Review For reprint orders, please contact [email protected]
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions Dual leucine zipper kinase (DLK) is a serine/threonine protein kinase that is a member of the mixed lineage kinase subfamily. Mixed lineage kinases are upstream MAP3Ks that activate the JNK pathway. DLK is primarily responsible for activating JNK and mediating the apoptotic stress response in various cell types, specifically neurons. Inhibition and knockdown of DLK has been demonstrated to have neuroprotective effects in cellular and animal models of Alzheimer’s disease, glaucoma, Parkinson’s disease and other neurodegenerative conditions. Several series of ATP-binding site inhibitors have been identified through profiling efforts providing launch points for future medicinal chemistry programs. Dual leucine zipper kinase is a serine/threonine protein kinase that was first characterized in 1994 as a member of the mixed lineage kinase subfamily [1]. Mixed lineage kinases are MAP3Ks that are part of a phosphorylation cascade, which results in the activation/phosphorylation of MKKs or MAP2Ks followed by JNKs, also known as SAPKs. JNKs are a family of serine/threonine protein kinases that phosphorylate, among other things, transcription factors such as c-Jun. This signaling is primarily responsible for mediating the stress response in various cell types and, in neurons, often culminates in apoptosis as depicted in Figure 1 [2]. Substantial evidence exists for JNK activation in Alzheimer’s disease [3] and other neurodegenerative conditions [4]. Because prolonged JNK activation induces apoptosis, inhibition of JNK has been a therapeutic focus for neuroprotection for over a decade, leading to several JNK inhibitors with CNS penetration [5,6]. However, there have been studies indicating that transient JNK activation promotes cell survival [7]. Thus, several of the upstream kinases (e.g., mixed lineage kinases such as DLK) of JNK may be better targets since they may have distinct, unique multiprotein complexes with JIPs, which result in distinct cellular responses such as neuronal cell death upon activation of JNK [8–12]. An analysis of mouse and human tissue indicates that DLK is expressed primarily in kidney and brain tissue, specifically in the synaptic terminal of neurons [13]. When neurons are stressed or injured, DLK dimerizes and is autophosphorylated/activated resulting in a series of phosphorylation events that activate the JNK pathway via phosphorylation of
MKK7 [14]. When it is activated, MKK7 can phosphorylate various JNK isoforms, priming transcription factors within the cell for either apoptosis or neuronal regeneration [15]. These disparate responses to injury both start with the activation of DLK. Clearly, some intrinsic or extrinsic factors are responsible for directing the cellular response. Despite the lack of a clearly defined and validated role for DLK in disease states, some intriguing new studies have demonstrated the importance of DLK in in vitro and in vivo models of neuronal cell death [16], axonal injury/degeneration [101], Parkinson’s disease [17] and glaucoma [18]. Given these studies, coupled with the fact that kinases are perceived to be very druggable targets [19], DLK will continue to attract more attention as a therapeutic target for neurodegeneration. This Review will summarize the functional role of DLK, some unique structural features of this protein and the effect that silencing or inhibiting its function has under neurodegenerative conditions. Furthermore, this review will showcase many of the known small-molecule DLK inhibitors and provide some insights into the inhibitory pharmacophore and some challenges associated with discovering and developing a specific DLK inhibitor for neurodegenerative diseases. Characterization & lineage of human DLK Human DLK is member of the TK-like group of protein kinases and the mixed lineage kinase subfamily (Figure 2). This subfamily is so named because some subdomains (I–VII) of the catalytic region resemble that of serine/threonine kinases such as CTR-1, STE11 and byr2 kinases,
10.4155/FMC.13.150 © 2013 Future Science Ltd
Future Med. Chem. (2013) 5(16), 1923–1934
Dana Ferraris*, Zhiyong Yang & Derek Welsbie John Hopkins University Brain Science Institute, 855 North Wolfe Street, Baltimore, MD 21205, USA *Author for correspondence: Tel.: +1 410 614 1107 Fax: +1 410 614 0659 E-mail: [email protected]
ISSN 1756-8919
1923
Review | Ferraris, Yang & Welsbie
Stress
P DLK DLK P
MKK7
P
JNK
P
Transcription factors (c-Jun, ATF)
Neuronal apoptosis P Neuronal regeneration
Figure 1. Activation of dual leucine zipper kinase and downstream phosphorylation cascade leading to neuronal apoptosis or neuronal regeneration. DLK: Dual leucine zipper kinase.
Key Terms JIPs: Scaffolding proteins that
have demonstrated the ability to interact and aggregate with all members of the JNK pathway.
JNK pathway:
Phosphorylation pathway that is activated under stress stimuli resulting in the activation of several transcription factors including c-Jun, giving rise to the name c-Jun N-terminal kinase pathway. Activation can lead to cellular responses such as apoptosis, cell differentiation, proliferation and inflammation depending on the type and length of stimuli, the specific JIP, and the JNK isoform involved.
Leucine zipper: A common
3D a-helical structural feature in proteins used primarily for dimerization by the interaction of hydrophobic residues running along one side of the zipper.
Small-molecule binder:
A compound that competes with a immobilized ligand or substrate for the active site of an enzyme. Binding is often a surrogate for inhibition, but not all small-molecule binders are necessarily inhibitors of the enzymatic reaction.
whereas some of the other subdomains (VIII– XI) bear homology to TK such as FGF receptors [13]. Within the mixed lineage kinase subfamily, DLK belongs to a subgroup with its closest related homologue, LZK (Figure 2). The catalytic region of DLK bears approximately 87% homology to LZK while only an approximate 30–35% homology to MLK1–4 [13]. The DLK protein has four characterized domains including a catalytic domain, a dual leucine zipper domain, glycine–serine–proline rich domains and a glycine–proline rich region (Figure 3). The catalytic domain in human DLK consists of amino acids 127–375 [13,20] with 11 conserved subdomains typical of protein kinases [21]. Subdomains (I–VII) resemble that of serine/threonine kinases, whereas subdomains (VIII–XI) are homologous to TKs. Within this catalytic region, there is a conserved lysine residue (Lys-185 in mouse and Lys-152 in human) that has been shown based on alignments with other serine/threonine protein kinases, to help stabilize ATP within the catalytic site. Indeed, DLK mutants of this residue lack catalytic activity [22]. There are also binding sites for JNKinteracting proteins (JIP1 in particular). These are located at the beginning and end of the catalytic domain (subdomains I and XI), corresponding to residues 142–147 and 356–368 (175–179 and 389–401 in murine DLK) [23]. Even though no structural data exist on human
MLK2
ZAK Tyrosine kinase-like (TKL) group
Mixed lineage kinase subfamily
MLK3 MLK1
DLK LZK
MLK4
Figure 2. Mixed lineage kinase subfamily. DLK: Dual leucine zipper kinase.
1924
Future Med. Chem. (2013) 5(16)
DLK or any of its homologs, an activation loop is most probably present between domains VII and VIII. This protein segment is usually identified by two consensus sequences flanking an approximate 20–40 residue stretch, in this case, a DFG motif (D254, F255 and G256), followed by an APE motif (A278, P279 and E280) [24]. Within this activation loop there are several potential sites for autophosphorylation, including Ser258, Ser262, Ser265, T266 and Ser269. Evidence exists for an autophosphorylating/self-activating mechanism for DLK, namely: n A conserved activation loop flanked by a DFG and APE motif [13]; Dimerization is necessary for DLK kinase activity (vide infra)and deletion of the leucine zipper region prohibits the phosphorylation and activation of downstream kinases [25];
n
Other proteins known to undergo this type of activation (e.g., SLK, STK10 and DAPK3) all contain coiled coil or leucine zipper sequences to promote dimerization [26].
n
Unlike most other members of the mixed lineage kinase subfamily, DLK contains two rather than one leucine zipper regions. While 3D structural data do not exist for DLK, leucine zipper regions of other proteins have been crystallized, as exemplified by the image of the c-Jun homodimerleucine zipper in Figure 4 [27]. This ‘zipper-like’ structure contains multiple hydrophobic residues that form two a-helices with the leucine and valine side chains acting as the ‘teeth’ of the zipper. The open end of this zipper would be part of the DLK sequence and the closed end contains two cysteine residues that can form intramolecular disulfide bridges as a monomer or intermolecular disulfide bonds to further stabilize the dimeric complex [22]. The two dual leucine zipper repeats of DLK share >70% similarity with MLK-1–3 [13]. The only other enzyme in this subfamily that contains this dual leucine zipper motif is LZK (MAP3K13) [28]. One characteristic feature of many kinases is the necessity for dimerization and auto phosphorylation in order to become active [24]. Deletion studies have demonstrated that both of the two leucine zipper regions are necessary for homodimerization and activation of the enzyme and downstream activation of the JNK pathway (Figures 1 & 5) [29]. In addition to the leucine zipper dimerization, a truncated version of murine DLK without the leucine zippers (amino acids 1–407) also had the ability to form future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions dimers suggesting that there are positive forces within the catalytic region coercing DLK into a dimer, consistent with the autophosphorylating/self-activating mechanism outlined above [23]. As a regulatory mechanism for activation, Whitmarsh and coworkers have demonstrated that in a basal state, JIP1 is bound to a catalytically inactive form of DLK, presumably via one or both of the JIP1 binding regions. Upon cellular stress JIP1 is dislodged from DLK by JNK allowing DLK to homodimerize and autophosphorylate (Figure 5). Phosphorylated DLK is the active form that activates the JNK pathway (Figure 1). It is also known that JIP3 interacts with DLK along a similar pathway that is directly related to neuronal degeneration [9]. The exact mechanism and utility of JIPs in the DLK/ JNK pathway remains a mystery, but it is known that JIPs act as scaffolding proteins to aggregate other mixed lineage kinases to facilitate signal transmission and activation of JNK [30]. DLK also contains a glycine–serine–proline rich domain (amino acids 523–859) and a smaller glycine–proline domain (residues 56–65), which are most likely responsible for mediating protein/protein interactions such as those with MKK7 [22]. Indeed, these interactions most likely provide some specificity along the JNK pathway when DLK is activated. Role of DLK in neuroprotection/axonal degeneration In vitro neuroprotection via suppression of DLK Several DLK knockdown studies were performed by a group at Genentech to support the hypothesis that DLK is integral in stressinduced neurodegeneration [101]. Knockdown was achieved using siRNA specific for DLK in rat dorsal root ganglia, cortical neurons and sympathetic neurons. The cells were stressed by either withdrawal of a growth factor (NGF) or treatment with vincristine to induce apoptosis. In each cell type, DLK knockdown resulted in protection of neurons against degeneration. Furthermore, in these same cell lines, DLK silencing in the absence of neuronal stressors had a trophic effect, causing significant increase the number of viable neurons in dorsal root ganglia, cortical and sympathetic nerve cultures. In order to determine the mechanism by which DLK is acting under stress, this group noted that DLK knockdown attenuated the activation of JNK, as evidenced by its phosphorylation state and the levels of its downstream target future science group
JIP binding sites 56–65 127 375 387–415
| Review
523
859
Human DLK 440–467 Glycine–proline rich domain
Kinase domain
Leucine zipper domain
Glycine–serine–proline rich domain
Figure 3. Composite structure of human dual leucine zipper bearing kinase. DLK: Dual leucine zipper kinase.
c-Jun. Interestingly, the silencing of the JNK2 and JNK3 isoforms most relevant to neuronal apoptosis and axon degeneration with siRNA did not produce a protective effect as profound as silencing DLK alone [31]. However, Bjorkblom et al. have demonstrated that simultaneous knockdown of JNKs1, 2 and 3 is necessary to confer neuroprotection, whereas knockdown of individual JNKs does not have the same protective effect [32]. Loss of retinal ganglion cells (RGCs) is the primary neurodegenerative event leading to glaucoma. Zack and colleagues performed a high-throughput siRNA screen against the full mouse kinome and identified two kinases that, when silenced, result in neuroprotection of RGCs in vitro [18]. Using a nanoparticle-based transfection system, RGCs were transfected with siRNAs against every kinase in the mouse kinome and assayed for their ability to promote survival in neurotrophin-deficient media. This technique identified DLK and its downstream substrate MKK7 that, when silenced, produced the most robust effect on RGC survival. Indeed, this group also demonstrated that RGCs carrying a floxed allele of DLK and transduced the Crerecombinase, demonstrated increased survival to a similar extent as DLK siRNA [18]. Furthermore, VX-680 (tozasertib), a small-molecule
Figure 4. Leucine zipper motif. Adapted from [27] .
www.future-science.com
1925
Review | Ferraris, Yang & Welsbie Key Terms Adenoassociated viral vector: A nonpathogenic viral
vector used to transfer genetic material into cells, particularly amenable as a delivery vehicle for inserting into non-dividing cells such as neurons.
DLK Stress
Hinge binder: A term used to describe the part of a molecule that binds to the ATP-binding cleft in a protein kinase. This cleft binds the adenine ring of ATP, hence most hinge binders are planar heterocycles.
Dimerization
Autophosphorylation/ activation
JNK JNK
JIP1
DLK
ATP
ADP
DLK P
P DLK
JNK JIP1
Figure 5. Activation of dual leucine zipper kinase by dissociation of JIP1 followed by dimerization and trans-autophosphorylation.
binder of DLK (Kd = 190 nM, vide infra)
[33], demonstrated dose-dependent improvement in RGC survival.
In vivo
neuroprotection via suppression of DLK In 2006, Hirai and coworkers discovered that knocking out the DLK gene in mice resulted in a lethal phenotype around birth with abnormal brain development [34], emphasizing that DLK plays a major role in embryogenesis and/or organogenesis. Since that time, most of the research efforts in vivo have focused on gene trap mutations or adenoassociated viral vector (AAV) transfer, to silence DLK in fruit flies and invertebrates. As outlined below, these studies demonstrate the importance of DLK in axonal growth and stressed/damaged neurons. Axonal degeneration is the major pathology in a variety of neurological disorders including mechanical injury, neuropathies, glaucoma, Alzheimer’s disease and Parkinson’s disease [35]. DLK was found to promote the degeneration of severed axons in Drosophila and mice via the JNK pathway [101]. The Drosophila ortholog of Dlk (wallenda, wnd ) was knocked-out in olfactory receptor neurons and compared with the wild-type after injury. The wild type olfactory receptor neurons degenerated within 24 h and the wnd knockouts were significantly preserved with degeneration delayed past 48 h. Furthermore, similar protective effects were seen in murine embryonic dorsal root ganglia that are DLK deficient after both axotomy and vincristine-induced degeneration. In this same study, JNK inhibition with SP600125, a broad spectrum JNK inhibitor, showed similar neuroprotection to the DLK-deficient cell 1926
DLK
Future Med. Chem. (2013) 5(16)
lines. These data support the hypothesis that activation of the JNK pathway is needed to signal axons to degenerate. Indeed, this protective effect was also seen in vivo as mice subjected to sciatic nerve transections demonstrated significant Wallerian degeneration after 52 h, while DLK-deficient mice demonstrated significant preservation over this time period with preserved axon profiles [101]. To support the potential role of DLK inhibition in Parkinson’s disease, two dominant negative forms of DLK were delivered to dopaminergic neurons in the substantia nigra of adult mice using A AV [17]. The two forms that were successfully transduced were a catalytically inactive form of DLK (DN-DLK[K152A]) and a DLK without the leucine zipper region (DN-DLK-LZ). Interestingly, the DN-DLK (K152A) displayed some trophic effects on the dopamine neurons in the substantia nigra, demonstrating a modest increase in cell number. These two dominant-negative constructs also resulted in attenuation of apoptosis and enhancement of long-term survival of dopamine neurons in the 6-hydroxydopamine neurotoxin model. Furthermore, the dominant-negative forms of DLK successfully reduced the amount of phosphorylated c-Jun to a similar degree to the level of AAV transduction. Evidence from this study supports a model where DLK mediates neuronal apoptosis through the JNK pathway and phosphorylation of c-Jun [17]. This type of study also supports the hypothesis that DLK inhibition can be neuroprotective in models of Parkinson’s disease. To investigate the role of DLK inhibition in optic neuropathies, an optic nerve crush model was performed with mice containing future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions a f loxed allele of Dlk (Dlk fl/fl). Injection of an AAV-2 expressing a Cre resulted in Cremediated deletion of Dlkin RGCs. Following optic nerve crush, the mice lacking Dlk showed a 75% reduction in RGC loss over the control mice. To further demonstrate the importance of DLK as a drug discovery target in optic neuropathies, an optic nerve transection model was used in Wistar rats with VX-680 (a kinase inhibitor with DLK activity), which was delivered intravitreally in slow-releasing microspheres as a therapeutic agent. After optic nerve transection, the number of surviving RGCs in VX-680-treated eyes was significantly higher than vehicle-treated rats. Finally, V X-680 treatment improved RGC survival over controls after laser-induced ocular hypertension in a rat model of glaucoma, by far the most common optic neuropathy [18]. Role in neuronal regeneration after injury Contrary to the multitude of neuroprotective effects of DLK outlined above, several studies have also outlined the necessity of DLK in neuronal regeneration after injury [36–38]. For example, in Caenorhabditis elegans, DLK-1 plays an important role in axonal regeneration. Specifically, DLK-1 acts through MKK4 and PMK (C. elegans homolog of p38). Knockout of dlk1 in C. elegans motor neurons resulted in axons without the ability to promote growth cone formation and regeneration toward the dorsal cord after axotomy. In addition, overexpression of DLK in this same system resulted in an improved time to growth cone formation over wild type and improved growth cone morphology. These findings suggest that in lower species DLK-1 is capable of activating the MKK4/PMK pathway and that this cascade is required for neuronal growth [39]. Recent studies were performed to clarify the role of DLK in neuronal injury. These studies demonstrate that upon neuronal injury, DLK is rapidly upregulated [18] and primes the expression of factors that ultimately lead to controlled cell death and neuronal/axonal regeneration [37]. Indeed, some intrinsic or extrinsic factors may ultimately control which pathway the cell undergoes, perhaps aborting the cell death pathway if regeneration is successful [15]. In many adult neurons (e.g., RGCs), the factors that control regeneration are largely absent; in addition, multiple factors are present that limit regeneration in the mammalian CNS [40], and for this reason apoptosis is generally the pathway taken. In addition, at least in future science group
| Review
the case of RGCs, the commitment to apoptosis is not immediate, but evidence suggests that a prolonged (days to weeks) stress signal (e.g., c-Jun phosphorylation) is needed for apoptosis to occur [15]. This type of chronic stressing of neurons is reflective of many neurodegenerative diseases, and for this reason DLK inhibition may be an attractive therapeutic strategy. Binders & inhibitors of DLK Many of the reported compounds that interact with DLK were discovered during a large-scale kinase profiling effort [41]. Many of the identified compounds are either FDA-approved drugs or were clinical candidates. The assay used to assess the potency of these compounds is a competition binding assay using an immobilized biotinylated ATP-competitive ligand. A phage-tagged kinase is bound to the immobilized ligand and the test inhibitors compete with the immobilized ligand to release the kinase protein [42]. The high-throughput nature of this binding assay makes it amenable for identifying potential fragments for drug discovery/medicinal chemistry optimization or identifying probe compounds to further elucidate the role of DLK inhibition in vivo. Compounds discovered by this method are referred to as ‘binders’ in this review while conventional competitive DLK ‘inhibitors’ will only be referred to as such if biochemical inhibition is demonstrated. From this profiling, several compounds from the classical type I ATP-binding kinase inhibitors [24] displayed potent binding potential for DLK. Unfortunately, there are no crystal structures of DLK or its closest relative LZK so one can only postulate at the binding mode of these various compounds. Several pharmacophore trends that are typical of many ATP binding site competitive kinase inhibitors are also apparent for DLK binders, namely: A classical ‘hinge binding’ heterocycle (e.g., pyrimidine, indazole, in red);
n
One or two substituted aryl groups in close proximity to the hinge binder (blue);
n
A chain of atoms connected to a tertiary amine (green).
n
Quinolines
The most potent DLK binder that has been published is XL-880 (Kd = 22 nM, Figure 6), a quinoline-based compound originally designed www.future-science.com
1927
Review | Ferraris, Yang & Welsbie O N
N
N
O
N
O O
O
CN
N H
N H
F
O
F
O
N
O
XL-880 (Kd = 22 nM)
O
Cl
Cl
SKI-606 (Kd = 670 nM)
N
O
CN
HN N
HN
HN
O
Cl N
O Neratinib, HKI-272 (Kd = 370 nM)
Figure 6. Quinoline-based dual leucine zipper kinase binders.
as a MET and KDR(VEGF2) inhibitor [43]. Other DLK binders in this series, such as SKI606 (Kd = 670 nM), a Src kinase inhibitor [44] and HKI-272 (Kd = 370 nM), an irreversible inhibitor of HER2/EGFR [45], are over an order of magnitude less potent than XL-880. It is possible that either the nitrile moiety or the smaller size of the side chain (black) of SKI-606 and HKI-272 H N HN
S
N
N
is responsible for this loss of binding potential, or the spirocyclopropylflurorophenyl amide moiety of XL-880 conveys a dramatic boost in potency. Despite the potency of XL-880 against DLK, the main limitation for using these compounds as a starting point for optimization is their size (MW of XL-880 = 632). An optimal DLK inhibitor would most likely be used for a CNS
O
N
O S
N H
N
O
HN
O
N
Cl
N
H N
N
N N
TAE-684 (Kd = 42 nM)
VX-680 (Kd = 190 nM)
N
O HN
S
H N
O HN
N
H N
N N
TG-101348 (Kd = 6300 nM)
N
N
N
O O
O
N N H
N H
CF3
AST-487 DLK Kd = 2500 nM MKK7 Kd = 27 nM JNK2 Kd = 56 nM JNK3 Kd = 750 nM
Figure 7. Pyrimidine-based dual leucine zipper kinase binders.
1928
Future Med. Chem. (2013) 5(16)
future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions indication necessitating its brain penetrability. The average MW of brain penetrable compounds is approximately 300–350 [46]. Pyrimidines VX-680 (F igure 7)
is a good DLK binder (Kd = 190 nM) and has been used in some validation studies by Zack and colleagues [18]; however, this pyrimidine also falls into the category of a pan kinase inhibitor with poor selectivity scores [41]. Nevertheless, VX-680, despite being primarily optimized for Aurora kinase activity [33] and tumor suppression, displayed promising neuroprotective effects [18]. Indeed, the neuroprotective effects seen with VX-680 are predominantly due to DLK since lower doses of compound were needed to achieve RGC survival when cells were DLK silenced and higher doses were needed when DLK was overexpressed. TAE-684(Kd = 42 nM), an ALK inhibitor [47] also shows dramatic DLK binding potential highlighting the importance of the tertiary amine sidechain (green), which is at least partly responsible for the two orders of magnitude increase in binding affinity over TG-101348 (Kd = 6300 nM), a JAK2 inhibitor [48]. AST-487, while not a very potent DLK binder, is intriguing as a tool compound since it displays the ability to bind other members of the DLK/JNK pathway. Namely, it has submicromolar ability to bind every other kinase downstream of DLK: MKK7 (27 nM), JNK2 (56 nM) and JNK3 (760 nM) [41]. Indazoles
KW-2449 (DLK K d = 67 nM, Figure 8) was designed as a multikinase inhibitor for leukemia with low nanomolar inhibitory potency against FLT3, ABL and Aurora kinases [49]. Interestingly, this compound has also demonstrated some binding potential against JNK2 (230 nM) and JNK3 (Kd = 51 nM), two JNK isoforms downstream of DLK along the neuroprotective pathway. This type of kinase profile, in combination with physicochemical properties that are more in line with CNS-based compounds (MW = 332, cLogP = 2.93, Serum total PSA = 57Å), are a prerequisite for DLK inhibitors. These characteristics make this compound a better staring point than many of the other binders and inhibitors outlined in this review. The related compound AG-013736 is 25-fold less potent than KW-2449, indicating that the piperazine (green) is beneficial for DLK binding and/or the thiobenzamide or pyridyl moiety (blue) is detrimental to binding. Given future science group
| Review
the smaller molecular weight of these compounds compared with other DLK binders, this series is certainly more likely to provide CNS-penetrable lead compounds. Indolocarbazoles
Cephalon (PA, USA) spent considerable effort optimizing indolocarbazoles for inhibition of mixed lineage kinases (F igure 9) . Although optimized for activity against MLK-1 and -2, some compounds within this series displayed some DLK off-target activity such as CEP-701 (Kd = 840 nM), which is also an 80 nM binder of MKK7, CEP-1347 (DLK IC50 = 114 nM),(+) K-252a (DLK IC50 = 360 nM), and CEP-5214 (DLK IC50 = 119 nM) [50]. CEP-1347 could be considered a pan MLK inhibitor and displays neuroprotective effects in several preclinical studies [4]. However, this compound was not effective in Phase II/III clinical trials for Parkinson’s disease, possibly due to the lack of target inhibition in the brain [51]. This group regularly measured biochemical inhibititory data for this series of compounds using a timeresolved fluorescence readout and MKK7 as a substrate, and for this reason they are referred to as inhibitors of DLK kinase activity [52]. Cephalon performed further modification of the sugar moiety and one of the indolo moieties, which led to diminished DLK potency [102]. This group has several recent patent publications outlining related compounds with ‘improved MLK and DLK activity’ without providing specific structure–activity relationship [102]. O N
N H
NH
N
KW-2449 (Kd = 67 nM)
O HN
N
S
N H
N
AG-013736 (Kd = 1700 nM)
Figure 8. Indazoles as dual leucine zipper kinase binders.
www.future-science.com
1929
Review | Ferraris, Yang & Welsbie H N
H N
O
N N
N
O
N
O
HO HO
an ROS-1 and AKT inhibitor. Both of these compounds have a better selectivity profile than, for example, the pyrimidines, but with a similar pharmacophore to other DLK binders.
O
Discovering
O O
OH
CEP-701 (Kd = 840 nM) H N
(+)K-252a (IC50 = 360 nM)
O
H N
O
S
S
O N
HO
O
N N
N H
O
HO
O
CEP-5214 (IC50 = 119 nM)
CEP-1347 (IC50 = 114 nM)
Figure 9. Indolocarbazole derivaties as dual leucine zipper kinase inhibitors. Miscellaneous
DLK binders Kinase profiling has also identified some miscellaneous binders of DLK (Figure 10), JNJ28312141 (Kd = 330 nM), a CSF-1 tyrosine kinase inhibitor [53], and crizotinib (Kd = 170 nM) [54],
HN
H N
N O
N
N O
JNJ-28312141 (Kd = 330 nM)
N
NH2 O
N
Cl F
N Cl HN Crizotinib (Kd = 170 nM)
Figure 10. Miscellaneous dual leucine zipper kinase binders.
1930
Future Med. Chem. (2013) 5(16)
CN
selective DLK inhibitors for neuroprotection One of the global problems in the kinase field is how to discover reasonably selective kinase inhibitors in order to adequately test the hypothesis of target inhibition leading to disease modification in a clinical setting. While there are several examples of DLK inhibitors and binders as outlined above, it would appear that all of these molecules interact with the ATP binding site of DLK. For this reason, it will be necessary to determine the desired kinase inhibition profile for a DLK inhibitor. For example, would it be desirable to have off target activity against other members of the JNK pathway? And to what extent? Or is it better to solely inhibit DLK, even teasing out selectivity among other MLKs [55]? The closest homologues to DLK are other members of the mixed lineage kinase subfamily, specifically LZK, indicating that selectivity between these two enzymes may be challenging. However, the homology between DLK and MLK1–4 is only approximately 40% between their respective catalytic regions, indicating that a modicum of selectivity may be achievable. Recent examples of kinase optimization efforts have demonstrated very systematic methods for achieving selectivity with ATP competitive inhibitors even over closely related isoforms [56]. Taken together, these studies indicate that there is a high probability of obtaining a DLK-selective ATP competitive inhibitor. Another possible strategy to discover selective DLK inhibitors is to target the dual leucine zipper regions of DLK. Several groups have demonstrated that dimerization is necessary for DLK activation. Logically, small-molecule disruption of this dimerization would curtail the autophosphorylation and activation of DLK, similar to inhibition of the catalytic machinery of the enzyme. This inhibition of protein/protein interactions has been demonstrated as a means to disrupt c-Myc/Max leucine zipper interaction leading to c-Myc-induced transcriptional activation [57]. Despite the paucity of examples of drugs that disrupt protein/protein interactions, it has been predicted that as much as 50% of these interactions can be druggable [58]. Whether or not the DLK dimerization is druggable, methods to screen for such small-molecule disruptors (e.g., fluorescence energy resonance transfer and future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions fluorescence polarization assays) are becoming more mainstream as methods of identifying hits. Another strategy to find selective DLK inhibitors is to screen for allosteric inhibitors. Allosteric inhibitors of kinases have been a topic of research for several years. The benefits of such inhibitors is that they are often unique to the target kinase, minimizing off-target interactions and thus improving the therapeutic window for such drugs [55]. Despite this promise, there are no documented drugs that are allosteric inhibitors of kinases and there have been no documented cases of allosteric DLK inhibitors, so this strategy would appear to have greater challenges than some of the others mentioned above. Even so, the technology for identifying allosteric inhibitors continues to improve [59] and much effort has been put forth to progress allosteric kinase inhibitors in the clinic [60]. Perhaps one of the biggest challenges in discovering a DLK selective inhibitor is to find one with the right balance of physicochemical properties. In general, kinase inhibitors are larger MW molecules that press the boundaries of oral availability and brain penetrability [61]. Both of these characteristics would be desirable for a DLK inhibitor for neuroprotection. Specifically, a DLK inhibitor would need lower molecular weight, lower polar surface area and fewer hydrogen bond donors than many of the compounds outlined in this review in order to penetrate the CNS [46]. Despite these hurdles and historically few examples of brainpenetrable kinase inhibitors [19], there have been recent success stories of brain-penetrable LRRK-2 inhibitors. These LRRK-2 inhibitors were discovered to be highly selective across the kinome, as well as displaying brain penetration when dosed orally across several species [62]. Summary To summarize, several groups have put forth data indicating that inhibition or knockdown of DLK is neuroprotective in animal models of neuronal injury, glaucoma, Parkinson’s disease and amyotrophic lateral sclerosis. However, there are still several challenges associated with targeting DLK for neuroprotection. Without crystallography data, little is known about isoforms of DLK [63]: are certain isoforms more prevalent in disease states? Is it possible to specifically target those isoforms? DLK is clearly a mediator of neuronal response to injury, but what factors tip the balance between apoptosis and neuroregeneration? And how does DLK inhibition affect these factors? DLK plays different roles depending on cell type, cell cycle, future science group
| Review
level of development, length and duration of injury and so forth. Do the cellular and animal models of these disease states adequately represent DLK as it would be in the human disease state? Several DLK binders and inhibitors are outlined above, but all were optimized for distinctly different kinases, many of which have anticancer activity, a fact that may prevent these compounds from being good chemical probes for assessing the neuroprotective ability of DLK inhibitors. DLK inhibition is acting primarily through the JNK pathway, which can involve the activation of a variety of transcription factors, scaffold proteins, adaptor proteins and other protein kinases [64]. What specific transcription factors/genes are affected when the DLK/JNK pathway is activated under neurological injury? Most of the compounds outlined in this review have liabilities such as low selectivity, low brain penetrability and limited chemistry space with regard to intellectual property. Future perspective Even taking into account the challenges outlined in this review, it is important to note that several facts make DLK a viable drug discovery target. Namely, both enzymatic binding and biochemical assays for screening potential inhibitors have been published. In addition, a few potent, tractable inhibitors and binders have been identified as outlined in this review. Furthermore, kinases are considered desirable, druggable therapeutic targets second only to G-protein coupled receptors. DLK, despite being a member of the mixed lineage kinase subfamily, only shares approximately 30–40% homology in the catalytic region with MLK1–3 implying that selectivity may be achievable. Because of these factors, medicinal chemistry groups interested in neuroprotective agents will start to discover more selective DLK inhibitors within the next 5 years. With selective inhibitors in hand, the tools will be available to further understand the nature of the DLK response to injury and its role in the JNK pathway under specific neurological conditions. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. www.future-science.com
1931
Review | Ferraris, Yang & Welsbie Executive summary Background Dual leucine zipper kinase (DLK) is a MAP3K primarily localized in neurons and plays a major mediating role in the JNK phosphorylation pathway priming the cell for either neuroregeneration or neuronal apoptosis. Characterization & lineage of human DLK
DLK is a mixed lineage kinase with several unique structural features such as dual leucine zippers that are necessary for homodimerization, autophosphorylation and activation of the kinase.
DLK shares 87% homology with LZK, but only 30–40% homology with other members of the mixed lineage kinase subfamily. Role of DLK in neuroprotection/axonal degeneration
Suppression of DLK leads to neuroprotective effects in vitro in retinal ganglion cells and dorsal root ganglia.
Suppression of DLK leads to neuroprotective effects in vivo in models of axonal degeneration and retinal degeneration.
Contrary to the multitude of neuroprotective effects of DLK, several studies have also outlined the necessity of DLK in neuronal regeneration after injury. Binders & inhibitors of DLK
Several different series of DLK binders and inhibitors such as quinolines, pyrimidines and indazoles have been identified, developing a preliminary pharmacophore for the enzyme and potential lead series for further optimization. Discovering selective DLK inhibitors
Obtaining brain penetrable and selective DLK inhibitors are two of the biggest challenges with this target. Selectivity may be obtained by allosteric inhibition or targeting the leucine zipper region.
References Papers of special note have been highlighted as: n of interest nn of considerable interest 1
n
2
3
4
5
6
7
Gallo Ka, Johnson Gl. Mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat. Rev. Mol. Cell Biol. 3(9), 663–672 (2002).
8
9
Outlines the members of the mixed lineage kinase family of which dual leucine zipper kinase (DLK) is a member.
Whitmarsh AJ, Davis RJ. Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals. Trends Biochem. Sci. 23(12), 481–485 (1998).
15 Watkins TA, Wang B, Huntwork-Rodriguez S
Ghosh AS, Wang B, Pozniak CD, Chen M, Watts RJ, Lewcock JW. DLK induces developmental neuronal degeneration via selective regulation of proapoptotic JNK activity. J. Cell Biol. 194(5), 751–764 (2013).
16 Miller BR, Press C, Daniels RW, Sasaki Y,
n
Zhu X, Raina AK, Rottkamp CA et al. Activation and redistribution of c-Jun N-terminal kinase/stress activated protein kinase in degenerating neurons in Alzheimer’s disease. J Neurochem. 76(2), 435–441 (2001).
11 Whitmarsh AJ, Cavanagh J, Tournier C,
Wang LH, Besirli CG, Johnson EM Jr. Mixedlineage kinases: a target for the prevention of neurodegeneration. Annu. Rev. Pharmacol. Toxicol. 44, 451–474 (2004).
12 Engstrom W, Ward A, Moorwood K. The role
Siddiqui MA, Reddy PA. Small molecule JNK (c-Jun N-terminal kinase) inhibitors. J. Med. Chem. 53(8), 3005–3012 (2010).
Identification, molecular cloning, and characterization of dual leucine zipper bearing kinase. A novel serine/threonine protein kinase that defines a second subfamily of mixed lineage kinases. J. Biol. Chem. 269(49), 30808–30817 (1994).
10 Tanoue T, Nishida E. Molecular recognitions
Milbrandt J, Diantonio A. A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration. Nat. Neurosci. 12(4), 387–389 (2009). 17 Chen X, Rzhetskaya M, Kareva T et al.
Antiapoptotic and trophic effects of dominantnegative forms of dual leucine zipper kinase in dopamine neurons of the substantia nigra in vivo. J. Neurosci. 28(3), 672–680 (2008).
in the MAP kinase cascades. Cell Signal. 15(5), 455–462 (2003). Yasuda J, Davis RJ. A mammalian scaffold complex that selectively mediates MAP kinase activation. Science 281(5383), 1671–1674 (1998). of scaffold proteins in JNK signalling. Cell Prolif. 43(1), 56–66 (2010).
18 Welsbie DS, Yang Z, GE Y et al. Functional
genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc. Natl Acad. Sci. USA 110(10), 4045–4050 (2013). nn
13 Holzman LB, Merritt SE, Fan G.
Graczyk PP. JNK inhibitors as antiinflammatory and neuroprotective agents. Future Med. Chem. 5(5), 539–551 (2013).
1932
et al. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury. Proc. Natl Acad. Sci. USA 110(10), 4039–4044 (2013).
Describes DLK signaling pathways for neuroregeneration and neuroprotection.
Behrens A, Sibilia M, Wagner EF. Aminoterminal phosphorylation of c-Jun regulates stress-induced apoptosis and cellular proliferation. Nat. Genet. 21(3), 326–329 (1999).
Ventura JJ, Hubner A, Zhang C, Flavell RA, Shokat KM, Davis RJ. Chemical genetic ana lysis of the time course of signal transduction by JNK. Mol. Cell 21(5), 701–710 (2006).
substrate. J. Biol. Chem. 274(15), 10195–10202 (1999).
n
First paper to fully characterize DLK.
14 Merritt SE, Mata M, Nihalani D, Zhu C, Hu
X, Holzman LB. The mixed lineage kinase DLK utilizes MKK7 and not MKK4 as
Future Med. Chem. (2013) 5(16)
The only manuscript utilizing a DLK inhibitor as a neuroprotectant.
19 Chico LK, Van Eldik LJ, Watterson DM.
Targeting protein kinases in central nervous system disorders. Nat. Rev. Drug Discov. 8(11), 892–909 (2009). nn
Excellent review profiling several kinase inhibitors that have been trialled in humans.
20 Choi S, Couture J-P, Blouin R. DLK (dual
leucine zipper-bearing kinase). In: Encyclopedia of Signaling Molecules. Choi S. (Ed.). Springer NY, USA, 522–526 (2012).
future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions 21 Hanks SK, Quinn AM, Hunter T. The protein
kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241(4861), 42–52 (1988).
critical for neuronal death. J. Biol. Chem. 283(28), 19704–19713 (2008). 33 Harrington EA, Bebbington D, Moore J et al.
VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat. Med. 10(3), 262–267 (2004).
22 Mata M, Merritt SE, Fan G, Yu GG, Holzman
LB. Characterization of dual leucine zipperbearing kinase, a mixed lineage kinase present in synaptic terminals whose phosphorylation state is regulated by membrane depolarization via calcineurin. J. Biol. Chem. 271(28), 16888–16896 (1996).
34 Hirai S, Cui De F, Miyata T et al. The c-Jun
N-terminal kinase activator dual leucine zipper kinase regulates axon growth and neuronal migration in the developing cerebral cortex. J. Neurosci. 26(46), 11992–12002 (2006).
23 Mooney LM, Whitmarsh AJ. Docking
interactions in the c-Jun N-terminal kinase pathway. J. Biol. Chem. 279(12), 11843–11852 (2004).
35 Lingor P, Koch JC, Tonges L, Bahr M. Axonal
degeneration as a therapeutic target in the CNS. Cell Tissue Res. 349(1), 289–311 (2012).
24 Cuny GD. Kinase inhibitors as potential
therapeutics for acute and chronic neurodegenerative conditions. Curr. Pharm. Des. 15(34), 3919–3939 (2009).
36 Xiong X, Collins CA. A conditioning lesion
protects axons from degeneration via the Wallenda/DLK MAP kinase signaling cascade. J. Neurosci. 32(2), 610–615 (2012).
25 Nihalani D, Merritt S, Holzman LB.
Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation. J. Biol. Chem. 275(10), 7273–7279 (2000). 26 Oliver AW, Knapp S, Pearl LH. Activation
37 Shin JE, Cho Y, Beirowski B, Milbrandt J,
Cavalli V, Diantonio A. Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration. Neuron 74(6), 1015–1022 (2012). 38 Tedeschi A, Bradke F. The DLK signalling
pathway-a double-edged sword in neural development and regeneration. EMBO Rep. doi:10.1038/embor.2013.64 (2013) (Epub ahead of print).
segment exchange: a common mechanism of kinase autophosphorylation? Trends Biochem. Sci. 32(8), 351–356 (2007). 27 Junius FK, O’Donoghue SI, Nilges M, Weiss
AS, King GF. High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer. J. Biol. Chem. 271(23), 13663–13667 (1996). 28 Sakuma H, Ikeda A, Oka S, Kozutsumi Y,
Zanetta JP, Kawasaki T. Molecular cloning and functional expression of a cDNA encoding a new member of mixed lineage protein kinase from human brain. J. Biol. Chem. 272(45), 28622–28629 (1997). 29 Nihalani D, Meyer D, Pajni S, Holzman LB.
Mixed lineage kinase-dependent JNK activation is governed by interactions of scaffold protein JIP with MAPK module components. EMBO J 20(13), 3447–3458 (2001). 30 Yasuda J, Whitmarsh AJ, Cavanagh J, Sharma
M, Davis RJ. The JIP group of mitogenactivated protein kinase scaffold proteins. Mol. Cell Biol. 19(10), 7245–7254 (1999). 31 Coffey ET, Smiciene G, Hongisto V et al.
c-Jun N-terminal protein kinase (JNK) 2/3 is specifically activated by stress, mediating c-Jun activation, in the presence of constitutive JNK1 activity in cerebellar neurons. J. Neurosci. 22(11), 4335–4345 (2002). 32 Bjorkblom B, Vainio JC, Hongisto V,
Herdegen T, Courtney MJ, Coffey ET. All JNKs can kill, but nuclear localization is
future science group
nn
Outlines the DLK signaling pathways in mammalian systems.
39 Hammarlund M, Nix P, Hauth L, Jorgensen
EM, Bastiani M. Axon regeneration requires a conserved MAP kinase pathway. Science 323(5915), 802–806 (2009). 40 Huebner EA, Strittmatter SM. Axon
regeneration in the peripheral and central nervous systems. Results Probl. Cell Differ. 48, 339–351 (2009). 41 Karaman MW, Herrgard S, Treiber DK et al.
A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26(1), 127–132 (2008). 42 Fabian MA, Biggs WH 3rd, Treiber DK et al.
A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23(3), 329–336 (2005). 43 Qian F, Engst S, Yamaguchi K et al.
Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res. 69(20), 8009–8016 (2009). 44 Vultur A, Buettner R, Kowolik C et al. SKI-
606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells. Mol. Cancer Ther. 7(5), 1185–1194 (2008).
www.future-science.com
| Review
45 Wissner A, Mansour TS. The development of
HKI-272 and related compounds for the treatment of cancer. Arch. Pharm. (Weinheim) 341(8), 465–477 (2008). 46 Hitchcock SA, Pennington LD. Structure-
brain exposure relationships. J. Med. Chem. 49(26), 7559–7583 (2006). 47 Galkin AV, Melnick JS, Kim S et al.
Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPMALK. Proc. Natl Acad. Sci. USA 104(1), 270–275 (2007). 48 Pardanani A, Gotlib JR, Jamieson C et al.
Safety and efficacy of TG101348, a selective JAK2 inhibitor, in myelofibrosis. J. Clin. Oncol. 29(7), 789–796 (2011). 49 Shiotsu Y, Kiyoi H, Ishikawa Y et al. KW-2449,
a novel multikinase inhibitor, suppresses the growth of leukemia cells with FLT3 mutations or T315I-mutated BCR/ABL translocation. Blood 114(8), 1607–1617 (2009). 50 Ruggeri B, Singh J, Gingrich D et al. CEP-
7055. a novel, orally active pan inhibitor of vascular endothelial growth factor receptor tyrosine kinases with potent antiangiogenic activity and antitumor efficacy in preclinical models. Cancer Res. 63(18), 5978–5991 (2003). 51 Parkinson Study Group PRECEPT
Investigators. Mixed lineage kinase inhibitor CEP-1347 fails to delay disability in early Parkinson disease. Neurology 69(15), 1480–1490 (2007). 52 Hudkins RL, Diebold JL, Tao M et al. Mixed-
lineage kinase 1 and mixed-lineage kinase 3 subtype-selective dihydronaphthyl(3,4-a) pyrrolo(3,4-c) carbazole-5-ones: optimization, mixed-lineage kinase 1 crystallography, and oral in vivo activity in 1-methyl-4phenyltetrahydropyridine models. J. Med. Chem. 51(18), 5680–5689 (2008). 53 Illig CR, Manthey CL, Wall MJ et al.
Optimization of a potent class of arylamide colony-stimulating factor-1 receptor inhibitors leading to anti-inflammatory clinical candidate 4-cyano-N-(2-[1-cyclohexen-1-yl]-4-(1[dimethylamino]acetyl)-4-piperidinyl)phenyl)-1H-imidazole-2-carboxamide (JNJ28312141). J. Med. Chem. 54(22), 7860–7883 (2011). 54 Cui JJ, Tran-Dube M, Shen H et al. Structure
based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J. Med. Chem. 54(18), 6342–6363 (2011). 55 Morphy R. Selectively nonselective kinase
inhibition: striking the right balance. J. Med. Chem. 53(4), 1413–1437 (2010).
1933
Review | Ferraris, Yang & Welsbie 56 Jimenez JM, Boyall D, Brenchley G et al.
Design and optimization of selective protein kinase C q(PKCq) inhibitors for the treatment of autoimmune diseases. J. Med. Chem. 56(5), 1799–1810 (2013). 57 Prochownik EV, Vogt PK. Therapeutic
targeting of Myc. Genes Cancer 1(6), 650–659 (2010). 58 Camacho CJ, Koes DR, Dömling AS.
Interactive technologies for leveraging the known chemistry of anchor residues to disrupt protein interactions. In: Protein–Protein Interactions in Drug Discovery. Dömling A. (Ed.). Wiley-VCH Verlag GmbH & Co. KGaA, Weinhelm, Germany, 85–100 (2013). 59 Lebakken CS, Reichling LJ, Ellefson JM,
Riddle SM. Detection of allosteric kinase
1934
inhibitors by displacement of active site probes. J. Biomol. Screen. 17(6), 813–821 (2012). 60 Fang Z, Grutter C, Rauh D. Strategies for the
selective regulation of kinases with allosteric modulators: exploiting exclusive structural features. ACS Chem. Biol. 8(1), 58–70 (2012). 61 Vieth M, Sutherland JJ, Robertson DH,
Campbell RM. Kinomics: characterizing the therapeutically validated kinase space. Drug Discov. Today 10(12), 839–846 (2005). 62 Estrada AA, Liu X, Baker-Glenn C et al.
Discovery of highly potent, selective, and brain-penetrable leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors. J. Med. Chem. 55(22), 9416–9433 (2012).
Future Med. Chem. (2013) 5(16)
63 Yan D, Jin Y. Regulation of DLK-1 kinase
activity by calcium-mediated dissociation from an inhibitory isoform. Neuron 76(3), 534–548 (2012). 64 Bogoyevitch MA, Kobe B. Uses for JNK: the
many and varied substrates of the c-Jun N-terminal kinases. Microbiol. Mol. Biol. Rev. 70(4), 1061–1095 (2006). Patents 101 Lewcock JW, Pozniak C, Senguota-Ghosh A:
US20120328609 (2012). 102 Becknell N, Brown PD, Dandu R et al.:
US8383813 (2013). 103 Hudkins RL, Dandu R, Tao M, Underiner
TL, Zulli AL: US8084472 (2011).
future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions Dual leucine zipper kinase (DLK) is a serine/threonine protein kinase that is a member of the mixed lineage kinase subfamily. Mixed lineage kinases are upstream MAP3Ks that activate the JNK pathway. DLK is primarily responsible for activating JNK and mediating the apoptotic stress response in various cell types, specifically neurons. Inhibition and knockdown of DLK has been demonstrated to have neuroprotective effects in cellular and animal models of Alzheimer’s disease, glaucoma, Parkinson’s disease and other neurodegenerative conditions. Several series of ATP-binding site inhibitors have been identified through profiling efforts providing launch points for future medicinal chemistry programs. Dual leucine zipper kinase is a serine/threonine protein kinase that was first characterized in 1994 as a member of the mixed lineage kinase subfamily [1]. Mixed lineage kinases are MAP3Ks that are part of a phosphorylation cascade, which results in the activation/phosphorylation of MKKs or MAP2Ks followed by JNKs, also known as SAPKs. JNKs are a family of serine/threonine protein kinases that phosphorylate, among other things, transcription factors such as c-Jun. This signaling is primarily responsible for mediating the stress response in various cell types and, in neurons, often culminates in apoptosis as depicted in Figure 1 [2]. Substantial evidence exists for JNK activation in Alzheimer’s disease [3] and other neurodegenerative conditions [4]. Because prolonged JNK activation induces apoptosis, inhibition of JNK has been a therapeutic focus for neuroprotection for over a decade, leading to several JNK inhibitors with CNS penetration [5,6]. However, there have been studies indicating that transient JNK activation promotes cell survival [7]. Thus, several of the upstream kinases (e.g., mixed lineage kinases such as DLK) of JNK may be better targets since they may have distinct, unique multiprotein complexes with JIPs, which result in distinct cellular responses such as neuronal cell death upon activation of JNK [8–12]. An analysis of mouse and human tissue indicates that DLK is expressed primarily in kidney and brain tissue, specifically in the synaptic terminal of neurons [13]. When neurons are stressed or injured, DLK dimerizes and is autophosphorylated/activated resulting in a series of phosphorylation events that activate the JNK pathway via phosphorylation of
MKK7 [14]. When it is activated, MKK7 can phosphorylate various JNK isoforms, priming transcription factors within the cell for either apoptosis or neuronal regeneration [15]. These disparate responses to injury both start with the activation of DLK. Clearly, some intrinsic or extrinsic factors are responsible for directing the cellular response. Despite the lack of a clearly defined and validated role for DLK in disease states, some intriguing new studies have demonstrated the importance of DLK in in vitro and in vivo models of neuronal cell death [16], axonal injury/degeneration [101], Parkinson’s disease [17] and glaucoma [18]. Given these studies, coupled with the fact that kinases are perceived to be very druggable targets [19], DLK will continue to attract more attention as a therapeutic target for neurodegeneration. This Review will summarize the functional role of DLK, some unique structural features of this protein and the effect that silencing or inhibiting its function has under neurodegenerative conditions. Furthermore, this review will showcase many of the known small-molecule DLK inhibitors and provide some insights into the inhibitory pharmacophore and some challenges associated with discovering and developing a specific DLK inhibitor for neurodegenerative diseases. Characterization & lineage of human DLK Human DLK is member of the TK-like group of protein kinases and the mixed lineage kinase subfamily (Figure 2). This subfamily is so named because some subdomains (I–VII) of the catalytic region resemble that of serine/threonine kinases such as CTR-1, STE11 and byr2 kinases,
10.4155/FMC.13.150 © 2013 Future Science Ltd
Future Med. Chem. (2013) 5(16), 1923–1934
Dana Ferraris*, Zhiyong Yang & Derek Welsbie John Hopkins University Brain Science Institute, 855 North Wolfe Street, Baltimore, MD 21205, USA *Author for correspondence: Tel.: +1 410 614 1107 Fax: +1 410 614 0659 E-mail: [email protected]
ISSN 1756-8919
1923
Review | Ferraris, Yang & Welsbie
Stress
P DLK DLK P
MKK7
P
JNK
P
Transcription factors (c-Jun, ATF)
Neuronal apoptosis P Neuronal regeneration
Figure 1. Activation of dual leucine zipper kinase and downstream phosphorylation cascade leading to neuronal apoptosis or neuronal regeneration. DLK: Dual leucine zipper kinase.
Key Terms JIPs: Scaffolding proteins that
have demonstrated the ability to interact and aggregate with all members of the JNK pathway.
JNK pathway:
Phosphorylation pathway that is activated under stress stimuli resulting in the activation of several transcription factors including c-Jun, giving rise to the name c-Jun N-terminal kinase pathway. Activation can lead to cellular responses such as apoptosis, cell differentiation, proliferation and inflammation depending on the type and length of stimuli, the specific JIP, and the JNK isoform involved.
Leucine zipper: A common
3D a-helical structural feature in proteins used primarily for dimerization by the interaction of hydrophobic residues running along one side of the zipper.
Small-molecule binder:
A compound that competes with a immobilized ligand or substrate for the active site of an enzyme. Binding is often a surrogate for inhibition, but not all small-molecule binders are necessarily inhibitors of the enzymatic reaction.
whereas some of the other subdomains (VIII– XI) bear homology to TK such as FGF receptors [13]. Within the mixed lineage kinase subfamily, DLK belongs to a subgroup with its closest related homologue, LZK (Figure 2). The catalytic region of DLK bears approximately 87% homology to LZK while only an approximate 30–35% homology to MLK1–4 [13]. The DLK protein has four characterized domains including a catalytic domain, a dual leucine zipper domain, glycine–serine–proline rich domains and a glycine–proline rich region (Figure 3). The catalytic domain in human DLK consists of amino acids 127–375 [13,20] with 11 conserved subdomains typical of protein kinases [21]. Subdomains (I–VII) resemble that of serine/threonine kinases, whereas subdomains (VIII–XI) are homologous to TKs. Within this catalytic region, there is a conserved lysine residue (Lys-185 in mouse and Lys-152 in human) that has been shown based on alignments with other serine/threonine protein kinases, to help stabilize ATP within the catalytic site. Indeed, DLK mutants of this residue lack catalytic activity [22]. There are also binding sites for JNKinteracting proteins (JIP1 in particular). These are located at the beginning and end of the catalytic domain (subdomains I and XI), corresponding to residues 142–147 and 356–368 (175–179 and 389–401 in murine DLK) [23]. Even though no structural data exist on human
MLK2
ZAK Tyrosine kinase-like (TKL) group
Mixed lineage kinase subfamily
MLK3 MLK1
DLK LZK
MLK4
Figure 2. Mixed lineage kinase subfamily. DLK: Dual leucine zipper kinase.
1924
Future Med. Chem. (2013) 5(16)
DLK or any of its homologs, an activation loop is most probably present between domains VII and VIII. This protein segment is usually identified by two consensus sequences flanking an approximate 20–40 residue stretch, in this case, a DFG motif (D254, F255 and G256), followed by an APE motif (A278, P279 and E280) [24]. Within this activation loop there are several potential sites for autophosphorylation, including Ser258, Ser262, Ser265, T266 and Ser269. Evidence exists for an autophosphorylating/self-activating mechanism for DLK, namely: n A conserved activation loop flanked by a DFG and APE motif [13]; Dimerization is necessary for DLK kinase activity (vide infra)and deletion of the leucine zipper region prohibits the phosphorylation and activation of downstream kinases [25];
n
Other proteins known to undergo this type of activation (e.g., SLK, STK10 and DAPK3) all contain coiled coil or leucine zipper sequences to promote dimerization [26].
n
Unlike most other members of the mixed lineage kinase subfamily, DLK contains two rather than one leucine zipper regions. While 3D structural data do not exist for DLK, leucine zipper regions of other proteins have been crystallized, as exemplified by the image of the c-Jun homodimerleucine zipper in Figure 4 [27]. This ‘zipper-like’ structure contains multiple hydrophobic residues that form two a-helices with the leucine and valine side chains acting as the ‘teeth’ of the zipper. The open end of this zipper would be part of the DLK sequence and the closed end contains two cysteine residues that can form intramolecular disulfide bridges as a monomer or intermolecular disulfide bonds to further stabilize the dimeric complex [22]. The two dual leucine zipper repeats of DLK share >70% similarity with MLK-1–3 [13]. The only other enzyme in this subfamily that contains this dual leucine zipper motif is LZK (MAP3K13) [28]. One characteristic feature of many kinases is the necessity for dimerization and auto phosphorylation in order to become active [24]. Deletion studies have demonstrated that both of the two leucine zipper regions are necessary for homodimerization and activation of the enzyme and downstream activation of the JNK pathway (Figures 1 & 5) [29]. In addition to the leucine zipper dimerization, a truncated version of murine DLK without the leucine zippers (amino acids 1–407) also had the ability to form future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions dimers suggesting that there are positive forces within the catalytic region coercing DLK into a dimer, consistent with the autophosphorylating/self-activating mechanism outlined above [23]. As a regulatory mechanism for activation, Whitmarsh and coworkers have demonstrated that in a basal state, JIP1 is bound to a catalytically inactive form of DLK, presumably via one or both of the JIP1 binding regions. Upon cellular stress JIP1 is dislodged from DLK by JNK allowing DLK to homodimerize and autophosphorylate (Figure 5). Phosphorylated DLK is the active form that activates the JNK pathway (Figure 1). It is also known that JIP3 interacts with DLK along a similar pathway that is directly related to neuronal degeneration [9]. The exact mechanism and utility of JIPs in the DLK/ JNK pathway remains a mystery, but it is known that JIPs act as scaffolding proteins to aggregate other mixed lineage kinases to facilitate signal transmission and activation of JNK [30]. DLK also contains a glycine–serine–proline rich domain (amino acids 523–859) and a smaller glycine–proline domain (residues 56–65), which are most likely responsible for mediating protein/protein interactions such as those with MKK7 [22]. Indeed, these interactions most likely provide some specificity along the JNK pathway when DLK is activated. Role of DLK in neuroprotection/axonal degeneration In vitro neuroprotection via suppression of DLK Several DLK knockdown studies were performed by a group at Genentech to support the hypothesis that DLK is integral in stressinduced neurodegeneration [101]. Knockdown was achieved using siRNA specific for DLK in rat dorsal root ganglia, cortical neurons and sympathetic neurons. The cells were stressed by either withdrawal of a growth factor (NGF) or treatment with vincristine to induce apoptosis. In each cell type, DLK knockdown resulted in protection of neurons against degeneration. Furthermore, in these same cell lines, DLK silencing in the absence of neuronal stressors had a trophic effect, causing significant increase the number of viable neurons in dorsal root ganglia, cortical and sympathetic nerve cultures. In order to determine the mechanism by which DLK is acting under stress, this group noted that DLK knockdown attenuated the activation of JNK, as evidenced by its phosphorylation state and the levels of its downstream target future science group
JIP binding sites 56–65 127 375 387–415
| Review
523
859
Human DLK 440–467 Glycine–proline rich domain
Kinase domain
Leucine zipper domain
Glycine–serine–proline rich domain
Figure 3. Composite structure of human dual leucine zipper bearing kinase. DLK: Dual leucine zipper kinase.
c-Jun. Interestingly, the silencing of the JNK2 and JNK3 isoforms most relevant to neuronal apoptosis and axon degeneration with siRNA did not produce a protective effect as profound as silencing DLK alone [31]. However, Bjorkblom et al. have demonstrated that simultaneous knockdown of JNKs1, 2 and 3 is necessary to confer neuroprotection, whereas knockdown of individual JNKs does not have the same protective effect [32]. Loss of retinal ganglion cells (RGCs) is the primary neurodegenerative event leading to glaucoma. Zack and colleagues performed a high-throughput siRNA screen against the full mouse kinome and identified two kinases that, when silenced, result in neuroprotection of RGCs in vitro [18]. Using a nanoparticle-based transfection system, RGCs were transfected with siRNAs against every kinase in the mouse kinome and assayed for their ability to promote survival in neurotrophin-deficient media. This technique identified DLK and its downstream substrate MKK7 that, when silenced, produced the most robust effect on RGC survival. Indeed, this group also demonstrated that RGCs carrying a floxed allele of DLK and transduced the Crerecombinase, demonstrated increased survival to a similar extent as DLK siRNA [18]. Furthermore, VX-680 (tozasertib), a small-molecule
Figure 4. Leucine zipper motif. Adapted from [27] .
www.future-science.com
1925
Review | Ferraris, Yang & Welsbie Key Terms Adenoassociated viral vector: A nonpathogenic viral
vector used to transfer genetic material into cells, particularly amenable as a delivery vehicle for inserting into non-dividing cells such as neurons.
DLK Stress
Hinge binder: A term used to describe the part of a molecule that binds to the ATP-binding cleft in a protein kinase. This cleft binds the adenine ring of ATP, hence most hinge binders are planar heterocycles.
Dimerization
Autophosphorylation/ activation
JNK JNK
JIP1
DLK
ATP
ADP
DLK P
P DLK
JNK JIP1
Figure 5. Activation of dual leucine zipper kinase by dissociation of JIP1 followed by dimerization and trans-autophosphorylation.
binder of DLK (Kd = 190 nM, vide infra)
[33], demonstrated dose-dependent improvement in RGC survival.
In vivo
neuroprotection via suppression of DLK In 2006, Hirai and coworkers discovered that knocking out the DLK gene in mice resulted in a lethal phenotype around birth with abnormal brain development [34], emphasizing that DLK plays a major role in embryogenesis and/or organogenesis. Since that time, most of the research efforts in vivo have focused on gene trap mutations or adenoassociated viral vector (AAV) transfer, to silence DLK in fruit flies and invertebrates. As outlined below, these studies demonstrate the importance of DLK in axonal growth and stressed/damaged neurons. Axonal degeneration is the major pathology in a variety of neurological disorders including mechanical injury, neuropathies, glaucoma, Alzheimer’s disease and Parkinson’s disease [35]. DLK was found to promote the degeneration of severed axons in Drosophila and mice via the JNK pathway [101]. The Drosophila ortholog of Dlk (wallenda, wnd ) was knocked-out in olfactory receptor neurons and compared with the wild-type after injury. The wild type olfactory receptor neurons degenerated within 24 h and the wnd knockouts were significantly preserved with degeneration delayed past 48 h. Furthermore, similar protective effects were seen in murine embryonic dorsal root ganglia that are DLK deficient after both axotomy and vincristine-induced degeneration. In this same study, JNK inhibition with SP600125, a broad spectrum JNK inhibitor, showed similar neuroprotection to the DLK-deficient cell 1926
DLK
Future Med. Chem. (2013) 5(16)
lines. These data support the hypothesis that activation of the JNK pathway is needed to signal axons to degenerate. Indeed, this protective effect was also seen in vivo as mice subjected to sciatic nerve transections demonstrated significant Wallerian degeneration after 52 h, while DLK-deficient mice demonstrated significant preservation over this time period with preserved axon profiles [101]. To support the potential role of DLK inhibition in Parkinson’s disease, two dominant negative forms of DLK were delivered to dopaminergic neurons in the substantia nigra of adult mice using A AV [17]. The two forms that were successfully transduced were a catalytically inactive form of DLK (DN-DLK[K152A]) and a DLK without the leucine zipper region (DN-DLK-LZ). Interestingly, the DN-DLK (K152A) displayed some trophic effects on the dopamine neurons in the substantia nigra, demonstrating a modest increase in cell number. These two dominant-negative constructs also resulted in attenuation of apoptosis and enhancement of long-term survival of dopamine neurons in the 6-hydroxydopamine neurotoxin model. Furthermore, the dominant-negative forms of DLK successfully reduced the amount of phosphorylated c-Jun to a similar degree to the level of AAV transduction. Evidence from this study supports a model where DLK mediates neuronal apoptosis through the JNK pathway and phosphorylation of c-Jun [17]. This type of study also supports the hypothesis that DLK inhibition can be neuroprotective in models of Parkinson’s disease. To investigate the role of DLK inhibition in optic neuropathies, an optic nerve crush model was performed with mice containing future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions a f loxed allele of Dlk (Dlk fl/fl). Injection of an AAV-2 expressing a Cre resulted in Cremediated deletion of Dlkin RGCs. Following optic nerve crush, the mice lacking Dlk showed a 75% reduction in RGC loss over the control mice. To further demonstrate the importance of DLK as a drug discovery target in optic neuropathies, an optic nerve transection model was used in Wistar rats with VX-680 (a kinase inhibitor with DLK activity), which was delivered intravitreally in slow-releasing microspheres as a therapeutic agent. After optic nerve transection, the number of surviving RGCs in VX-680-treated eyes was significantly higher than vehicle-treated rats. Finally, V X-680 treatment improved RGC survival over controls after laser-induced ocular hypertension in a rat model of glaucoma, by far the most common optic neuropathy [18]. Role in neuronal regeneration after injury Contrary to the multitude of neuroprotective effects of DLK outlined above, several studies have also outlined the necessity of DLK in neuronal regeneration after injury [36–38]. For example, in Caenorhabditis elegans, DLK-1 plays an important role in axonal regeneration. Specifically, DLK-1 acts through MKK4 and PMK (C. elegans homolog of p38). Knockout of dlk1 in C. elegans motor neurons resulted in axons without the ability to promote growth cone formation and regeneration toward the dorsal cord after axotomy. In addition, overexpression of DLK in this same system resulted in an improved time to growth cone formation over wild type and improved growth cone morphology. These findings suggest that in lower species DLK-1 is capable of activating the MKK4/PMK pathway and that this cascade is required for neuronal growth [39]. Recent studies were performed to clarify the role of DLK in neuronal injury. These studies demonstrate that upon neuronal injury, DLK is rapidly upregulated [18] and primes the expression of factors that ultimately lead to controlled cell death and neuronal/axonal regeneration [37]. Indeed, some intrinsic or extrinsic factors may ultimately control which pathway the cell undergoes, perhaps aborting the cell death pathway if regeneration is successful [15]. In many adult neurons (e.g., RGCs), the factors that control regeneration are largely absent; in addition, multiple factors are present that limit regeneration in the mammalian CNS [40], and for this reason apoptosis is generally the pathway taken. In addition, at least in future science group
| Review
the case of RGCs, the commitment to apoptosis is not immediate, but evidence suggests that a prolonged (days to weeks) stress signal (e.g., c-Jun phosphorylation) is needed for apoptosis to occur [15]. This type of chronic stressing of neurons is reflective of many neurodegenerative diseases, and for this reason DLK inhibition may be an attractive therapeutic strategy. Binders & inhibitors of DLK Many of the reported compounds that interact with DLK were discovered during a large-scale kinase profiling effort [41]. Many of the identified compounds are either FDA-approved drugs or were clinical candidates. The assay used to assess the potency of these compounds is a competition binding assay using an immobilized biotinylated ATP-competitive ligand. A phage-tagged kinase is bound to the immobilized ligand and the test inhibitors compete with the immobilized ligand to release the kinase protein [42]. The high-throughput nature of this binding assay makes it amenable for identifying potential fragments for drug discovery/medicinal chemistry optimization or identifying probe compounds to further elucidate the role of DLK inhibition in vivo. Compounds discovered by this method are referred to as ‘binders’ in this review while conventional competitive DLK ‘inhibitors’ will only be referred to as such if biochemical inhibition is demonstrated. From this profiling, several compounds from the classical type I ATP-binding kinase inhibitors [24] displayed potent binding potential for DLK. Unfortunately, there are no crystal structures of DLK or its closest relative LZK so one can only postulate at the binding mode of these various compounds. Several pharmacophore trends that are typical of many ATP binding site competitive kinase inhibitors are also apparent for DLK binders, namely: A classical ‘hinge binding’ heterocycle (e.g., pyrimidine, indazole, in red);
n
One or two substituted aryl groups in close proximity to the hinge binder (blue);
n
A chain of atoms connected to a tertiary amine (green).
n
Quinolines
The most potent DLK binder that has been published is XL-880 (Kd = 22 nM, Figure 6), a quinoline-based compound originally designed www.future-science.com
1927
Review | Ferraris, Yang & Welsbie O N
N
N
O
N
O O
O
CN
N H
N H
F
O
F
O
N
O
XL-880 (Kd = 22 nM)
O
Cl
Cl
SKI-606 (Kd = 670 nM)
N
O
CN
HN N
HN
HN
O
Cl N
O Neratinib, HKI-272 (Kd = 370 nM)
Figure 6. Quinoline-based dual leucine zipper kinase binders.
as a MET and KDR(VEGF2) inhibitor [43]. Other DLK binders in this series, such as SKI606 (Kd = 670 nM), a Src kinase inhibitor [44] and HKI-272 (Kd = 370 nM), an irreversible inhibitor of HER2/EGFR [45], are over an order of magnitude less potent than XL-880. It is possible that either the nitrile moiety or the smaller size of the side chain (black) of SKI-606 and HKI-272 H N HN
S
N
N
is responsible for this loss of binding potential, or the spirocyclopropylflurorophenyl amide moiety of XL-880 conveys a dramatic boost in potency. Despite the potency of XL-880 against DLK, the main limitation for using these compounds as a starting point for optimization is their size (MW of XL-880 = 632). An optimal DLK inhibitor would most likely be used for a CNS
O
N
O S
N H
N
O
HN
O
N
Cl
N
H N
N
N N
TAE-684 (Kd = 42 nM)
VX-680 (Kd = 190 nM)
N
O HN
S
H N
O HN
N
H N
N N
TG-101348 (Kd = 6300 nM)
N
N
N
O O
O
N N H
N H
CF3
AST-487 DLK Kd = 2500 nM MKK7 Kd = 27 nM JNK2 Kd = 56 nM JNK3 Kd = 750 nM
Figure 7. Pyrimidine-based dual leucine zipper kinase binders.
1928
Future Med. Chem. (2013) 5(16)
future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions indication necessitating its brain penetrability. The average MW of brain penetrable compounds is approximately 300–350 [46]. Pyrimidines VX-680 (F igure 7)
is a good DLK binder (Kd = 190 nM) and has been used in some validation studies by Zack and colleagues [18]; however, this pyrimidine also falls into the category of a pan kinase inhibitor with poor selectivity scores [41]. Nevertheless, VX-680, despite being primarily optimized for Aurora kinase activity [33] and tumor suppression, displayed promising neuroprotective effects [18]. Indeed, the neuroprotective effects seen with VX-680 are predominantly due to DLK since lower doses of compound were needed to achieve RGC survival when cells were DLK silenced and higher doses were needed when DLK was overexpressed. TAE-684(Kd = 42 nM), an ALK inhibitor [47] also shows dramatic DLK binding potential highlighting the importance of the tertiary amine sidechain (green), which is at least partly responsible for the two orders of magnitude increase in binding affinity over TG-101348 (Kd = 6300 nM), a JAK2 inhibitor [48]. AST-487, while not a very potent DLK binder, is intriguing as a tool compound since it displays the ability to bind other members of the DLK/JNK pathway. Namely, it has submicromolar ability to bind every other kinase downstream of DLK: MKK7 (27 nM), JNK2 (56 nM) and JNK3 (760 nM) [41]. Indazoles
KW-2449 (DLK K d = 67 nM, Figure 8) was designed as a multikinase inhibitor for leukemia with low nanomolar inhibitory potency against FLT3, ABL and Aurora kinases [49]. Interestingly, this compound has also demonstrated some binding potential against JNK2 (230 nM) and JNK3 (Kd = 51 nM), two JNK isoforms downstream of DLK along the neuroprotective pathway. This type of kinase profile, in combination with physicochemical properties that are more in line with CNS-based compounds (MW = 332, cLogP = 2.93, Serum total PSA = 57Å), are a prerequisite for DLK inhibitors. These characteristics make this compound a better staring point than many of the other binders and inhibitors outlined in this review. The related compound AG-013736 is 25-fold less potent than KW-2449, indicating that the piperazine (green) is beneficial for DLK binding and/or the thiobenzamide or pyridyl moiety (blue) is detrimental to binding. Given future science group
| Review
the smaller molecular weight of these compounds compared with other DLK binders, this series is certainly more likely to provide CNS-penetrable lead compounds. Indolocarbazoles
Cephalon (PA, USA) spent considerable effort optimizing indolocarbazoles for inhibition of mixed lineage kinases (F igure 9) . Although optimized for activity against MLK-1 and -2, some compounds within this series displayed some DLK off-target activity such as CEP-701 (Kd = 840 nM), which is also an 80 nM binder of MKK7, CEP-1347 (DLK IC50 = 114 nM),(+) K-252a (DLK IC50 = 360 nM), and CEP-5214 (DLK IC50 = 119 nM) [50]. CEP-1347 could be considered a pan MLK inhibitor and displays neuroprotective effects in several preclinical studies [4]. However, this compound was not effective in Phase II/III clinical trials for Parkinson’s disease, possibly due to the lack of target inhibition in the brain [51]. This group regularly measured biochemical inhibititory data for this series of compounds using a timeresolved fluorescence readout and MKK7 as a substrate, and for this reason they are referred to as inhibitors of DLK kinase activity [52]. Cephalon performed further modification of the sugar moiety and one of the indolo moieties, which led to diminished DLK potency [102]. This group has several recent patent publications outlining related compounds with ‘improved MLK and DLK activity’ without providing specific structure–activity relationship [102]. O N
N H
NH
N
KW-2449 (Kd = 67 nM)
O HN
N
S
N H
N
AG-013736 (Kd = 1700 nM)
Figure 8. Indazoles as dual leucine zipper kinase binders.
www.future-science.com
1929
Review | Ferraris, Yang & Welsbie H N
H N
O
N N
N
O
N
O
HO HO
an ROS-1 and AKT inhibitor. Both of these compounds have a better selectivity profile than, for example, the pyrimidines, but with a similar pharmacophore to other DLK binders.
O
Discovering
O O
OH
CEP-701 (Kd = 840 nM) H N
(+)K-252a (IC50 = 360 nM)
O
H N
O
S
S
O N
HO
O
N N
N H
O
HO
O
CEP-5214 (IC50 = 119 nM)
CEP-1347 (IC50 = 114 nM)
Figure 9. Indolocarbazole derivaties as dual leucine zipper kinase inhibitors. Miscellaneous
DLK binders Kinase profiling has also identified some miscellaneous binders of DLK (Figure 10), JNJ28312141 (Kd = 330 nM), a CSF-1 tyrosine kinase inhibitor [53], and crizotinib (Kd = 170 nM) [54],
HN
H N
N O
N
N O
JNJ-28312141 (Kd = 330 nM)
N
NH2 O
N
Cl F
N Cl HN Crizotinib (Kd = 170 nM)
Figure 10. Miscellaneous dual leucine zipper kinase binders.
1930
Future Med. Chem. (2013) 5(16)
CN
selective DLK inhibitors for neuroprotection One of the global problems in the kinase field is how to discover reasonably selective kinase inhibitors in order to adequately test the hypothesis of target inhibition leading to disease modification in a clinical setting. While there are several examples of DLK inhibitors and binders as outlined above, it would appear that all of these molecules interact with the ATP binding site of DLK. For this reason, it will be necessary to determine the desired kinase inhibition profile for a DLK inhibitor. For example, would it be desirable to have off target activity against other members of the JNK pathway? And to what extent? Or is it better to solely inhibit DLK, even teasing out selectivity among other MLKs [55]? The closest homologues to DLK are other members of the mixed lineage kinase subfamily, specifically LZK, indicating that selectivity between these two enzymes may be challenging. However, the homology between DLK and MLK1–4 is only approximately 40% between their respective catalytic regions, indicating that a modicum of selectivity may be achievable. Recent examples of kinase optimization efforts have demonstrated very systematic methods for achieving selectivity with ATP competitive inhibitors even over closely related isoforms [56]. Taken together, these studies indicate that there is a high probability of obtaining a DLK-selective ATP competitive inhibitor. Another possible strategy to discover selective DLK inhibitors is to target the dual leucine zipper regions of DLK. Several groups have demonstrated that dimerization is necessary for DLK activation. Logically, small-molecule disruption of this dimerization would curtail the autophosphorylation and activation of DLK, similar to inhibition of the catalytic machinery of the enzyme. This inhibition of protein/protein interactions has been demonstrated as a means to disrupt c-Myc/Max leucine zipper interaction leading to c-Myc-induced transcriptional activation [57]. Despite the paucity of examples of drugs that disrupt protein/protein interactions, it has been predicted that as much as 50% of these interactions can be druggable [58]. Whether or not the DLK dimerization is druggable, methods to screen for such small-molecule disruptors (e.g., fluorescence energy resonance transfer and future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions fluorescence polarization assays) are becoming more mainstream as methods of identifying hits. Another strategy to find selective DLK inhibitors is to screen for allosteric inhibitors. Allosteric inhibitors of kinases have been a topic of research for several years. The benefits of such inhibitors is that they are often unique to the target kinase, minimizing off-target interactions and thus improving the therapeutic window for such drugs [55]. Despite this promise, there are no documented drugs that are allosteric inhibitors of kinases and there have been no documented cases of allosteric DLK inhibitors, so this strategy would appear to have greater challenges than some of the others mentioned above. Even so, the technology for identifying allosteric inhibitors continues to improve [59] and much effort has been put forth to progress allosteric kinase inhibitors in the clinic [60]. Perhaps one of the biggest challenges in discovering a DLK selective inhibitor is to find one with the right balance of physicochemical properties. In general, kinase inhibitors are larger MW molecules that press the boundaries of oral availability and brain penetrability [61]. Both of these characteristics would be desirable for a DLK inhibitor for neuroprotection. Specifically, a DLK inhibitor would need lower molecular weight, lower polar surface area and fewer hydrogen bond donors than many of the compounds outlined in this review in order to penetrate the CNS [46]. Despite these hurdles and historically few examples of brainpenetrable kinase inhibitors [19], there have been recent success stories of brain-penetrable LRRK-2 inhibitors. These LRRK-2 inhibitors were discovered to be highly selective across the kinome, as well as displaying brain penetration when dosed orally across several species [62]. Summary To summarize, several groups have put forth data indicating that inhibition or knockdown of DLK is neuroprotective in animal models of neuronal injury, glaucoma, Parkinson’s disease and amyotrophic lateral sclerosis. However, there are still several challenges associated with targeting DLK for neuroprotection. Without crystallography data, little is known about isoforms of DLK [63]: are certain isoforms more prevalent in disease states? Is it possible to specifically target those isoforms? DLK is clearly a mediator of neuronal response to injury, but what factors tip the balance between apoptosis and neuroregeneration? And how does DLK inhibition affect these factors? DLK plays different roles depending on cell type, cell cycle, future science group
| Review
level of development, length and duration of injury and so forth. Do the cellular and animal models of these disease states adequately represent DLK as it would be in the human disease state? Several DLK binders and inhibitors are outlined above, but all were optimized for distinctly different kinases, many of which have anticancer activity, a fact that may prevent these compounds from being good chemical probes for assessing the neuroprotective ability of DLK inhibitors. DLK inhibition is acting primarily through the JNK pathway, which can involve the activation of a variety of transcription factors, scaffold proteins, adaptor proteins and other protein kinases [64]. What specific transcription factors/genes are affected when the DLK/JNK pathway is activated under neurological injury? Most of the compounds outlined in this review have liabilities such as low selectivity, low brain penetrability and limited chemistry space with regard to intellectual property. Future perspective Even taking into account the challenges outlined in this review, it is important to note that several facts make DLK a viable drug discovery target. Namely, both enzymatic binding and biochemical assays for screening potential inhibitors have been published. In addition, a few potent, tractable inhibitors and binders have been identified as outlined in this review. Furthermore, kinases are considered desirable, druggable therapeutic targets second only to G-protein coupled receptors. DLK, despite being a member of the mixed lineage kinase subfamily, only shares approximately 30–40% homology in the catalytic region with MLK1–3 implying that selectivity may be achievable. Because of these factors, medicinal chemistry groups interested in neuroprotective agents will start to discover more selective DLK inhibitors within the next 5 years. With selective inhibitors in hand, the tools will be available to further understand the nature of the DLK response to injury and its role in the JNK pathway under specific neurological conditions. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. www.future-science.com
1931
Review | Ferraris, Yang & Welsbie Executive summary Background Dual leucine zipper kinase (DLK) is a MAP3K primarily localized in neurons and plays a major mediating role in the JNK phosphorylation pathway priming the cell for either neuroregeneration or neuronal apoptosis. Characterization & lineage of human DLK
DLK is a mixed lineage kinase with several unique structural features such as dual leucine zippers that are necessary for homodimerization, autophosphorylation and activation of the kinase.
DLK shares 87% homology with LZK, but only 30–40% homology with other members of the mixed lineage kinase subfamily. Role of DLK in neuroprotection/axonal degeneration
Suppression of DLK leads to neuroprotective effects in vitro in retinal ganglion cells and dorsal root ganglia.
Suppression of DLK leads to neuroprotective effects in vivo in models of axonal degeneration and retinal degeneration.
Contrary to the multitude of neuroprotective effects of DLK, several studies have also outlined the necessity of DLK in neuronal regeneration after injury. Binders & inhibitors of DLK
Several different series of DLK binders and inhibitors such as quinolines, pyrimidines and indazoles have been identified, developing a preliminary pharmacophore for the enzyme and potential lead series for further optimization. Discovering selective DLK inhibitors
Obtaining brain penetrable and selective DLK inhibitors are two of the biggest challenges with this target. Selectivity may be obtained by allosteric inhibition or targeting the leucine zipper region.
References Papers of special note have been highlighted as: n of interest nn of considerable interest 1
n
2
3
4
5
6
7
Gallo Ka, Johnson Gl. Mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat. Rev. Mol. Cell Biol. 3(9), 663–672 (2002).
8
9
Outlines the members of the mixed lineage kinase family of which dual leucine zipper kinase (DLK) is a member.
Whitmarsh AJ, Davis RJ. Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals. Trends Biochem. Sci. 23(12), 481–485 (1998).
15 Watkins TA, Wang B, Huntwork-Rodriguez S
Ghosh AS, Wang B, Pozniak CD, Chen M, Watts RJ, Lewcock JW. DLK induces developmental neuronal degeneration via selective regulation of proapoptotic JNK activity. J. Cell Biol. 194(5), 751–764 (2013).
16 Miller BR, Press C, Daniels RW, Sasaki Y,
n
Zhu X, Raina AK, Rottkamp CA et al. Activation and redistribution of c-Jun N-terminal kinase/stress activated protein kinase in degenerating neurons in Alzheimer’s disease. J Neurochem. 76(2), 435–441 (2001).
11 Whitmarsh AJ, Cavanagh J, Tournier C,
Wang LH, Besirli CG, Johnson EM Jr. Mixedlineage kinases: a target for the prevention of neurodegeneration. Annu. Rev. Pharmacol. Toxicol. 44, 451–474 (2004).
12 Engstrom W, Ward A, Moorwood K. The role
Siddiqui MA, Reddy PA. Small molecule JNK (c-Jun N-terminal kinase) inhibitors. J. Med. Chem. 53(8), 3005–3012 (2010).
Identification, molecular cloning, and characterization of dual leucine zipper bearing kinase. A novel serine/threonine protein kinase that defines a second subfamily of mixed lineage kinases. J. Biol. Chem. 269(49), 30808–30817 (1994).
10 Tanoue T, Nishida E. Molecular recognitions
Milbrandt J, Diantonio A. A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration. Nat. Neurosci. 12(4), 387–389 (2009). 17 Chen X, Rzhetskaya M, Kareva T et al.
Antiapoptotic and trophic effects of dominantnegative forms of dual leucine zipper kinase in dopamine neurons of the substantia nigra in vivo. J. Neurosci. 28(3), 672–680 (2008).
in the MAP kinase cascades. Cell Signal. 15(5), 455–462 (2003). Yasuda J, Davis RJ. A mammalian scaffold complex that selectively mediates MAP kinase activation. Science 281(5383), 1671–1674 (1998). of scaffold proteins in JNK signalling. Cell Prolif. 43(1), 56–66 (2010).
18 Welsbie DS, Yang Z, GE Y et al. Functional
genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc. Natl Acad. Sci. USA 110(10), 4045–4050 (2013). nn
13 Holzman LB, Merritt SE, Fan G.
Graczyk PP. JNK inhibitors as antiinflammatory and neuroprotective agents. Future Med. Chem. 5(5), 539–551 (2013).
1932
et al. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury. Proc. Natl Acad. Sci. USA 110(10), 4039–4044 (2013).
Describes DLK signaling pathways for neuroregeneration and neuroprotection.
Behrens A, Sibilia M, Wagner EF. Aminoterminal phosphorylation of c-Jun regulates stress-induced apoptosis and cellular proliferation. Nat. Genet. 21(3), 326–329 (1999).
Ventura JJ, Hubner A, Zhang C, Flavell RA, Shokat KM, Davis RJ. Chemical genetic ana lysis of the time course of signal transduction by JNK. Mol. Cell 21(5), 701–710 (2006).
substrate. J. Biol. Chem. 274(15), 10195–10202 (1999).
n
First paper to fully characterize DLK.
14 Merritt SE, Mata M, Nihalani D, Zhu C, Hu
X, Holzman LB. The mixed lineage kinase DLK utilizes MKK7 and not MKK4 as
Future Med. Chem. (2013) 5(16)
The only manuscript utilizing a DLK inhibitor as a neuroprotectant.
19 Chico LK, Van Eldik LJ, Watterson DM.
Targeting protein kinases in central nervous system disorders. Nat. Rev. Drug Discov. 8(11), 892–909 (2009). nn
Excellent review profiling several kinase inhibitors that have been trialled in humans.
20 Choi S, Couture J-P, Blouin R. DLK (dual
leucine zipper-bearing kinase). In: Encyclopedia of Signaling Molecules. Choi S. (Ed.). Springer NY, USA, 522–526 (2012).
future science group
Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions 21 Hanks SK, Quinn AM, Hunter T. The protein
kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241(4861), 42–52 (1988).
critical for neuronal death. J. Biol. Chem. 283(28), 19704–19713 (2008). 33 Harrington EA, Bebbington D, Moore J et al.
VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat. Med. 10(3), 262–267 (2004).
22 Mata M, Merritt SE, Fan G, Yu GG, Holzman
LB. Characterization of dual leucine zipperbearing kinase, a mixed lineage kinase present in synaptic terminals whose phosphorylation state is regulated by membrane depolarization via calcineurin. J. Biol. Chem. 271(28), 16888–16896 (1996).
34 Hirai S, Cui De F, Miyata T et al. The c-Jun
N-terminal kinase activator dual leucine zipper kinase regulates axon growth and neuronal migration in the developing cerebral cortex. J. Neurosci. 26(46), 11992–12002 (2006).
23 Mooney LM, Whitmarsh AJ. Docking
interactions in the c-Jun N-terminal kinase pathway. J. Biol. Chem. 279(12), 11843–11852 (2004).
35 Lingor P, Koch JC, Tonges L, Bahr M. Axonal
degeneration as a therapeutic target in the CNS. Cell Tissue Res. 349(1), 289–311 (2012).
24 Cuny GD. Kinase inhibitors as potential
therapeutics for acute and chronic neurodegenerative conditions. Curr. Pharm. Des. 15(34), 3919–3939 (2009).
36 Xiong X, Collins CA. A conditioning lesion
protects axons from degeneration via the Wallenda/DLK MAP kinase signaling cascade. J. Neurosci. 32(2), 610–615 (2012).
25 Nihalani D, Merritt S, Holzman LB.
Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation. J. Biol. Chem. 275(10), 7273–7279 (2000). 26 Oliver AW, Knapp S, Pearl LH. Activation
37 Shin JE, Cho Y, Beirowski B, Milbrandt J,
Cavalli V, Diantonio A. Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration. Neuron 74(6), 1015–1022 (2012). 38 Tedeschi A, Bradke F. The DLK signalling
pathway-a double-edged sword in neural development and regeneration. EMBO Rep. doi:10.1038/embor.2013.64 (2013) (Epub ahead of print).
segment exchange: a common mechanism of kinase autophosphorylation? Trends Biochem. Sci. 32(8), 351–356 (2007). 27 Junius FK, O’Donoghue SI, Nilges M, Weiss
AS, King GF. High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer. J. Biol. Chem. 271(23), 13663–13667 (1996). 28 Sakuma H, Ikeda A, Oka S, Kozutsumi Y,
Zanetta JP, Kawasaki T. Molecular cloning and functional expression of a cDNA encoding a new member of mixed lineage protein kinase from human brain. J. Biol. Chem. 272(45), 28622–28629 (1997). 29 Nihalani D, Meyer D, Pajni S, Holzman LB.
Mixed lineage kinase-dependent JNK activation is governed by interactions of scaffold protein JIP with MAPK module components. EMBO J 20(13), 3447–3458 (2001). 30 Yasuda J, Whitmarsh AJ, Cavanagh J, Sharma
M, Davis RJ. The JIP group of mitogenactivated protein kinase scaffold proteins. Mol. Cell Biol. 19(10), 7245–7254 (1999). 31 Coffey ET, Smiciene G, Hongisto V et al.
c-Jun N-terminal protein kinase (JNK) 2/3 is specifically activated by stress, mediating c-Jun activation, in the presence of constitutive JNK1 activity in cerebellar neurons. J. Neurosci. 22(11), 4335–4345 (2002). 32 Bjorkblom B, Vainio JC, Hongisto V,
Herdegen T, Courtney MJ, Coffey ET. All JNKs can kill, but nuclear localization is
future science group
nn
Outlines the DLK signaling pathways in mammalian systems.
39 Hammarlund M, Nix P, Hauth L, Jorgensen
EM, Bastiani M. Axon regeneration requires a conserved MAP kinase pathway. Science 323(5915), 802–806 (2009). 40 Huebner EA, Strittmatter SM. Axon
regeneration in the peripheral and central nervous systems. Results Probl. Cell Differ. 48, 339–351 (2009). 41 Karaman MW, Herrgard S, Treiber DK et al.
A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26(1), 127–132 (2008). 42 Fabian MA, Biggs WH 3rd, Treiber DK et al.
A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23(3), 329–336 (2005). 43 Qian F, Engst S, Yamaguchi K et al.
Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res. 69(20), 8009–8016 (2009). 44 Vultur A, Buettner R, Kowolik C et al. SKI-
606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells. Mol. Cancer Ther. 7(5), 1185–1194 (2008).
www.future-science.com
| Review
45 Wissner A, Mansour TS. The development of
HKI-272 and related compounds for the treatment of cancer. Arch. Pharm. (Weinheim) 341(8), 465–477 (2008). 46 Hitchcock SA, Pennington LD. Structure-
brain exposure relationships. J. Med. Chem. 49(26), 7559–7583 (2006). 47 Galkin AV, Melnick JS, Kim S et al.
Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPMALK. Proc. Natl Acad. Sci. USA 104(1), 270–275 (2007). 48 Pardanani A, Gotlib JR, Jamieson C et al.
Safety and efficacy of TG101348, a selective JAK2 inhibitor, in myelofibrosis. J. Clin. Oncol. 29(7), 789–796 (2011). 49 Shiotsu Y, Kiyoi H, Ishikawa Y et al. KW-2449,
a novel multikinase inhibitor, suppresses the growth of leukemia cells with FLT3 mutations or T315I-mutated BCR/ABL translocation. Blood 114(8), 1607–1617 (2009). 50 Ruggeri B, Singh J, Gingrich D et al. CEP-
7055. a novel, orally active pan inhibitor of vascular endothelial growth factor receptor tyrosine kinases with potent antiangiogenic activity and antitumor efficacy in preclinical models. Cancer Res. 63(18), 5978–5991 (2003). 51 Parkinson Study Group PRECEPT
Investigators. Mixed lineage kinase inhibitor CEP-1347 fails to delay disability in early Parkinson disease. Neurology 69(15), 1480–1490 (2007). 52 Hudkins RL, Diebold JL, Tao M et al. Mixed-
lineage kinase 1 and mixed-lineage kinase 3 subtype-selective dihydronaphthyl(3,4-a) pyrrolo(3,4-c) carbazole-5-ones: optimization, mixed-lineage kinase 1 crystallography, and oral in vivo activity in 1-methyl-4phenyltetrahydropyridine models. J. Med. Chem. 51(18), 5680–5689 (2008). 53 Illig CR, Manthey CL, Wall MJ et al.
Optimization of a potent class of arylamide colony-stimulating factor-1 receptor inhibitors leading to anti-inflammatory clinical candidate 4-cyano-N-(2-[1-cyclohexen-1-yl]-4-(1[dimethylamino]acetyl)-4-piperidinyl)phenyl)-1H-imidazole-2-carboxamide (JNJ28312141). J. Med. Chem. 54(22), 7860–7883 (2011). 54 Cui JJ, Tran-Dube M, Shen H et al. Structure
based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J. Med. Chem. 54(18), 6342–6363 (2011). 55 Morphy R. Selectively nonselective kinase
inhibition: striking the right balance. J. Med. Chem. 53(4), 1413–1437 (2010).
1933
Review | Ferraris, Yang & Welsbie 56 Jimenez JM, Boyall D, Brenchley G et al.
Design and optimization of selective protein kinase C q(PKCq) inhibitors for the treatment of autoimmune diseases. J. Med. Chem. 56(5), 1799–1810 (2013). 57 Prochownik EV, Vogt PK. Therapeutic
targeting of Myc. Genes Cancer 1(6), 650–659 (2010). 58 Camacho CJ, Koes DR, Dömling AS.
Interactive technologies for leveraging the known chemistry of anchor residues to disrupt protein interactions. In: Protein–Protein Interactions in Drug Discovery. Dömling A. (Ed.). Wiley-VCH Verlag GmbH & Co. KGaA, Weinhelm, Germany, 85–100 (2013). 59 Lebakken CS, Reichling LJ, Ellefson JM,
Riddle SM. Detection of allosteric kinase
1934
inhibitors by displacement of active site probes. J. Biomol. Screen. 17(6), 813–821 (2012). 60 Fang Z, Grutter C, Rauh D. Strategies for the
selective regulation of kinases with allosteric modulators: exploiting exclusive structural features. ACS Chem. Biol. 8(1), 58–70 (2012). 61 Vieth M, Sutherland JJ, Robertson DH,
Campbell RM. Kinomics: characterizing the therapeutically validated kinase space. Drug Discov. Today 10(12), 839–846 (2005). 62 Estrada AA, Liu X, Baker-Glenn C et al.
Discovery of highly potent, selective, and brain-penetrable leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors. J. Med. Chem. 55(22), 9416–9433 (2012).
Future Med. Chem. (2013) 5(16)
63 Yan D, Jin Y. Regulation of DLK-1 kinase
activity by calcium-mediated dissociation from an inhibitory isoform. Neuron 76(3), 534–548 (2012). 64 Bogoyevitch MA, Kobe B. Uses for JNK: the
many and varied substrates of the c-Jun N-terminal kinases. Microbiol. Mol. Biol. Rev. 70(4), 1061–1095 (2006). Patents 101 Lewcock JW, Pozniak C, Senguota-Ghosh A:
US20120328609 (2012). 102 Becknell N, Brown PD, Dandu R et al.:
US8383813 (2013). 103 Hudkins RL, Dandu R, Tao M, Underiner
TL, Zulli AL: US8084472 (2011).
future science group
