International Journal of Alzheimer's Disease

Volume 2013 (2013), Article ID 809386, 8 pages

http://dx.doi.org/10.1155/2013/809386

Research Article

Preferential Selectivity of Inhibitors with Human Tau Protein Kinase Gsk3 Elucidates Their Potential Roles for Off-Target Alzheimer’s Therapy

Jagadeesh Kumar Dasappa and H. G. Nagendra

Department of Biotechnology, Sir M. Visvesvaraya Institute of Technology, Hunasamaranahalli, Via Yelahanka, Bangalore 562157, India

Received 29 June 2013; Accepted 30 August 2013

Academic Editor: Jesús Avila

Copyright © 2013 Jagadeesh Kumar Dasappa and H. G. Nagendra. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid beta peptides (A) and neurofibrillary tangles (NFTs). The abnormal phosphorylation of tau leads to the formation of NFTs produced by the action of tau kinases, resulting in the loss of neurons and synapse, leading to dementia. Hence, tau kinases have become potential drug target candidates for small molecule inhibitors. With an aim to explore the identification of a common inhibitor, this investigation was undertaken towards analyzing all 10 tau kinases which are implicated in phosphorylation of AD. A set of 7 inhibitors with varied scaffolds were collected from the Protein Data Bank (PDB). The analysis, involving multiple sequence alignment, 3D structural alignment, catalytic active site overlap, and docking studies, has enabled elucidation of the pharmacophoric patterns for the class of 7 inhibitors. Our results divulge that tau protein kinases share a specific set of conserved structural elements for the binding of inhibitors and ATP, respectively. The scaffold of 3-aminopyrrolidine (inhibitor 6) exhibits high preferential affinity with GSK3. Surprisingly, the PDB does not contain the structural details of GSK3 with this specific inhibitor. Thus, our investigations provide vital clues towards design of novel off-target drugs for Alzheimer’s.

1. Introduction

Alzheimer’s disease is the most common form of neurodegenerative disorders [1] characterized by the formation of extracellular deposits composed of amyloid beta peptide (A) [2] and masses of paired, helically wound protein filaments in the cytoplasm of neuronal cell bodies and neuritic processes called neurofibrillary tangles [3]. These NFTs are formed as a result of hyperphosphorylation of tau protein [4]. The tau proteins are phosphoproteins whose levels of phosphorylation are regulated by tau kinases and phosphatases [5]. Substantial evidence reveals the increased activity of glycogen synthase kinase 3 (GSK3) (also known as human tau protein kinase I) during AD. Similarly, P25/cyclin-dependent kinase 5 (Cdk5), dual-specific tyrosine [Y] regulated kinase 1A (Dyrk1A), and mitogen-activated protein kinases (MAPK) also possess higher activity in AD brain [6].

Thus, our work focuses on the chosen 10 kinases involved in hyperphosphorylation of tau and elevated responses in AD [7]. Kinase holds a large gene family and these domains are alike in sequence and structure. Developments of discriminating inhibitors are a key task in drug discovery and development, and appreciating the basis of kinase inhibitor selectivity is critical to the design of effective drugs.

GSK3 is composed of three domains: an N-terminal domain consisting of a closed -barrel structure, a C-terminal domain containing a “kinase fold” structure, and a small extradomain subsequent to the C-terminal domain. The catalytic site is between the two major domains and has an ATP analogue molecule in its ATPbinding site. The adenine ring is buried in the hydrophobic pocket and interacts specifically with the main-chain atoms of the hinge loop [8]. The structure of GSK3 is known to have a catalytically active dimer conformation that progressively phosphorylates substrates with Ser/Thr penta repeats [9]. It is known that the inhibitors compete with the ATP binding sites of GSK3 [10, 11]. Realizing the need to design the inhibitors for these kinases, with the hope that it could affect the hyperphosphorylation of tau, the investigations have been undertaken. The studies reveal shared conservations across sequences, structural homologies, and ATP binding site geometries across the ten tau kinases. Interestingly, the inhibitor 3-aminopyrrolidine scaffold exhibits high preferential affinity with GSK3. Though the literature on AD indicates overexpression and activity of GSK3 during pathogenesis of AD, surprisingly, the PDB does not contain the structural details of GSK3 with these specific inhibitors. Consequently, our explorations provide vital clues towards design of novel off-target drugs for AD.

2. Materials and Methods

The analysis of the structures of the 10 tau kinases, along with their respective ligands, was carried out as illustrated in the flow chart (Figure 1). To begin with, the structures of the 10 tau kinases, along with their respective ligands, were retrieved from the PDB and their details are provided in Table 1. A Phylogenetic tree was generated for the amino acid sequences of 10 tau kinases using CLUSTALW [12] and is illustrated in Figure 2. The dendrogram reveals that GSK3, CDK5, MAPK such as p38, extracellular signal-regulated kinases 1 & 2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and dual-specificity tyrosine-[Y]-phosphorylation-regulated kinase 1A (DYRK1A) are closely related (as in cluster 1); protein kinase A (PKA), protein kinase B (AKT/PKB), and protein kinase C (PKC) form the closely related second cluster. Interestingly, casein kinase 1 delta (CK1d) stands out as an independent taxis, indicating characteristic sequence variations from the group.

Table 1: Details of 10 tau kinases used in the analysis.

Sl. number
Protein name
UniProt ID
PDB ID
Number of residues
Ligand present in the PDB structure
Nomenclature of inhibitors in the study

(1)
Glycogen synthase kinase 3 beta (GSK3β)
P49841
1J1C
420
Adenosine-5′-diphosphate
—

(2)
Cyclin-dependent kinase 5 (CDK5)
Q00535
3O0G
292
4-Amino-2-4-chlorophenylamino-1,3-thiazol-5-yl3-nitrophenylmethanone
Inhibitor 1

(3)
p38 delta kinase (p38)
O15264
3COI
365
No Ligand
—

(4)
Mitogen-activated protein kinase 1 (Erk1/2)
P28482
1TVO
360
5-2-Phenylpyrazolo1,5-a]pyridin-3-yl-1h-pyrazolo3,4-c]pyridazin-3-amine Drug Bank ID DB07794
Inhibitor 2

(5)
Mitogen-activated protein kinase 10 (JNK3)
P53779
2O0U
464
N-{3-Cyano-6-3-1-piperidinyl]propanoyl-4,5,6,7-tetrahydrothieno2,3-cpyridin-2-yl}1-naphthalenecarboxamide
Inhibitor 3

(6)
Casein kinase 1 delta (CK1d)
P48730
3UYT
415
4-1-Cyclohexyl-4-4-fluorophenyl-1H-imidazol-5-ylpyrimidin-2-amine
Inhibitor 4

(7)
Dual-specificity tyrosine-Y-phosphorylation-regulated kinase 1A (DYRK1A)
Q13627
3ANR
763
7-Methoxy-1-methyl-9h-beta-carboline harmine complex Drug Bank ID DB07919
Inhibitor 5

(8)
Protein kinase A (PKA)
P17612
3MVJ
351
3R-1-5-Methyl-7H-pyrrolo2,3-dpyrimidin-4-ylpyrrolidin-3-amine
Inhibitor-6 (3-aminopyrrolidine scaffold)

(9)
Protein kinase B (PKB/AKT)
P31749
3MV5
480
3R-1-5-Methyl-7H-pyrrolo2,3-dpyrimidin-4-ylpyrrolidin-3-amine
Inhibitor-6 (3-aminopyrrolidine scaffold)

(10)
Protein kinase C alpha (PKC)
P17252
3IW4
672
3-1H-Indol-3-yl-4-2-4-methylpiperazin-1-ylquinazolin-4-yl-1H-pyrrole-2,5-dione
Inhibitor 7

Figure 1: Work flow chart.

Figure 2: Phylogenetic tree generated for sequences of the tau kinases using CLUSTALW software.

To observe the conservation of residues in the ATP binding region, a multiple sequence alignment was carried out amongst the 10 kinases, using the tool MultAlin [13]. The alignment is depicted in Figure 3(a), which reveals that the sequences differ significantly at the positions of gate-keeper residues and the surface residues, while they are well conserved at the subsites that interact directly with ATP. The ATP binding amino acids LYS 85 & ASP 200, ASP 133 & VAL 135 and GLU 185 are conserved across these kinases, and lie in the phosphate binding region, adenine binding region and in the sugar binding regions respectively (refer to Figure 3(b)). Overall sequence identity and similarity amongst the sequences and RMSD between the 3D geometries are highlighted in Table 2. The identity (and similarity) amongst the kinase sequences lies in the broad range of 22.8% (48.1%) and 49% (77.4%). The closest set appears between ERK1/2 and P38, which share the highest identity of 49%, while the least values of 22.8% exist between 2 sets, namely, CK1d and CDK5 and AKT and ERK1/2, respectively. The structural alignment to calculate the RMSD values was carried out using MultiProt [14]. It is interesting to note that the RMSD between all pairs of 10 tau kinases for C atoms range between 1.07 and 1.82 Å (refer to Table 2). Interestingly, these lowest and highest values of RMSD are related to the molecule PKC. However as expected, for the molecule CK1d which stands out in the phylogeny, the identities with all other kinases are less than 30%. Similarly, the RMSD values of all the kinases with CK1d lie in the higher range of 1.57 to 1.76 Å. Further, the structural alignments revealed that the active site occurs between two lobes of kinases showing the conserved overlap of the ATP binding regions. Thus, the RMSD values for the entire ATP binding region amongst the kinases lie between 0.63 and 1.25 Å. However, the RMSD values for the phosphate binding region lie between 0.23 and 0.46 Å, and the nucleotide binding region is in-between 0.16 and 1.25 Å respectively. Hence, docking the binding site of kinases with ATP was attempted to appreciate the nature of interactions. Table 3 provides the molecular details and properties of the various inhibitors used in the study. Their relative binding affinities in terms of IC50 and values are also indicated for each inhibitor (Table 3). The set of chosen inhibitors does contain two small molecules which are deposited in the drug bank, that is, inhibitor 2 (Drug Bank ID DB07794) and inhibitor 5 (Drug Bank ID DB07919). In order to appreciate the structural similarity of these inhibitors with ATP, 3D alignments were carried out across the 7 kinase inhibitors and ATP. The outcome of these structural comparisons revealed that the RMSD between ATP and the 7 ligands lie between 0.37 and 0.67 Å, while the inhibitor-to-inhibitor RMSD values are in the range of 0.32 to 0.67 Å. All the RMSD values are consolidated in Table 4.

Table 2: Percentage identity, similarity, and RMSD values (for the overlapping number of atoms).

GSK3β
CDK5
P38
ERK1/2
JNK
CK1d
DYRK1A
AKT
PKA

CDK5
#35.6%

@66.0%

*1.25 (238)
—
—
—
—
—
—
—
—

P38
32.3%

62.0%

*1.45 (221)
36.4%

64.9%

*1.52 (202)
—
—
—
—
—
—
—

ERK1/2
32.8%

64.5%

*1.43 (252)
37.6%

68.0%

*1.42 (233)
49.0%

77.4%

*1.60 (277)
—
—
—
—
—
—

JNK
28.0%

54.8%

*1.59 (238)
35.4%

64.3%

*1.61 (228)
47.7%

75.3%

*1.65 (265)
43.1%

73.8%

*1.46 (287)
—
—
—
—
—

CK1d
25.3%

49.6%

*1.64 (205)
22.8%

48.1%

*1.60 (200)
25.6%

54.0%

*1.72 (167)
29.5%

50.4%

*1.75 (195)
24.6%

50.8%

*1.76 (188)
—
—
—
—

DYRK1A
28.4%

53.2%

*1.29 (253)
30.4%

7.5%

*1.34 (243)
29.1%

53.6%

*1.52 (218)
28.3%

57.1%

*1.39 (246)
28.9%

52.3%

*1.72 (247)
24.5%

51.8%

*1.57 (213)
—
—
—

AKT
27.0%

48.4%

*1.55 (222)
25.4%

57.4%

*1.47 (214)
26.1%

56.7%

*1.61 (205)
22.8%

53.1%

*1.48 (231)
26.3%

52.5%

*1.64 (221)
25.8%

54.5%

*1.68 (207)
26.0%

56.4%

*1.36 (230)
—
—

PKA
26.4%

59.2%

*1.56 (213)
23.4%

58.9%

*1.50 (207)
26.6%

59.4%

*1.65 (183)
24.1%

53.9%

*1.65 (214)
26.3%

52.9%

*1.72 (194)
26.1%

53.8%

*1.76 (207)
26.0%

57.1%

*1.50 (219)
45.6%

77.2%

*1.21 (292)
—

PKC
24.8%

51.8%

*1.52 (223)
27.8%

61.9%

*1.45 (214)
27.9%

56.4%

*1.65 (203)
23.9%

56.2%

*1.68 (228)
24.6%

50.8%

*1.82 (221)
23.5%

55.8%

*1.70 (208)
25.9%

56.7%

*1.43 (224)
47.4%

79.2%

*1.07 (285)
32.8%

60.3%

*1.37 (279)

Identity, @similarity, and *RMSD (total number of atoms involved).

Table 3: Structures of 7 potential small molecule inhibitors and their relative binding affinities (IC₅₀ and ) values.

Sl. number
Ligand
Molecular formula
Molecular weight (Da)
IC₅₀-median inhibition concentration value
-inhibition constant value

(1)

C₁₆ H₁₁ Cl N₄ O₃ S 

Drug Bank ID DB07794
374.80
2000 nM
600 nM

(2)

C₁₈ H₁₃ N₇
327.34
1900 nM
140 nM

(3)

C₂₇ H₂₈ N₄ O₂ S
472.60
3200 nM
3200 nM

(4)

C₁₉ H₂₀ F N₅
337.39
130 nM
n/a

(5)

C₁₃ H₁₂ N₂ O  

Drug Bank ID DB07919
212.25
350 nM
n/a

(6)

C₁₁ H₁₅ N₅
217.27
3200 nM
n/a

(7)

C₂₅ H₂₂ N₆ O₂
438.48
2.1 nM
n/a

Table 4: RMSD values of ATP and the seven kinase inhibitors amongst each other.

Inhibitor 1
Inhibitor 2
Inhibitor 3
Inhibitor 4
Inhibitor 5
Inhibitor 6
Inhibitor 7

Inhibitor 1
—
—
—
—
—
—
—

Inhibitor 2
0.57
—
—
—
—
—
—

Inhibitor 3
0.46
0.32
—
—
—
—
—

Inhibitor 4
0.56
0.66
0.54
—
—
—
—

Inhibitor 5
0.43
0.41
0.33
0.52
—
—
—

Inhibitor 6
0.51
0.39
0.46
0.49
0.49
—
—

Inhibitor 7
0.53
0.54
0.41
0.54
0.41
0.55
—

ATP
0.37
0.64
0.58
0.54
0.51
0.67
0.48

(a)

(b)

Figure 3: (a) Alignment of tau kinases showing the conserved residues in the ATP binding region. (b) Highlight of sequence conservation across ATP binding residues.

The ATP binding site of GSK3 spans a long stretch containing 22 residues. Table 5 lists the various residues forming the ATP binding pocket and exhibiting favorable interactions with ATP. In order to appreciate the conservation of this ATP binding site across all 10 tau kinases, ATP molecule was docked and all plausible interactions within 6 Å were tabulated. Docking studies were carried out using the Discovery Studio software Version 3.5 (Accelrys Software Inc., USA) and Lead IT tool of FlexX 2.1.2. It is clear from Table 5 that G63, A83, K85, E97, D/E133, N186, and D200 form the set of well-conserved residues in the ATP binding pocket and interact with ATP in all the 10 kinases. Though the molecules JNK and ERK1/2 exhibit less number of interactions with ATP, key contacts of conserved residues are indeed present. Residue corresponding to V110 exhibits least number of interactions across kinases. Though the residue corresponding to R141 appears changed in all the kinases, its interactions with ATP, across the receptors, are well conserved.

Table 5: Interaction of binding site residues amongst the 10 tau kinases with ATP.

GSK3β
CDK5
P38
ERK1/2
JNK
CK1d
DYRK1A
AKT
PKA
PKC
ATP binding site conservation across the 10 kinases

#I 62
I10
V31
I 31
I 70
I 15-
I 165-
L156
L49
L345
I/V/L

#G 63
G11
G32
G32 >6
G71
G16
G166
G157
G50
G346
G-well conserved

#N 64
—
S33
E33 >6
—
—
K167
K158-
T51
K347
Not conserved

#G 65
—
G34
Y36
—
—
G168
G159
G52
G348 >6
Not conserved

#S 66
—
A35
—
—
—
S169 >6
—
S53
S349
Not conserved

#F 67
—
Y36
—
—
F20
F170 >6
F161
F54
—
Not conserved

#V 70
V18-
V39
V39
V78 >6
I 23
V173 >6
V164
V57
V353
V/I

#A 83
A31
A52
A52
A91
A 36
A186
A177
A70
A366
A-well conserved

#K 85
K33
K54-
K54-
K93
K38
K188
I179
K72-
K368-
K-well conserved

#E 97
E51 >6
E72
E71
E111
E52
E203
E198
E91
E387
E-well conserved

#V 110
V64 >6
I85 >6
I84 >6
I124 >6
P66 >6
V222
T211
V104
T401
V/I/P/T

#L 132
F80
M107
Q105-
M146-
M82-
F238
M227
M120
M417
F/Q/M

#D 133
E81-
P108-
D106-
E147
E-83-
E239-
E228-
E121-
E418-
D/E-well conserved

#Y 134
F82
F109
L107
L148
L84
M240
Y229
Y122
Y419
F/L/M/Y

#V 135
C83-
M110-
M108-
M149-
L85-
L241-
A230-
V123-
V420-
C/M/L/A/V

#T 138
D86-
D113-
D111-
N152-
S88-
N244
E 234-
E127-
D424-
D/N/S/E

#R 141
K89
K116
K 114
Q 155
D 91
D247
F237
S130
Y427 >6
R/K/Q/D/F/S/Y

#Q 185
Q130-
G 154
S153-
S193
D132
E291
E278-
E170
D467-
Q/G/S/D/E

#N 186
N131
N155 >6
N154
N194
N133
N292
N279
N171
N468
N-well conserved

#L 188
L133
A157
L156 >6
V196 >6
L135
L294 >6
M281
L173
M470
L/A/V/M

#C 199
A143
L167 >6
C166
L206
I148
V306
T291
T183
A480
C/A/L/I/V/T

#D 200
N144
D168
D167
D207
D149
D307-
D292
D184
D481-
D-well conserved

22
16
19
16
15
18
18
21
22
18
Number of interacting residues with ATP within 6 

Indicates residue type and number; *indicates distances in Angstroms.

Our aim to study the efficacies of select inhibitors with GSK3 triggered the need to dock these ligands to the key tau kinase GSK3. Identifying the ATP binding site, the molecular docking studies of kinase inhibitors were carried out using the Discovery Studio software Version 3.5 and Lead IT tool of FlexX 2.1.2. GSK3 receptor structure was docked to all of the seven ligands to the ATP binding pocket by the rigid receptor-flexible ligand docking competencies of FlexX and the interactions are tabulated in Table 6. The docking examination revealed that the inhibitor-3-aminopyrrolidine scaffold exhibits high preferential affinity with GSK3. While all inhibitors appear to sit in the ATP pocket of GSK3, the most favorable is inhibitor 6 and the least probable is inhibitor 5.

Table 6: Binding site residues of GSK3 interacting with 7 inhibitors when docked in the ATP Pocket.

Residues in the ATP binding site of GSK3β
ATP
Inhibitor 1
Inhibitor 2
Inhibitor 3
Inhibitor 4
Inhibitor 5
Inhibitor 6
Inhibitor 7
Number of interactions each residue has across inhibitors (within 6 )

I 62
4.4
—
3.62
2.35
4.09
3.98
2.90
2.84
6

G 63
5.4
—
—
—
—
—
4.50
3.20
3

N 64
5.24
—
—
—
—
—
—
4.56
4

G 65
4.03
—
—
—
—
—
—
—
1

S 66
2.82
—
—
—
—
—
—
—
1

F 67
3.21
—
—
—
—
—
—
—
1

V 70
5.9
4.09
—
5.20
—
—
5.37
—
4

A 83
5.46
4.96
5.51
—
5.80
—
5.03
—
5

K 85
2.64
3.03
—
3.21
5.02
—
—
2.84
5

E 97
4.59
—
—
—
—
—
—
—
1

V 110
5.1
—
3.35
—
—
—
—
—
2

L 132
5.7
—
4.41
5.69
—
—
5.11
—
4

D 133
2.58
2.62
2.88
3.21
2.87
—
3.03
—
6

Y 134
3.9
—
—
—
2.77
—
5.65
—
3

V 135
3.05
2.39
2.90
2.65
2.72
4.81
2.59
5.72
8

T 138
5.6
—
—
—
—
—
—
—
1

R 141
3.12
4.91
2.17
—
3.75
4.51
5.15
3.81
7

Q 185
2.98
—
—
2.99
5.89
—
2.94
2.90
5

N 186
3.45
—
—
4.64
—
—
4.368
—
3

L 188
5.8
4.03
5.12
—
—
—
—
—
3

C 199
4.63
4.05
—
—
—
—
—
—
2

D 200
4.84
2.70
4.57
3.45
—
—
—
—
4

Number of interacting residues across various inhibitors (within 6 )
22
9
9
9
8
3
11
7
—

3. Results and Discussion

In the present study, the analysis of 10 tau kinases implicated in AD has been performed to elucidate the conservation of the binding site and selectivity of the 7 inhibitors. Multiple sequence alignment, 3D structural alignment, catalytic active site overlap, and docking studies of inhibitors with GSK3 have been carried out to fingerprint the interactions with the key/gatekeeper residues in the ATPbinding pocket. The results highlight that tau protein kinases share common structural elements for the binding of the inhibitors and ATP. Comparatively, the inhibitor 3-aminopyrrolidine (inhibitor 6) exhibits high preferential affinity with GSK3. Interestingly, the literature on AD indicates the overexpression and activity of GSK3 during pathogenesis of AD, and surprisingly, the PDB does not contain the structural details of GSK3 with this specific inhibitor. Our studies disclose that regions of the active site which found high conservation across tau kinases may form the determinants for binding to the ligand. Interactions of 7 inhibitors with the remaining 9 tau kinases in AD are also being explored.

4. Conclusions

Our results indicate that the binding pocket of the 10 tau kinases is structurally conserved and offer a common feature of determinants with the 7 inhibitors investigated. Specific analysis with GSK3 reveals preferential binding to 3-aminopyrrolidine scaffold. This study highlights that suitable therapeutics can be successfully developed from the available chemical space, thus facilitating inhibitor design and off-target effects for AD.

Conflict of Interests

The authors declare that this paper has no conflict of interests with FlexX 2.1.2. and Accelrys Software Inc., USA, version 3.5 software, which is a licensed version.

Acknowledgments

The authors gratefully acknowledge the generous support and facilities extended by Sir M. Visvesvarya Institute of Technology and Sri Krishnadevaraya Educational Trust, Bangalore, to this project.

References

M. L. Hemming, J. E. Elias, S. P. Gygi, and D. J. Selkoe, “Identification of beta-secretase (BACE1) substrates using quantitative proteomics,” PloS ONE, vol. 4, no. 12, Article ID e8477, 2009.

D. J. Selkoe, “Amyloid β-protein and the genetics of Alzheimer's disease,” The Journal of Biological Chemistry, vol. 271, no. 31, pp. 18295–18298, 1996.

D. J. Selkoe, “Cell biology of protein misfolding: the examples of Alzheimer's and Parkinson's diseases,” Nature Cell Biology, vol. 6, no. 11, pp. 1054–1061, 2004.

K. Iqbal, F. Liu, C.-X. Gong, and I. Grundke-Iqbal, “Tau in Alzheimer disease and related tauopathies,” Current Alzheimer Research, vol. 7, no. 8, pp. 656–664, 2010.

L. Martina, X. Latypovac, C. M. Wilsona et al., “Tau protein kinases: involvement in Alzheimer’s disease,” Ageing Research Reviews, vol. 12, no. 1, pp. 289–309, 2013.

S.-H. Chung, “Aberrant phosphorylation in the pathogenesis of Alzheimer's disease,” BMB Reports, vol. 42, no. 8, pp. 467–474, 2009.

I. Ferrer, T. Gomez-Isla, B. Puig et al., “Current advances on different kinases involved in tau phosphorylation, and implications in Alzheimer's disease and tauopathies,” Current Alzheimer Research, vol. 2, no. 1, pp. 3–18, 2005.

M. Aoki, T. Yokota, I. Sugiura et al., “Structural insight into nucleotide recognition in tau-protein kinase I/glycogen synthase kinase 3β,” Acta Crystallographica D, vol. 60, no. 3, pp. 439–446, 2004.

W. Ji and I. Ha, “Drug development for Alzheimer’s disease: recent progress,” Experimental Neurology, vol. 19, no. 3, pp. 120–131, 2010.

F. Liu, Z. Liang, J. Shi et al., “PKA modulates GSK-3β- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners,” FEBS Letters, vol. 580, no. 26, pp. 6269–6274, 2006.

A. Martinez, C. Gil, and D. I. Perez, “Glycogen synthase kinase 3 inhibitors in the next horizon for Alzheimer's disease treatment,” International Journal of Alzheimer's Disease, vol. 2011, Article ID 280502, 7 pages, 2011.

http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/

F. Corpet, “Multiple sequence alignment with hierarchical clustering,” Nucleic Acids Research, vol. 16, no. 22, pp. 10881–10890, 1988.

M. Shatsky, R. Nussinov, and H. J. Wolfson, “A method for simultaneous alignment of multiple protein structures,” Proteins, vol. 56, no. 1, pp. 143–156, 2004.