZK, which contributes to downstream signaling events. The MAP4 kinases MAP4K4, Mink1, and TNIK as well as Akt also contribute to DLK activation.

these factors phosphorylate the activation loop, and the observation that dimerization of DLK via the leucine zipper domains can result in autophosphorylation of these sites in the absence of upstream factors13 suggests that DLK dimerization may be necessary and sufficient to induce activation and initiate downstream signaling events. 8079



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Figure 2. Cephalon mixed-lineage kinase inhibitors.

Table 1. Overview of in Vitro and in Vivo Models in Which Loss of DLK Signaling Is Neuroprotective mechanism of degeneration

disease modeled

effects of DLK loss

Loss of trophic support/axonal lesion Excitotoxicity Loss of trophic support

Developmental neurodegeneration/ peripheral neuropathy Stroke/seizure ALS

Reduced axon degeneration and neuronal apoptosis

Microtubule toxin

Glaucoma

Addition of ApoE isoforms Axonal die-back

Alzheimer’s

Kainic acid Traumatic brain injury SOD1

Excitotoxicity Combination of mechanisms SOD1 mediated toxicity

Stroke/seizure Traumatic brain Injury

PS2/APP

Abeta

Alzheimer’s

Tau

Phospho-Tau

Alzheimer’s/FTD/PSP

MPTP

Mitochondrial toxin

Parkinson’s

model In vitro DRG In vitro NMDA In vitro motor neurons In vitro retinal ganglion cells iPSC derived cortical neurons Nerve crush

Acute nerve injury

ALS

readouts following inhibitor treatment

refs 8, 12

Reduction in p-c-Jun Not tested

p-c-Jun and neuroprotection p-c-Jun Neuroprotection

Reduction in p-c-Jun, gene expression, neuroprotection Reduction in Abeta production

p-c-Jun and neuroprotection Not tested

9, 45

Reduction in p-c-Jun, gene expression, long lasting protection of RGCs and axons Reduction in p-c-Jun, neuroprotection Reduction in p-c-Jun, neuroprotection

PK/PD (p-c-Jun), neuroprotection Not tested Not tested

7, 9

Reduction in p-c-Jun, protection of motor neurons, survival Reduction in p-c-Jun, synapse protection, improved cognition Maintenance of hippocampal, cortical volume (MRI) Reduction in p-c-Jun, neuroprotection

PK/PD (p-c-Jun), NMJ preservation PK/PD (p-c-Jun)

10 10

PK/PD (p-c-Jun)

10

PK/PD (p-c-Jun), neuroprotection

74

11 9

48

11 47

ities. This suggests that loss of DLK signaling following inhibitor treatment would be well tolerated. More recently, knockdown of DLK via siRNA was demonstrated to be protective in a model of traumatic brain injury (TBI), suggesting that DLK inhibition may provide therapeutic benefit.47 These observations were extended by the Genentech team to models of Alzheimer’s and ALS.10 In this study, genetic deletion or pharmacological inhibition of DLK demonstrated benefit in mouse models of Alzheimer’s based on amyloid precursor protein (APP) and Tau, as well as SOD1and TDP-43-based models of ALS. Loss of DLK signaling in these models corresponded not only with the protection of neurons but also a functional benefit, indicating the neurons that were preserved in dlk null animals maintained synaptic connectivity. DLK pathway activation was also observed in brains of patients with Alzheimer’s and ALS, implying that these results may be translatable to the clinic. Importantly, the functional rescue in models of Alzheimer’s disease could be achieved even when DLK signaling was attenuated well after the onset of disease progression, a paradigm that more accurately reflects when patients would begin treatment. Taken together, these results suggest that DLK inhibition

to a new wave of preclinical studies examining the potential utility of targeting this kinase more specifically (Table 1). This approach would have specific advantages over pan-MLK or JNK inhibition based approaches as targeting DLK would impact JNK mediated injury signaling in neurons while not affecting JNK pathway activity in other cell types where it has distinct functions.6 Through the use of genetically engineered mice in which the dlk gene had been deleted, DLK was shown to be required for degeneration of neurons following axotomy, vincristine treatment, or trophic factor withdrawal in vitro.8,12 Similar results were obtained in vivo using an optic nerve crush model by teams at Johns Hopkins University and Genentech, where neurons lacking DLK expression were protected from neurodegeneration.7,9 Another study published shortly thereafter demonstrated that loss of DLK signaling protected neurons from excitotoxicity induced degeneration in vitro and in vivo, indicating that DLK function is not limited to axonal injury and is instead required for the response to a range of neuronal insults.11 As part of this work, mice in which the dlk gene had been deleted in all tissues were characterized 3 months after dlk deletion and were found to have no histological, electrophysiological, or gross behavioral abnormal8080



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and attributed the beneficial effect to DLK inhibition.9 Despite the promiscuity of tozasertib,51 the hypothesis of DLK inhibition is rationalized by the results of separate cell experiments with siRNA knockdown of DLK and adenovirustransduced overexpression of DLK in which corresponding shifts in the ability of tozasertib to protect RGCs were observed.9 Furthermore, tozasertib (delivered via ocular injection with intravitreal microspheres) promoted RGC survival in a rat optic nerve crush model of axonal injury and a rat diode laser-induced IOP model of glaucoma. In a similar fashion, the Johns Hopkins group demonstrated that the multiple receptor tyrosine kinase inhibitor sunitinib (5, DLK Ki = 0.0063 μM, Figure 4) improved retinal ganglion cell survival upon ocular delivery in the same models.52 As accessibility of DLK in standard kinase panels such as SelectScreen kinase broad assay panel has been historically limited,53 other existing kinase inhibitors may have potent DLK activity. Although marketed and clinical kinase inhibitors are instructive in revealing potential starting points for medicinal chemistry efforts, significant improvements in potency, kinase selectivity, and CNS penetration are necessary. Toward this effort, novel targeted DLK inhibitors have been disclosed in both the patent54−60 and primary literature from Genentech. Additionally, inhibitors from Faming Zhuanli Shenqing are claimed in a series of patent applications in China.60−67 This Miniperspective will primarily focus on selective DLK inhibitors that have contributed to our understanding of the role of DLK in stress-induced JNK/c-Jun signaling in vivo. The physicochemical properties of approved kinase inhibitors are, as a whole, not compatible with efficient CNSpenetration. As described in a recent brain cancer kinase inhibitor review,68 marketed kinase inhibitors are generally larger (median MW = 483), more lipophilic (median cLogP = 4.2, median cLogD7.4 = 3.6), and composed of more polar atoms (median tPSA = 91) and hydrogen bond donors (median HBD = 2) as compared to CNS drugs (median values, cLogP = 2.8, cLogD7.4 = 1.7, tPSA = 45, HBD = 1, MW = 305).69 Accordingly, a disciplined approach to maintaining CNS drug-like properties for brain penetration is necessary when targeting a kinase in the CNS, a difficult yet achievable task as exemplified by recent progress in CNS-penetrant ALK,70 LRRK2,71 and PI3K72 inhibitors. A review outlining the design considerations and physicochemical properties of kinase inhibitors for neurodegenerative diseases has recently been published that provides additional detail on this topic.73 In 2014, Genentech reported the development of a highthroughput screening (HTS) hit 6 to 7 (GNE-3511, Figure 5), a potent brain-penetrant inhibitor with selectivity over

may be broadly beneficial in indications ranging from glaucoma to ALS. Recent work has demonstrated that inhibition of the closely related kinase leucine zipper kinase (LZK) in addition to DLK may result in more robust neuroprotection than DLK alone.45 Interestingly, genetic deletion or siRNA mediated knockdown of lzk alone does not substantially impact neurodegeneration and a combination of both kinases is required for this benefit. In a second study, it was shown that LZK may also play an important role in mediating the response to CNS injury in astrocytes, which are support cells that are thought to influence neuronal health and function.49 This work suggests that broad LZK inhibition may be detrimental in the context of acute spinal cord injury. Future studies will be required to determine the net benefit of compounds that inhibit LZK in addition to DLK in models of chronic neurodegenerative disease.

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SMALL MOLECULE INHIBITORS OF DLK As the mechanistic understanding of DLK function continues to strengthen, progress in the identification of inhibitors to pharmacologically interrogate the role and function of DLK has ensued. Cephalon, with experience on developing indolocarbazole MLK/DLK inhibitors, has further optimized their molecules by replacing the indole subscaffold with a tetrahydroindazole leading to hexahydroindazolo[5,4-a]pyrrolo[3,4-c]carbazole-4-ones as exemplified by 3 (Figure 3) with potent activity against DLK.50 At this time, detailed optimization of SAR and in vivo studies has not been disclosed for this series of DLK inhibitors.

Figure 3. Second generation Cephalon DLK inhibitor.

In 2013, Welsbie et al. reported the DLK activity of several marketed and clinical kinase inhibitors to highlight the feasibility of inhibiting DLK.43 The Johns Hopkins group showed that the aurora kinase inhibitor tozasertib (4, DLK Ki = 0.0358 μM, Figure 4) promoted retinal ganglion cell survival

Figure 4. Kinase inhibitors utilized by Johns Hopkins group to examine DLK inhibition in vivo. 8081



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Figure 5. Genentech DLK inhibitors showing the evolution of high throughput screening hit 6 to DLK inhibitor 7, scaffold hop to 8 to improve pharmacokinetic properties, and further hinge binder/substituent modifications to 9 and 10.

Figure 6. X-ray structures of DLK kinase domain bound with 7 (blue, PDB code 5CEO) and 8 (orange, PDB code 5CEP). (a) DLK binding mode showing the P-loop (upper right) with an overlay of 7 and 8. The slightly larger difluoropyrrolidine of 7 engages the P-loop to create a slightly outward trajectory (ivory). (b) A cut-away top−down view of the ligands with the DLK hinge at upper left shows the precise cyanopyridine alignment and conserved hinge interactions of both ligands. The five-membered pyrazole versus six-membered pyridine core ring provides altered vectors, though the outer substituents converge into shared spatial position. The figure was reproduced from Patel et al.,75 2015, with permission from the Journal of Medicinal Chemistry. Copyright 2015 American Chemical Society.

Å2) and one hydrogen bond donor led to DLK inhibitor 7. This potent, oral brain-penetrant inhibitor of DLK has selectivity exceeding kinase inhibitors used in prior studies and displayed potent protection of primary neurons in an in vitro axon degeneration assay. More importantly, oral dosing of inhibitor 7 reduced phosphorylation of the downstream transcription factor c-Jun, a marker of neuronal injury and DLK activation, in both the optic nerve crush and MPTP mouse models of neurodegeneration. In a subsequent publication, Genentech reported a shapebased scaffold hopping approach to arrive at a pyrazole scaffold with improved physiochemical properties.75 Despite a reduc-

homologous mixed lineage kinases (MLK1/2/3) and kinases in the JNK/c-Jun signaling pathway (MKK4/7, JNK2/3).74 The starting point for optimization (HTS hit) was specifically chosen for its low topological polar surface area (tPSA) and number of hydrogen bond donors (HBD). Important to the optimization of the HTS hit was the discovery of the most efficient use of the polar atoms in the scaffold given the limitations to brain penetration. Specifically, modification of the hinge binder (2-aminothiazole to 2-amino-4-cyanopyridine), core (pyrimidine to pyridine), and core substituents (methyl to 3,3-difluoropyrrolidine and N-benzoyl-2-piperidine to Noxetanyl-4-piperidine) maintained reasonable tPSA (71 → 77 8082



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Table 2. Data Summary of Genentech DLK Inhibitors 774 log D/tPSA DLK Ki (μM) pJNK IC50 (μM) (10% FBS) DRG EC50 (μM) MDR1 efflux ratio rat PK CLp (mL min‑1 kg‑1) CLu (mL min‑1 kg‑1) Vdss (L kg‑1) t1/2 (h) F (%) Kpuu,brain (AUC) mouse PK CLp (mL min−1 kg−1) CLu (mL min−1 kg−1) Vdss (L kg−1) t1/2 (h) F (%) Kpuu,brain (at 6 h) LZK Kd (μM) MLK1 IC50 (μM) MLK2 IC50 (μM) MLK3 IC50 (μM) JNK1 IC50 (μM) JNK2 IC50 (μM) JNK3 IC50 (μM) kinase selectivity53 (kinase >80% inh)

2

875

976

2

2

1076

4/77 Å 10 >10 >10 1.04 5 2.1 0/58 at 1 μM (24 × DLK Ki)

9 113 2.7 3.7 93 0.81 0.120 3.5 5.15 >10 >10 >10 >10 0/220 at 1 μM (250 × DLK Ki)

25 186 3.6 2.0 60 0.54 0.460 5.92 7.88 >10 >10 >10 >10 0/220 at 1 μM (333 × DLK Ki)

56 4667 2.5 0.6 45 0.24 0.0013 0.068 0.767 0.602 0.129 0.514 0.364 7/298 at 0.1 μM (>200 × DLK Ki)

lipophilicity without the addition of polar atoms (tPSA), the exo-3-azabicyclo[3.1.0]hexane group was introduced in order to maintain CNS pharmacokinetic properties. This modification along the hinge toward the solvent exposed region in DLK significantly improved potency and kinase selectivity within the pyrazole scaffold leading to the identification of 9 (GNE-8505) and 10 (Figure 5). The University of Texas has recently disclosed similar imidazole derivatives of these pyrazole inhibitors that also display potent DLK inhibition.77 The selectivity of compounds 9 and 10 for DLK enabled inhibition of stress induced JNK signaling in neurons without an effect on other JNK dependent signaling events in other tissues, which represented a more targeted therapeutic approach as compared to previous efforts targeting the pathway. Consistent with this, compound 10 displayed a favorable in vivo safety profile, enabling interrogation of DLK inhibition in animal models of chronic neurodegeneration.76 The inhibitors 7, 8, 9, and 10 form the basis of the pharmacological in vivo studies that complemented genetic mouse studies using siRNA and conditional gene knock out. Important to their utility as tool molecules are the potency and selectivity for DLK in addition to rodent pharmacokinetic properties, especially the ability to penetrate the blood−brain barrier (Table 2). With these inhibitors in hand, Genentech has been able to interrogate the role of DLK in stress induced neurodegeneration models for optic neuropathy/glaucoma (optic nerve crush),10,74,75 Parkinson’s (MPTP),74 Alzheimer’s (PS2APP, Tau),10,76 and ALS (SOD1).10 Pharmacological inhibition of DLK in these models leads to rapid reduction of pc-Jun following a single dose in the areas of neuronal injury: optic nerve (optic nerve crush), substantial nigra (MPTP),

tion in potency compared to DLK inhibitor 7, the authors argued that an inhibitor with improved pharmacokinetics (free drug exposure) had better potential for further optimization to a small molecule therapeutic. This assertion was supported by the ability of pyrazole 8 to elicit similar pharmacodynamics effect as pyridine 7 in the mouse optic nerve crush model at the same dose. Notable in this work was the use of structure-based design enabled by the X-ray crystal structures of the DLK kinase domain bound with inhibitors. The DLK inhibitors 7 and 8 are ATP-competitive inhibitors, and their crystal structures show the characteristic polar hydrogen bond donor−acceptor−donor interactions between the 2-aminopyridine with the active site hinge residues (Figure 6). Additionally, van der Waals interactions between the cyanosubstituted pyridine hinge binder with the Met gatekeeper residue and between the 3,3-difluoropyrrolidine of 7 and cyclopentane of 8 to the glycine rich P-loop contribute to the overall binding affinities. Furthermore, the N-oxetanylpiperidine of both inhibitor 7 and 8 orients toward the solvent exposed opening of the active site. Most recently, Genentech further optimized the pyrazole scaffold using structure- and property-based design (tPSA < 80 and HBD ≤ 1).76 Specific modifications toward the hinge residue Phe 192, gatekeeper Met 190, and Gln 197 were targeted for selectivity as the combination of these residues are distinct from related kinases. Structure-guided adjustments of the key polar contacts with the DLK hinge residues led to the modification of the N-substituted 2-amino-4-cyanopyridine of 8 to a 2-amino-3,5-substituted pyridine of 9 and 10 with specific 3-substitutents to mask the additional HBD of the primary amino group. Additionally, with the goal of decreasing 8083



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Biographies

cortex (PS2APP, Tau), and spinal cord (SOD1) of mice. In these studies p-c-Jun repression was strongly correlated with the concentration of DLK inhibitor, and the resulting free drug levels required for 50% inhibition of p-c-Jun in vivo were consistent with the in vitro cell-based p-JNK IC50 assay when corrected for serum binding78 (9, in vitro free pJNK IC50 = 0.078 μM, in vivo optic nerve crush p-c-Jun free IC50 = 0.081 μM; 10, in vitro free pJNK IC50 = 0.129 μM, in vivo PSAPP2 pc-Jun free IC50 = 0.190 μM).10,76 This PK/PD relationship guided dose selection for in vivo studies that demonstrated neuroprotection in a subset of these models. In addition, the rapid reversal of c-Jun phosphorylation with DLK inhibitor treatment demonstrates that continuous DLK activity is required to maintain c-Jun phosphorylation and suggests that individual neurons can still be rescued from degeneration following DLK/JNK pathway activation. This observation, taken along with the finding that only a subset of neurons are pc-Jun positive in chronic neurodegeneration models, provides further evidence that DLK inhibitors may still be efficacious even if dosed after the onset of disease. Indeed, the reversal of injury-induced DLK signaling by 7 is sufficient to elicit neuroprotection in the retina when dosed after optic nerve crush and to preserve neuromuscular junctions when dosed after disease onset in SOD1 model of ALS.10 These DLK inhibitor studies in injury models along with models of genetic deletion of DLK in mice have advanced our knowledge of the mechanisms of DLK inhibition and our efforts to progress DLK inhibitors toward clinical studies.

Michael Siu is a Senior Scientist in Discovery Chemistry at Genentech, Inc. He received his B.S. in Chemistry from the University of California, Berkeley where he performed undergraduate research in the group of Professor Paul A. Bartlett and received his Ph.D. in Chemistry from Harvard University under the guidance of Professor Andrew G. Myers. Prior to joining Genentech, Michael was a member of the Medicinal Chemistry group at Pfizer La Jolla. Arundhati Sengupta Ghosh is a Senior Scientific Researcher at the Neuroscience Department at Genenetch, Inc. (South San Francisco). She received a B.Sc. in Zoology from University of Delhi, India, and a B.A. and M.A. in Natural Sciences from Cambridge University, U.K. Her current research focus includes understanding DLK biology in neurodegenerative diseases. Arundhati has coauthored various papers related to DLK biology and patents related to DLK. Joseph W. Lewcock is the Head of Biology Discovery at Denali Therapeutics, a neurodegeneration focused company in South San Francisco, CA. He received a B.S. from the University of California, San Diego and Ph.D. in Molecular Biology and Genetics from John’s Hopkins University. Prior to joining Denali, Joe completed a postdoctoral fellowship at the Salk Institute and worked in the Department of Neuroscience at Genentech where he led the efforts to understand DLK biology. Joe has a broad interest in developing effective therapeutics for the treatment of chronic neurodegenerative disease. He has coauthored approximately 27 research papers and 4 published patent applications

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ACKNOWLEDGMENTS We thank Martin Larhammar for the artwork in Figure 1 and the table of contents graphic.

CONCLUSIONS The requirement of DLK signaling for degeneration downstream of diverse insults suggests the signaling through this kinase represents a central mechanism by which neurons respond to injury. As shown by multiple research groups, DLK reduction/inhibition reduces both cell death and axonal degeneration in multiple preclinical models that reflect diverse medical conditions (Table 1). These cumulative data from multiple disease and injury models support DLK as a viable drug target for treatment of neurodegenerative diseases with severe unmet medical need and limited therapeutic options. Toward this effort, Genentech/Roche have progressed the small molecule DLK inhibitor GDC-0134 (RG6000) to clinical investigation of this target (currently in phase 1 for ALS). With the strong biological rationale for the role of DLK in neurodegeneration and the ability to discover potent, selective, and brain-penetrant inhibitors, there is increased optimism for the development of DLK inhibitors for the treatment of neurodegenerative diseases.

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ABBREVIATIONS USED ALS, amyotrophic lateral sclerosis; cLogP, calculated logarithm of partition coefficient; DLK, dual leucine zipper kinase; DRG, dorsal root ganglion; FBS, fetal bovine serum; FTD, frontotemporal dementia; HBD, hydrogen bond donor; JNK, c-Jun N-terminal kinase; Kpuu, (unbound partition coefficient); LipE, lipophilic ligand efficiency; log D, logarithm of distribution coefficient; MAP3K12, mitogen-activated protein kinase kinase kinase 12; MKK, mitogen-activated protein kinase kinase; MLK, mixed-lineage kinase; MDR1, multidrug resistance protein 1; MDCK, Madin−Darby canine kidney; NGF, nerve growth factor; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NMDA, N-methyl-D-aspartate; P-gp, P-glycoprotein; NMJ, neuromuscular junction;; PSP, progressive supranuclear palsy; RGC, retinal ganglion cell; SOD1, superoxide dismutase 1; TPSA, topological polar surface area.

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REFERENCES

(1) Mattson, M. Pathways towards and away from Alzheimer’s disease. Nature 2004, 430, 631−639. (2) Dawson, T. M.; Dawson, V. L. Molecular pathways of neurodegeneration in Parkinson’s disease. Science 2003, 302, 819−822. (3) Parikshak, N. N.; Gandal, M. J.; Geschwind, D. H. Systems biology and gene networks in neurodevelopmental and neurodegenerative disorders. Nat. Rev. Genet. 2015, 16, 441−458. (4) Scott, L.; Dawson, V. L.; Dawson, T. M. Trumping neurodegeneration: Targeting common pathways regulated by autosomal recessive Parkinson’s disease genes. Exp. Neurol. 2017, 298, 191−201. (5) Menzies, F. M.; Fleming, A.; Caricasole, A.; Bento, C. F.; Andrews, S. P.; Ashkenazi, A.; Fullgrabe, J.; Jackson, A.; Sanchez, M. J.; Karabiyik, C.; Licitra, F.; Ramirez, A. L.; Pavel, M.; Puri, C.; Renna, M.; Ricketts, T.; Schlotawa, L.; Vicinanza, M.; Won, H.; Zhu, H.;

AUTHOR INFORMATION

Corresponding Authors

*M.S.: e-mail, [email protected]; phone, 650-467-7764. *J.W.L.: e-mail, [email protected]; phone, 650-745-5247. ORCID

Michael Siu: 0000-0002-2822-6584 Notes

The authors declare the following competing financial interest(s): M.S. and A.S.G. are employees of Genentech, part of the Roche Group, and are shareholders of Roche. J.W.L. is a former employee of Genentech, part of the Roche Group. 8084



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