Microglia-Based Phenotypic Screening Identifies a ... - ACS Publications

Subscriber access provided by United Arab Emirates University | Libraries Deanship

Article

Microglia-based phenotypic screening identifies a novel inhibitor of neuroinflammation effective in Alzheimer’s disease models Wei Zhou, Guifa Zhong, Sihai Fu, Hui Xie, Tianyan Chi, Luyi Li, Xiurong Rao, Shaogao Zeng, Dengfeng Xu, Hao Wang, Guoqing Sheng, Xing Ji, Xiaorong Liu, XueFei Ji, Donghai Wu, Libo Zou, Micky D. Tortorella, Kejian Zhang, and Wenhui Hu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00125 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 9, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

1

Microglia-based phenotypic screening identifies a novel inhibitor of

2

neuroinflammation effective in Alzheimer’s disease models

3

Wei Zhou1,5,#, Guifa Zhong1,# , Sihai Fu2, Hui Xie3, Tianyan Chi4, Luyi Li4, Xiurong Rao1,

4

Shaogao Zeng1, Dengfeng Xu1, Hao Wang1, Guoqing Sheng1, Xing Ji2, Xiaorong Liu1, Xuefei

5

Ji4, Donghai Wu1, Libo Zou4, Micky Tortorella1, Kejian Zhang2 & Wenhui Hu1, *

6

1

7

Chinese Academy of Sciences, Guangzhou 510530, People's Republic of China

8

2

9

510663, People’s Republic of China

State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health,

Department of Pharmacy, South China Center of Innovative Pharmaceuticals, Guangzhou

10

3

11

Republic of China

12

4

13

People's Republic of China

14

5

15

Engineering, Guangdong University of Technology, Guangzhou 510003, People’s Republic of

16

China

17

#

18

*

The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, People's

Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016,

Institute of Natural Products and Green Chemistry, School of Light Industry and Chemical

These authors contributed equally to this work. Corresponding author W.H. (E-mail: [email protected]).

19

1 / 37

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 42

20

ABSTRACT

21

Currently, anti-AD drug discovery using target-based approaches is extremely

22

challenging due to unclear aetiology of AD and absence of validated therapeutic

23

protein targets. Neuronal death, regardless of causes, plays a key role in AD

24

progression, and it is directly linked to neuroinflammation. Meanwhile, phenotypic

25

screening is making a resurgence in drug discovery process as an alternative to

26

target-focused approaches. Herein, we employed microglia-based phenotypic

27

screenings to search for small molecules that modulate the release of detrimental

28

proinflammatory cytokines. The identified novel pharmacological inhibitor of

29

neuroinflammation (named GIBH-130) was validated to alter phenotypes of

30

neuroinflammation in AD brains. Notably, this molecule exhibited comparable in

31

vivo efficacy of cognitive impairment relief to donepezil and memantine

32

respectively in both β amyloid-induced and APP/PS1 double transgenic Alzheimer’s

33

murine models at a substantially lower dose (0.25 mg/kg). Therefore, GIBH-130

34

constitutes a unique chemical probe for pathogenesis research and drug development

35

of AD, and it also suggests microglia-based phenotypic screenings that target

36

neuroinflammation as an effective and feasible strategy to identify novel anti-AD

37

agents.

38

KEYWORDS

39

Alzheimer’s disease, neuroinflammation, inhibitor, animal models;

2 / 37


Page 3 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40

INTRODUCTION

41

Alzheimer’s disease (AD), a complex neurodegenerative disorder in the central

42

nervous system (CNS), is clinically characterized with progressive impairments in

43

memory and cognition functions1. However, insufficient understanding of the

44

pathophysiology limits available therapies to symptomatic relief, rather than

45

disease-modifying. Currently, available drugs for AD, e.g. acetylcholinesterase

46

inhibitor donepezil and N-methyl-D-aspartic acid (NMDA) receptor antagonist

47

memantine, which modulate neuronal signal transduction, only attenuate cognitive

48

decline and other negative symptoms of the disease. No new anti-AD drugs has been

49

approved for decades. Increasing life expectancy of humans and the rising incidence

50

of AD provoke a pressing demand for innovative therapies that can either slow or

51

stop the progression of this neurodegenerative disorder2.

52

The complexity and unclear aetiology of AD make anti-AD drug discovery a

53

challenging task3. Recently, the most popular target-centric paradigm was

54

questioned because of the consecutive failures of some promising candidate drugs in

55

phase III clinical trials. The perceived decline of productivity in drug research and

56

development focusing on target-based enzyme screenings compels us to seek

57

alternative strategies. Meanwhile, the advent of the cholesterol-lowering drug

58

ezetimibe and the drug of intermittent claudication, cilostazol, reminds us that

59

conventional approaches based on phenotypic screenings should not be marginalized

60

and it may preferentially suit poorly understood complex diseases, like AD4, 5. 3 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 42

61

Therefore, we switched our strategy to the conventional drug discovery approach

62

that employs phenotypic screenings to identify small molecules with in vivo

63

therapeutic potential against AD.

64

Microglia-dominated neuroinflammation is a prominent feature of AD and it

65

probably plays a substantial role in AD progression6. Chronic and sustained

66

microglia activation results in over production of neurotoxic factors, like nitric oxide

67

(NO), and proinflammatory cytokines, e.g. tumour necrosis factor α (TNF-α) and

68

interleukin 1β (IL-1β)7, 8. These mediators may directly induce neuronal apoptosis

69

or amplify the local inflammatory response, which leads to possible synaptic

70

dysfunction or neuronal loss9. An inflammatory environment might activate the tau

71

hyperphosphorylation kinases to promote the formation of neurofibrillary tangles.

72

The loss of tau’s normal microtube-stabilizing function would compromise axonal

73

transport and thus lead to synaptic dysfunction and neurodegeneration10. Neuronal

74

damage or death may also induce microglial activation, which facilitate the

75

propagation of a localized, detrimental cycle of neuroinflammation11-13. However,

76

this pathway has not been explored in depth in previous anti-AD drug discovery

77

studies. The accumulating evidence has already revealed the feasibility of modifying

78

AD through intervention of neuroinflammation14, 15, but few small molecules with

79

promising in vivo efficacy were developed to support this concept and confirm their

80

disease-modifying effects16. Therefore, it would be valuable attempt to search for

4 / 37


Page 5 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


81

novel compounds that alter the phenotypes of neuroinflammation and bear favorable

82

pharmacological efficacy in vivo to develop potential anti-AD agents.

83

We previously identified an anti-neuroinflammatory molecule (compound 1,

84

Fig. 1) that showed preliminary effects of memory restoration in an Aβ-induced AD

85

mouse model17. Poor pharmacokinetic (PK) properties of the agent, especially

86

insufficient brain-blood barrier (BBB) permeability and short half-life of oral

87

administration, prevented further investigation of this compound. Here, we reported

88

the identification of a pyridazine-3-carboxamide derivative, named GIBH-130

89

(compound 4, Fig. 1), as a novel anti-neuroinflammatory agent that was identified

90

through microglia-based phenotypic screenings. We also characterized its

91

anti-neuroinflammatory efficacy and therapeutic potential in AD animal models.

92

RESULTS AND DISCUSSION

93

Identification of GIBH-130 and its anti-neuroinflammatory effect in vitro.

94

Lipopolysaccharides (LPS) stimulated microglia was adopted as the phenotypic

95

screening assay to search for small molecules that suppress microglia activation. In

96

the screenings, IL-1β, one of the main proinflammatory cytokines produced by

97

activated microglia, was set as the phenotypic marker, reducing levels of which were

98

used to assess anti-neuroinflammation potency of compounds.

99

With this model, we developed an anti-neuroinflammatory molecule

100

(compound 1, Fig. 1)17. Further attempt to topically modify compound 1 was

101

unsuccessful. Therefore we used a scaffold-hopping strategy, which employed 5 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 42

102

different bioisosteres to replace the original pharmacophore the pyridazine of

103

compound 1, to search for novel chemical structures. The new scaffold of

104

quinazoline introduced in compound 2 (Fig. 1) endowed it with wide chemical space

105

for further structural modification, while retaining its anti-neuroinflammatory

106

efficacy (IC50 against IL-1β on N9 microglia, 8.3 µM compared to 0.87 µM of

107

compound 1). Compound 2 was devoid of metabolic vulnerability of the thiophene

108

motif, so it had longer half-life in mice. The insertion of a carbonyl group between

109

the scaffold and the pharmacophore of compound 2, which led to the synthesis of

110

compound 3, greatly enhanced its potency in reducing IL-1β secretion from

111

microglia (IC50 0.43 µM). However, a large conjugate plane formed within

112

compound 3 (Fig. 1), which may result in poor solubility in water or blood, made it

113

difficult to absorb or penetrate the BBB to enter the brain. So compound 3 was less

114

likely to become a drug that was supposed to use in CNS. Inspired by the great

115

improvement of activity by the insertion of a carbonyl group between the

116

pharmacophore and the scaffold, another series of compounds based on compound 1

117

were prepared to improve in vitro efficacy. Compound 4 (Fig. 1, named GIBH-130,

118

IC50 3.4 nM) was identified in screenings as one of the most effective inhibitors with

119

an acceptable half-life.

120

Figure 1

121

LPS-stimulated microglia possess the phenotypes of cytotoxicity and

122

inflammation, both of which are considered as major contributors to neuronal 6 / 37


Page 7 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


123

damage in inflammation-mediated neurological disorders, such as AD18,

124

Therefore, we investigated the inhibitory effect of GIBH-130 on the production of

125

NO, TNF-α and IL-1β by LPS-stimulated microglial cells. GIBH-130 showed no

126

significant impact on N9 microglial cells viability below concentration of 20 µM

127

(Fig. S2a,b of Supporting Information). Pretreatment of microglia with GIBH-130

128

significantly reduced the production of these factors in response to LPS stimulation,

129

and the extent of the reduction was dependent on the concentrations of GIBH-130

130

(Fig. 2a,b). The IC50 values of GIBH-130 for NO and TNF-α inhibition were 46.24

131

and 40.82 µM, respectively. Notably, pretreatment with GIBH-130 significantly

132

suppressed the IL-1β secretion by activated microglia (Fig. 2c, IC50 = 3.4 nM).

133

GIBH-130 showed an over 200-folds improvement in the potency of IL-1β

134

suppression compared to our lead compound 1 (IL-1β, IC50 = 2.4 µM on BV2, 0.87

135

µM on N9)17. These results showed that GIBH-130 restrained the secretion of

136

cytotoxic and proinflammatory factors by LPS-activated microglia in vitro, and it

137

might

138

neuroinflammation cycle at early stages, which were mediated by activated

139

microglia in AD brains.

alleviate

neurotoxic

oxidative

stress and

prevent

the

19

.

deleterious

140

Figure 2

141

Microglia adopt classical (M1) activation after LPS20 or Aβ21 stimulation, and

142

release pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-12, IL-23 and

143

corresponding toxic intermediates. Minocycline is an extensively studied inhibitor of 7 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 42

144

microglia activation that selectively inhibits M1 polarization of microglia22. The

145

inhibitory efficiency of GIBH-130 at 20 nM was comparable to 20 µM minocycline

146

against IL-1β release. IL-1β is one of the major cytokines during neuroinflammatory

147

progression of AD. So, it is meaningful to explain the selectivity of GIBH-130

148

against IL-1β (IC50 value 3.4 nM) over NO and TNF-α (IC50 value 46.24 and 40.82

149

µM, respectively). The selectivity is presumably due to that GIBH-130 may also

150

interfere the maturation of IL-1β from inactive IL-1β precursor23 or microglia

151

activation amplification by IL-1β itself24.

152

Druglikeness Assessment of GIBH-130. The pharmacokinetic properties of GIBH-130

153

were assessed in Sprague Dawley rats. As a potential drug candidate targeting in

154

CNS, GIBH-130 was found to be orally bioavailable in rats, with 74.91%

155

bioavailability and 4.32 h half-life (Table 1 and Table S1 of Supporting Information).

156

In addition, GIBH-130 displayed good penetration ability across blood-brain barrier

157

(AUCBrain/Plasma = 0.21, Table 1, Fig. 2S c,d of Supporting Information). Moreover,

158

GIBH-130 may present a low risk of heart QT interval prolongation, with an IC50

159

value of over 100 µM against hERG (a potassium voltage-gated channel).

160

Furthermore, consecutive administration of GIBH-130 up to 250 mg/kg for 7 days did

161

not alter body weight and behavioural flexibility (Fig. S2e of Supporting

162

Information). From the PK and safety analyses, we considered that oral administration

163

of GIBH-130 could maintain plasma and brain concentration without causing obvious

164

toxic effect, which could serve as a guide of in vivo assessment. 8 / 37


Page 9 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


165

Table 1

166

GIBH-130 in an Aβ β 25-35-injected rat model of AD. The inhibition of

167

neuroinflammation by GIBH-130 is supposed to reduce the detrimental neurotoxic

168

proinflammatory factors and neuroinflammation amplification, both of which are

169

associated with neuronal degeneration25,

170

neuronal degeneration. Therefore, we speculated that GIBH-130 could decelerate

171

AD progression by attenuating the microglial overproduction of various neurotoxic

172

and proinflammatory factors.

26

. AD is characterized by progressive

173

To establish a rat model of AD, we injected Aβ25-35 into the hippocampus of

174

rats to induce an AD-like disorder and then performed behavioural performance

175

tests to assess the beneficial effect of daily oral administration of GIBH-130 in these

176

animals. Five groups of Aβ25-35-injected rats received 3 different doses of GIBH-130

177

(high, 0.18 mg/kg; middle, 0.02 mg/kg; low, 0.0022 mg/kg) and anti-AD drug

178

donepezil (0.9 mg/kg) and memantine (1.8 mg/kg). Their memory performances

179

were compared with those in the non-treated model group and the sham-operated

180

group. Behavioural changes in each group were comprehensively examined using

181

novel object recognition test (NORT), Morris water maze test (MWMT), Y maze

182

test (YMT) and step-through test (STT), which reflected visual recognition memory,

183

spatial recognition memory, short-term memory and long-term memory impairment,

184

respectively (Fig. 3a).

9 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 42

185

The retention session was performed 24 h after the training session of NORT,

186

and rats in the sham-operated group spent a relatively longer time exploring the

187

novel object than those in the model group, which resulted in a significantly higher

188

discrimination index (Fig. 3b) and preference index (Fig. 3c) in the sham group than

189

in the model group. Similar to the donepezil and memantine groups, oral

190

administration of GIBH-130 (0.18 mg/kg) improved visual recognition memory in

191

Aβ25-35-injected rats, which was indicated by the restoration of discrimination index

192

(Fig. 3b) and preference index (Fig. 3c). Moreover, treatment of Aβ25-35-injected rats

193

with GIBH-130 restored the discrimination and preference indexes 1 hour after the

194

training, indicating a beneficial effect of GIBH-130 on short-term visual recognition

195

memory (Fig. S3a,b of Supporting Information).

196

Next, MWMT (Fig. 3a) was performed to assess the effect of GIBH-130

197

treatment on impairments in spatial recognition memory in Aβ25-35-injected rats. The

198

model group rats swam longer distances until they reached a hidden safety platform

199

than the sham-operated group during all 4 days of training for reference memory

200

task. Notably, decreases in the swimming distance (Fig. 3d) and time (Fig. S3c of

201

Supporting Information) were observed from the 3rd day on, not only in the

202

donepezil-treated

203

GIBH-130-treated groups. The results suggested that GIBH-130 showed a

204

therapeutic effect against deficits in spatial reference memory in Aβ25-35-injected rats

205

similar to donepezil and memantine. Although Aβ25-35-injected rats swam for

and

memantine-treated

groups,

but

also

in

all

three

10 / 37


Page 11 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


206

significantly less time (Fig. 3e) and less distance (Fig. S3d of Supporting

207

Information) than sham-operated rats in the target quadrant during a following 90-s

208

probe trial task, donepezil and GIBH-130 (0.18 mg/kg) treatment improved this

209

performance. These results demonstrated that GIBH-130 treatment relatively

210

alleviated spatial working memory impairment induced by Aβ25-35 injection.

211

Partial restoration of the short-term and long-term memory of Aβ25-35-injected

212

rats was also observed following GIBH-130 treatment in the YMT (Fig. S3e of

213

Supporting Information) and the STT (Fig. S3f of Supporting Information),

214

respectively. These results exhibited that donepezil and memantine symptomatically

215

alleviated impairments in different aspects of memory induced by Aβ25-35, which

216

was consistent with effects of the two drugs on impairments induced by

217

Aβ1-40-injection27, 28. The therapeutic effect produced by GIBH-130 (0.18 mg/kg)

218

was comparable to donepezil (0.9 mg/kg) and memantine (1.8 mg/kg), which

219

suggested that GIBH-130 was more potent than the previously described

220

anti-neuroinflammatory compound 115.

221

Figure 3

222

GIBH-130 in an APP/PS1 transgenic Alzheimer mouse model. We assessed the

223

ability of GIBH-130 to improve behavioural performance in an APP/PS1 transgenic

224

mouse model (Fig. 4a) to further validate its therapeutic effects. Because transgenic

225

mice possesses features of early AD, including impairments in synaptic plasticity,

226

increased levels Aβ in brain and abnormal short-term memory29, 30. 11 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 42

227

The effects of GIBH-130 on APP/PS1 transgenic mice in the MWMT was

228

consistent with the effect on Aβ25-35 hippocampal-injected rats. The swimming

229

distance (Fig. 4b) and swimming time (Fig. S4a of Supporting Information) of

230

APP/PS1 transgenic mice to the safety platform in the Morris water maze was

231

significantly longer than the control C57BL/6J mice beginning on day 3 of the

232

reference memory task. The daily oral administration of donepezil (1.3 mg/kg),

233

memantine (2.6 mg/kg) or GIBH-130 (0.028 or 0.25 mg/kg) reversed this

234

impairment in spatial reference memory in a dose-dependent manner (Fig. 4b, Fig.

235

S4a of Supporting Information).

236

Then, in the 90-s probe trial task, the APP/PS1 transgenic mice spent less time

237

(Fig. 4c) swimming in the target quadrant where the safety platform was previously

238

located than control mice. The percentage of path length (Fig. S4b of Supporting

239

Information) in the target quadrant was also reduced in the transgenic mouse group.

240

Treatment of APP/PS1 mice with donepezil or GIBH-130 (0.028 or 0.25 mg/kg)

241

significantly prolonged the swimming time in the target quadrant. Moreover, the

242

time needed by control mice to escape to a randomly-located safety platform in a

243

repeated acquisition task was reduced from the 3rd trial onwards, whereas the

244

escape latency of APP/PS1 transgenic mice was significantly prolonged. Treatment

245

with donepezil (1.3 mg/kg), memantine (2.6 mg/kg) or GIBH-130 (0.0031-0.25

246

mg/kg) caused a dose-dependent reversal of impairments in spatial working memory

247

(Fig. 4c) in the 90-s probe trial task and repeated acquisition task. Treatment of these 12 / 37


Page 13 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


248

mice with GIBH-130 significantly attenuated the impairment of visual recognition

249

memory in the NORT (Fig. S4c, d of Supporting Information), as well as short-term

250

memory in the YMT (Fig. S4e, f of Supporting Information). and long-term memory

251

in the STT (Fig. S4f, f of Supporting Information).

252

In this study, donepezil and memantine effectively improved both spatial

253

reference and working memory in APP/PS1 transgenic AD mice as previously

254

reported31,

255

approximately one 50th doses of donepezil and memantine, possibly via a

256

mechanism that may be distinguishable with the two anti-AD drugs. The overall

257

cognitive and functional improvements of GIBH-130 in two AD animal models

258

confirmed its potential for further drug development.

259

Figure 4

260

Phenotypes of neuroinflammation suppression in vivo. We then investigated

261

phenotype alterations of neuroinflammation oby GIBH-130 in Aβ1-42-injected mice.

262

An intrahippocampal injection of Aβ1-42, which stimulated the Aβ burden in brain,

263

was performed to establish an AD mouse model that also exhibited phenotypes of

264

neuroinflammation. The surgery altered the levels of inflammatory cytokines, such

265

as the down-regulation of IL-4 (Fig. 5a) and the up-regulation of IL-6 (Fig. 5b),

266

compared to control or sham-operated mice. GIBH-130 therapy (oral administration

267

dose of 0.025 mg/kg or 0.25 mg/kg daily) for 8 consecutive days after the surgery

268

partially restored the IL-4 and IL-6 levels in the cortex and hippocampus of

32

. GIBH-130 provided comparable memory restoration effects at

13 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 42

269

Aβ1-42-injected mice (Fig. 5a,b). We then examined changes of CD11b in frozen

270

brain sections of mice in each group using immunohistochemical staining. CD11b in

271

the Aβ1-42-injected model group was remarkably increased compared to the

272

sham-operated group. We observed significant reduction of CD11b (Fig. 5c) in

273

groups received GIBH-130 orally for 8 days (0.025 mg/kg or 0.25 mg/kg daily).

274

Moreover, a dose-dependent response was observed in groups treated with different

275

doses of GIBH-130.

276

IL-4,

known as one of the main anti-inflammatory cytokines in

277

neuroinflammation, has been reported to attenuate the neuroinflammation induced

278

by Aβ and Alzheimer’s disease-like pathogenesis in APP/PS1 double transgenic

279

mice33,34. In our assay, IL-4 levels were upregulated significantly in Aβ1-42-injected

280

mice after GIBH-130 treatment, approximately to normal levels. Similarly, IL-6,

281

which acted as an important proinflammatory cytokine in microglia-mediated

282

neuroinflammation,

283

specifically, CD11b, a typical protein on the membrane of activated microglia, was

284

reduced by GIBH-130 dose-dependently. These observations indicated that

285

GIBH-130 suppress the neuroinflammation in the brains of AD mice.

was

decreased

following

GIBH-130

treatment.

More

286

We also used transmission electron microscopy to assess the impact of

287

GIBH-130 treatment on the ultrastructure of hippocampal CA1 neurons in

288

Aβ1-42-injected mice (Fig. 5d). Images of control or sham-operated mice showed

289

good neuronal form, including a clear outline of nuclear membranes, 14 / 37


Page 15 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


290

well-distributed chromatin and abundant organelles. However, mice in the AD

291

model group demonstrated degenerative morphologies of neurons in hippocampus

292

CA1 region, such as heterochromatin aggregation, reduction of organelles and

293

nuclear envelope breakdown. Normal neuronal morphology was partially restored in

294

Aβ1-42 injected mice treated with GIBH-130, especially in the high dose group (0.25

295

mg/kg). Therefore, GIBH-130 reversed morphology alterations of neurons, and it

296

may protect neurons from degeneration caused by Aβ burden.Transmission electron

297

microscopy revealed that GIBH-130 may maintain normal neuronal morphology in

298

hippocampus. The facts that GIBH-130 protected neurons and controlled

299

neuroinflammation in brain may provide some explanation for the alleviation of

300

behavioural deficits by GIBH-130.

301

Figure 5

302

CONCLUSIONS

303

GIBH-130 was an effective inhibitor of neuroinflammation and a drug

304

potential candidate for AD treatment. It has been approved by China Food and Drug

305

Administration for clinical trials.The molecular mechanism of action of this agent

306

are under investigation. Though we have precluded some typical targets that are

307

reportedly involved in AD pathogenesis, inflammation or memory enhancement,

308

such as γ secretase, BACE1, cholinesterase, p38-MAPK and acetylcholine receptors.

309

We also tried chemical probes for target fishing and this work is ongoing.

15 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 42

310

In general, our data suggested that a pyridazine-3-carboxamide compound

311

(GIBH-130) with fine druglikeness properties suppressed neuroinflammation and

312

ameliorated memory impairments in two AD animal models. Our studies provide

313

some evidence to support neuroinflammation as an alternative viable target for the

314

treatment of AD14. Moreover, neuroinflammation is widely implicated in the

315

pathogenesis of other CNS disorders, such as Parkinson's disease35, Huntington's

316

disease36 and ischemic stroke37, which may provide additional therapeutic potential

317

to the agent. On the other hand, possible risks of side effects induced by improper

318

modulation of microglia activation or undesired inhibition of peripheral

319

inflammation require careful consideration. Further insight into the molecular

320

mechanisms of GIBH-130 in neuroinflammation inhibition will help identify new

321

chemical structures for the drug development of neuroinflammation-implicated

322

diseases.

323

METHODS

324

Synthesis

325

3-bromo-4-methyl-6-phenylpyridazine as described in the Supporting Information.

326

Chemical characterization data are also presented (Fig. S1a,b,c,d of Supporting

327

Information).

328

Cell culture. The murine microglial cell line N9 (N9 cells) were purchased from

329

Shanghai Bioleaf Biotech Co., Ltd (China). N9 cells were cultured in DMEM

330

supplemented with 10% FBS, 1 mM sodium pyruvate, 100 U/ml penicillin and 100

of

GIBH-130.

GIBH-130

was

prepared

starting

from

16 / 37


Page 17 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


331

µg/ml streptomycin. All cultures were maintained in a humidified CO2 incubator

332

(Sanyo, Japan) at 37 °C and 5% CO2. Stock N9 cells were passaged 2-3 times/week

333

with a 1:4 split ratio and used within 8 passages.

334

Nitric oxide production assay. N9 cells (5×104 cells/well) were plated into 96-well

335

microtiter plates, followed by the treatment of minocycline (20 µM) or various

336

concentrations of the GIBH-130 (25, 50 and 100 µM) for 18 h. The NO production

337

was stimulated by incubation with LPS (1 µg/ml) for 48 h. The levels of NO in the

338

culture medium were measured using the nitric oxide detection kit according to the

339

manufacturer's instructions.

340

TNF-α α and IL-1β β production assay. Cells (5×104 cells/well) were plated into 96-well

341

microtiter plates and pre-treated (18 h) with minocycline (20 µM) or various

342

concentrations of GIBH-130 (25, 50 and 100 µM of TNF-α assay; 0.8, 4 and 20 nM

343

of IL-1β assay) in the presence of LPS (1 µg/ml) for 24 h (TNF-α production assay)

344

or 48 h (IL-1β production assay), respectively. The levels of TNF-α and IL -1β in

345

the culture medium were measured using ELISA kits according to the

346

manufacturer's instructions.

347

Pharmacokinetic study. A reliable and selective quantification method using

348

HPLC/MS was established to determine the GIBH-130 concentration in blood

349

samples using propranolol hydrochloride (No. 100783-200401) as an internal

350

standard. SD rats (180-220 g) were obtained from Southern Medical University

351

(SCXK 2006-0015) and housed in Guangzhou Institutes of Biomedicine and Health. 17 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 42

352

Rats were divided into two groups (n = 6, bisexual each half). Each group was

353

treated with a 5 mg/ml i.v. injection and 20 mg/ml p.o. administration after 12 h

354

fasting with free access to water. Blood samples were drained through the orbit at 2,

355

10 min (i.v.), 5, 15 min (p.o.), and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24 h into

356

heparinized tubes. Upper plasma was collected after 6000 rpm centrifugation for 10

357

m and stored under 4 °C until quantification. The animal experiments was approved

358

by our institutional Animal Care and Use Committees.

359

Blood brain barrier penetration test. Male Wistar rats (180-220 g) were obtained

360

from Southern Medical University (SCXK 2006-0015) and housed in Guangzhou

361

Institutes of Biomedicine and Health. Rats were divided into several groups

362

(positive control drug midazolam, negative drug lincomycin, reference candidate

363

minozac, and GIBH-130) and fasted for 12 h. the midazolam group was treated with

364

a 10 mg/ml i.v. injection. The lincomycin groups were treated with a 20 mg/ml i.v.

365

injection and 25 mg/ml p.o. administration. Minozac and GIBH-130 groups were

366

treated with a 25 mg/ml and 20 mg/ml p.o. administration, respectively. Blood,

367

cerebrospinal fluid (CSF), and brain tissue samples were obtained at 0.083, 0.25,

368

0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h and stored in -20 °C until processed for

369

quantification. The animal experiments was approved by our institutional Animal

370

Care and Use Committees.

371

Animal. Sprague-Dawley (SD) rats (half male and half female, 250~270 g),

372

APP/PS1 transgenic mice (half male and half female, 14 weeks age) and 18 / 37


Page 19 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


373

age-and-strained-matched wild type mice (C57BL/6J) were obtained from Beijing

374

Huafukang biotechnology Co. Ltd (China). Animals were housed at Shenyang

375

Pharmaceutical University and kept in a regulated environment (23 ± 1 °C, 50 ± 5 %

376

humidity) with a 12 h light/dark cycle and were provided with ad libitum access to

377

standard laboratory food and water. Behavioral experiments were carried out in a

378

sound-attenuated and air-regulated experimental room, and animals were habituated

379

for ≥ 1 h. All animal studies were performed in strict accordance with the National

380

Institutes of Health Guide for the use and care of laboratory animals and the

381

guidelines established by the Chinese Society of Laboratory Animal Sciences. The

382

animal behavioral experiments was approved by Animal Care and Use Committees

383

of Shenyang Pharmaceutical University.

384

Aβ β 25-35 injected rat model. The rats were anaesthetized using intraperitoneal

385

injection of 350 mg per kg body weight chloral hydrate and bilaterally injected into

386

each side of hippocampal CA1 region with a solution containing 10 µg (3 µL)

387

aggregated Aβ25-35 (Sigma-Aldrich, St. Louis, MO, USA) or physiological saline

388

(sham-operated group) of the same volume. The saline–diluted Aβ25-35 was

389

incubated for aggregation at 37 °C for 5 days before injection. Solutions were

390

administered using stereotaxic injections according to a previous study38.

391

Rats exposed to Aβ25-35 received oral (p.o.) GIBH-130 (0.0022, 0.02 or 0.18 mg/kg),

392

donepezil hydrochloride (0.9 mg/kg), memantine hydrochloride (1.8 mg/kg) or

393

distilled water via gavage after the Aβ25-35 injection. The sham-operated group 19 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 42

394

received a p.o. administration of distilled water (n = 10 per group). All compounds

395

were systemically administered in a volume of 0.01 ml per g body weight once daily

396

after the day of surgery until the end of the behavioral testing. The behavioral tests

397

started 8 days after the Aβ25–35 injection and were performed sequentially according

398

to the experimental schedule shown in Fig. 3a.

399

APP/PS1 transgenic mouse model. APP/PS1 transgenic mice were randomly

400

divided into six groups: model group, GIBH-130 0.0031, 0.028 and 0.25 mg/kg

401

group, donepezil hydrochloride 1.3 mg/kg group, and memantine hydrochloride 2.6

402

mg/kg group (n = 10 per group). C57BL/6J mice were used as normal control group

403

(n = 10). The doses of GIBH-130 and the positive drugs used in this mouse model

404

were equivalent to the rat model. All mice received oral administration at 15 weeks

405

of age and continuously for 25 weeks until the end of the behavioral tests. The

406

model group and normal control group received the same volume of distilled water

407

(0.01 ml per g body weight). The experimental schedule is shown in Fig. 4a.

408

Behavioral tests. The Y-maze test was performed according to previous reports39, 40.

409

Alternation behavior (%) was used to reflect short-term memory ability. The novel

410

object recognition test was performed according to previous reports40, 41, with minor

411

modifications. The preference index and discrimination index for the novel object of

412

1 h and 24 h after the training session was used to measure short-term and long-term

413

visual recognition memory during the retention session. A preference index is a ratio

414

of the amount of time spent exploring the novel object over the total time spent 20 / 37


Page 21 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


415

exploring both objects, and the discrimination index is a ratio of the difference in

416

time spent exploring the two objects over the total time spent exploring both objects.

417

The Morris water maze test was performed according to previous reports41, 42. In the

418

APP/PS1 mice model, the repeated acquisition task was conducted for 3 consecutive

419

days and consisted of 5 trials (one session) per day after the 90-s probe trial task.

420

The procedure was similar to the training for the standard water maze task, except

421

that the location of the platform was changed for each session43. The step-through

422

(passive avoidance) test were performed according to previous reports44, 45.

423

ELISA analyses of cytokines in vivo. The concentrations of IL-6 and IL-4 in the

424

supernatants were determined from freshly frozen hippocampal tissues. The samples

425

were homogenized in 0.1 M PBS solution. Cell extracts were centrifuged at 3,000×g

426

for 20 min at 4 °C after sonication. The levels of IL-6 and IL-4 were quantified

427

using ELISA (R&D, Systems) according to the manufacturer’s protocol.

428

Absorbance of the plates was read at 450 nm using a SPECTRA (shell) Reader

429

(TECAN, Grödig, Austria). Standard curves were established using a variety of

430

concentrations (1-2000 ng/ml) of IL-6 and IL-4.

431

Immunohistochemical staining of CD11b.Immunohistochemical staining was

432

performed as previously described46. Briefly, sections were incubated with rabbit

433

anti-BDNF antibody (1:100, Santa Cruz), mouse anti-NeuN antibody (1:100,

434

Millipore) at 4 °C overnight. Sections were washed (3 times) with PBS, and

435

incubated with biotin-labelled goat anti-rabbit/mouse antibody (1:200, Santa Cruz) 21 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 42

436

at 37 °C for 30 min. The sections were treated with avidin-biotin enzyme reagent

437

(Santa Cruz) and DAB was used to visualize positive signals. The intensity of each

438

section was quantified using Image Pro-Plus 6.0 software and an Olympus IX 71.

439

Transmission electron microscopy of neuronal ultrastructure. After the behavioral

440

tests, Aβ1-42-injected mice were anaesthetized and the brains were dissected.

441

Neuronal ultrastructure of the brain hippocampal CA1 region was examined using

442

electron microscopy. The micrograph magnification was 4000×.

443

Statistical analyses. All analyses were performed using “SPSS Statistics 17.0”

444

software. All data are the means ± s.e.m. Significance differences between groups

445

were examined using one-way ANOVA or two-way ANOVA followed by the LSD

446

post hoc test. Values of P < 0.05 were considered statistically significant.

447

ASSOCIATED CONTENT

448

Supporting Information

449

Synthesis of GIBH-130 and its chemical characterization data, Fig. S1a,b,c,d;

450

cellular toxicity, BBB permeability and maximum tolerance dose of GIBH-130, Fig.

451

S2; pharmacokinetic parameters of GIBH-130 in Sprague Dawley rats, Table S1;

452

pharmacokinetic parameters and distribution of GIBH-130 in Wistar rats, Table S2;

453

effect of GIBH-130 on the memory impairments of rats with bilateral hippocampal

454

injection of Aβ25-35 in the novel object recognition test, Fig. S3; effect of GIBH-130

455

on the memory impairments of APP/PS1 transgenic mice in the Morris water maze

456

test, Fig. S4.

457

AUTHOR INFORMATION

458

Corresponding Author 22 / 37


Page 23 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


459

*(W.H.)

Phone:

+86

20-32015211.

Fax:

+86

20-32015299.

E-mail:

460

[email protected]

461

Author Contributions

462

G.Z., S.Z. and D.X. designed and constructed the compound library, and G.Z.

463

identified GIBH-130. W.Z. was involved in planning and conducting all aspects of

464

the research, including chemical synthesis, data analysis and figure preparation. W.Z.

465

also wrote the manuscript. T.C., X.R., L.L., H.W., G.S., X.J. and L.Z. conducted the

466

cell assays and animal behavioural tests, and interpreted the results. H.X. and X.L.

467

were responsible for pharmacokinetic and toxicity experiments. M.T. and D.W.

468

performed some biological mechanism studies. W.H. conceived the study idea and

469

was responsible of overseeing the study, including all aspect of study design, data

470

analysis, interpretation of results and manuscript reviewing. All authors discussed

471

the results presented in the manuscript.

472

Funding

473

The authors received funding from the Drug Discovery Pipeline of Guangzhou

474

Institutes of Biomedicine and Health, the Natural Science Foundation of Guangdong

475

Province, China (10251066302000000), Major Science and Technology Program of

476

Guangdong Province, China (2012A080201013) and National Natural Science

477

Foundation of China (81502911). GIBH-130 has been selected for drug

478

development supported by South China Center for Innovative Pharmaceuticals.

479

Notes 23 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 42

480

The authors declare no competing financial interest.

481

ABBREVIATIONS

482

AD,

483

N-methyl-D-aspartic acid; Aβ, β amyloid; NO, Nitric oxide; TNF-α necrosis factor

484

α; IL-1β, interleukin 1β ; PK, pharmacokinetic ; BBB, brain-blood barrier; LPS,

485

lipopolysaccharides; NORT, novel object recognition test; MWMT, Morris water

486

maze test; YMT, Y maze test; STT, step-through test; CSF, cerebrospinal fluid.

487

REFERENCES

488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511

[1] Walsh, D. M., and Selkoe, D. J. (2004) Deciphering the molecular basis of memory failure in Alzheimer's disease, Neuron 44, 181-193. [2] (2011) Alzheimer's therapy: a BACE in the hand?, Nature Medicine 17, 932-933. [3] Mangialasche, F., Solomon, A., Winblad, B., Mecocci, P., and Kivipelto, M. (2010) Alzheimer's disease: clinical trials and drug development, Lancet Neurology 9, 702-716. [4] Ross-Macdonald, P. (2007) Drug discovery without a molecular target: the road less traveled, Expert Review Mollecular Diagnosis 7, 1-4. [5] Swinney, D. C., and Anthony, J. (2011) How were new medicines discovered?, Nature Reviews Drug Discovery 10, 507-519. [6] McGeer, E. G., and McGeer, P. L. (2010) Neuroinflammation in Alzheimer's Disease and Mild Cognitive Impairment: A Field in Its Infancy, Journal of Alzheimers Disease 19, 355-361. [7] Cacquevel, M., Lebeurrier, N., Cheenne, S., and Vivien, D. (2004) Cytokines in neuroinflammation and Alzheimer's disease, Current Drug Targets 5, 529-534. [8] Hensley, K. (2010) Neuroinflammation in Alzheimer's Disease: Mechanisms, Pathologic Consequences, and Potential for Therapeutic Manipulation, Journal of Alzheimer's Disease 21, 1-14. [9] Agostinho, P., Cunha, R. A., and Oliveira, C. (2010) Neuroinflammation, Oxidative Stress and the Pathogenesis of Alzheimer's Disease, Current Pharmaceutical Design 16, 2766-2778. [10] Ballatore, C., Lee, V. M., and Trojanowski, J. Q. (2007) Tau-mediated neurodegeneration in Alzheimer's disease and related disorders, Nature Review Neuroscience 8, 663-672.

Alzheimer’s

disease;

CNS,

central

nervous

system;

NMDA,

24 / 37


Page 25 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553

[11] Gonzalez-Scarano, F., and Baltuch, G. (1999) Microglia as mediators of inflammatory and degenerative diseases, Annual Review Neuroscience 22, 219-240. [12] Akiyama, H., Barger, etal., (2000) Inflammation and Alzheimer's disease, Neurobiology of Aging 21, 383-421. [13] Wyss-Coray, T. (2006) Inflammation in Alzheimer disease: driving force, bystander or beneficial response?, Nature Medicine 12, 1005-1015. [14] Hu, W., Ralay Ranaivo, H., Craft, J. M., Van Eldik, L. J., and Watterson, D. M. (2005) Validation of the neuroinflammation cycle as a drug discovery target using integrative chemical biology and lead compound development with an Alzheimer's disease-related mouse model, Current Alzheimer Research 2, 197-205. [15] O'Hare, E., Scopes, etal., (2011) Novel Anti-Inflammatory Compound SEN1176 Alleviates Behavioral Deficits Induced Following Bilateral Intrahippocampal Injection of Aggregated Amyloid-beta(1-42), Journal Alzheimers Disease 25, 219-229. [16] Zhou, W., and Hu, W. (2013) Anti-neuroinflammatory agents for the treatment of Alzheimer’s disease, Future Medicinal Chemistry 5, 1559-1571. [17] Zhou, W., Zhong, G. F., Rao, X. R., Xie, H., Zeng, S. G., Chi, T. Y., Zou, L. B., Wu, D. H., and Hu, W. H. (2012) Identification of Aminopyridazine-Derived Antineuroinflammatory Agents Effective in an Alzheimer's Mouse Model, ACS Medicinal Chemistry Letter 3, 903-907. [18] Hanisch, U. K., and Kettenmann, H. (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain, Nature Neuroscience 10, 1387-1394. [19] Schwartz, M., Butovsky, O., Bruck, W., and Hanisch, U. K. (2006) Microglial phenotype: is the commitment reversible?, Trends of Neuroscience 29, 68-74. [20] Varnum, M. M., and Ikezu, T. (2012) The classification of microglial activation phenotypes on neurodegeneration and regeneration in Alzheimer's disease brain, Archivum immunologiae et therapiae experimentalis 60, 251-266. [21] Reed-Geaghan, E. G., Savage, J. C., Hise, A. G., and Landreth, G. E. (2009) CD14 and Toll-Like Receptors 2 and 4 Are Required for Fibrillar Aβ-Stimulated Microglial Activation, The Journal of Neuroscience 29, 11982-11992. [22] Kobayashi, K., Imagama, S., Ohgomori, T., Hirano, K., Uchimura, K., Sakamoto, K., Hirakawa, A., Takeuchi, H., Suzumura, A., Ishiguro, N., and Kadomatsu, K. (2013) Minocycline selectively inhibits M1 polarization of microglia, Cell Death & Disease 4, e525. [23] Yamin, T. T., Ayala, J. M., and Miller, D. K. (1996) Activation of the native 45-kDa precursor form of interleukin-1-converting enzyme, Journal of Biological Chemistry 271, 13273-13282. [24] Kell, D. B., and Pretorius, E. (2015) On the translocation of bacteria and their lipopolysaccharides between blood and peripheral locations in chronic, 25 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594

Page 26 of 42

inflammatory diseases: the central roles of LPS and LPS-induced cell death, Integr Biol-Uk 7, 1339-1377. [25] McCoy, M. K., and Tansey, M. G. (2008) TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease, J Neuroinflammation 5, 45. [26] Simi, A., Tsakiri, N., Wang, P., and Rothwell, N. J. (2007) Interleukin-1 and inflammatory neurodegeneration, Biochemical Society transactions 35, 1122-1126. [27] Yamada, K., Takayanagi, M., etal., (2005) Effects of memantine and donepezil on amyloid beta-induced memory impairment in a delayed-matching to position task in rats, Behavioural Brain Research 162, 191-199. [28] Takayanagi, M., Nagai, T., Dohniwa, M., Kobayashi, K., Kamei, H., Takuma, K., Nabeshima, T., and Yamada, K. (2005) Effects of memantine and donepezil on A beta 1-40-induced memory impairment in 3-lever operant task, Journal Pharmacological Science 97, 90p-90p. [29] Trinchese, F., Liu, S., Battaglia, F., Walter, S., Mathews, P. M., and Arancio, O. (2004) Progressive age-related development of Alzheimer-like pathology in APP/PS1 mice, Annual Neurology 55, 801-814. [30] Arendash, G. W., King, D. L., Gordon, M. N., Morgan, D., Hatcher, J. M., Hope, C. E., and Diamond, D. M. (2001) Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes, Brain Research 891, 42-53. [31] Nagakura, A., Shitaka, Y., Yarimizu, J., and Matsuoka, N. (2013) Characterization of cognitive deficits in a transgenic mouse model of Alzheimer's disease and effects of donepezil and memantine, European Journal of Pharmacology 703, 53-61. [32] Minkeviciene, R., Banerjee, P., and Tanila, H. (2004) Memantine improves spatial learning in a transgenic mouse model of Alzheimer's disease, Journal of Pharmacological Experiment and Therapy 311, 677-682. [33] Lyons, A., Griffin, R. J., Costelloe, C. E., Clarke, R. M., and Lynch, M. A. (2007) IL-4 attenuates the neuroinflammation induced by amyloid-beta in vivo and in vitro, Journal of Neurochemistry 101, 771-781. [34] Kiyota, T., Okuyama, S., Swan, R. J., Jacobsen, M. T., Gendelman, H. E., and Ikezu, T. (2010) CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer's disease-like pathogenesis in APP+PS1 bigenic mice, FASEB J 24, 3093-3102. [35] Qian, L., Flood, P. M., and Hong, J. S. (2010) Neuroinflammation is a key player in Parkinson's disease and a prime target for therapy, Journal of Neural Transmission 117, 971-979. [36] Moller, T. (2010) Neuroinflammation in Huntington's disease, Journal of Neural Transmission 117, 1001-1008.

26 / 37


Page 27 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632

[37] Lakhan, S. E., Kirchgessner, A., and Hofer, M. (2009) Inflammatory mechanisms in ischemic stroke: therapeutic approaches, Journal of Translational Medicine 7. [38] Liu, R. T., Zou, L. B., Fu, J. Y., and Lu, Q. J. (2010) Effects of liquiritigenin treatment on the learning and memory deficits induced by amyloid beta-peptide (25-35) in rats, Behavioural Brain Research 210, 24-31. [39] Olariu, A., Tran, M. H., Yamada, K., Mizuno, M., Hefco, V., and Nabeshima, T. (2001) Memory deficits and increased emotionality induced by beta-amyloid (25-35) are correlated with the reduced acetylcholine release and altered phorbol dibutyrate binding in the hippocampus, Journal of Neural Transmission 108, 1065-1079. [40] Mouri, A., Noda, Y., Hara, H., Mizoguchi, H., Tabira, T., and Nabeshima, T. (2007) Oral vaccination with a viral vector containing A beta cDNA attenuates age-related A beta accumulation and memory deficits without causing inflammation in a mouse Alzheimer model, Faseb Journal 21, 2135-2148. [41] Mouri, A., Zou, L. B., Iwata, N., Saido, T. C., Wang, D. Y., Wang, M. W., Noda, Y., and Nabeshima, T. (2006) Inhibition of neprilysin by thiorphan (i.c.v.) causes an accumulation of amyloid beta and impairment of learning and memory, Behavioural Brain Research 168, 83-91. [42] Liu, R. T., Tang, J. T., Zou, L. B., Fu, J. Y., and Lu, Q. J. (2011) Liquiritigenin attenuates the learning and memory deficits in an amyloid protein precursor transgenic mouse model and the underlying mechanisms, European Journal of Pharmacology 669, 76-83. [43] Frick, K. M., Baxter, M. G., Markowska, A. L., Olton, D. S., and Price, D. L. (1995) Age-related spatial reference and working memory deficits assessed in the water maze, Neurobiology of Aging 16, 149-160. [44] Miyamoto, Y., Yamada, K., Noda, Y., Mori, H., Mishina, M., and Nabeshima, T. (2001) Hyperfunction of dopaminergic and serotonergic neuronal systems in mice lacking the NMDA receptor epsilon1 subunit, Journal of Neuroscience 21, 750-757. [45] Chi, T. Y., Wang, L. H., Qu, C., Yang, B. Z., Ji, X. F., Wang, Y., Okuyama, T., Yoshihito, O., and Zou, L. B. (2009) Protective effects of xanthoceraside on learning and memory impairment induced by Abeta(25-35) in mice, Journal of Asian Natural Products Research 11, 1019-1027. [46] Andsberg, G., Kokaia, Z., Klein, R. L., Muzyczka, N., Lindvall, O., and Mandel, R. J. (2002) Neuropathological and behavioral consequences of adeno-associated viral vector-mediated continuous intrastriatal neurotrophin delivery in a focal ischemia model in rats, Neurobiology of Disease 9, 187-204.

633

27 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

634

[Displays and footnotes]

635

Figure 1

636 637 638 639 640 641 642 643 644 645 646 647 648 649

Page 28 of 42

Figure 1 GIBH-130 is identified among compounds structurally derived from compound 1 using phenotypic screening assays. LPS-stimulated N9 microglial line was adopted as the phenotypic screening assays to search for small molecules that can suppress proinflammatory cytokine production, with IL-1β as the phenotypic marker to assess the anti-neuroinflammation potency of compounds. Structural optimization started from compound 1, which was an effective inhibitor of microglia activation. Introduction of a quinazoline scaffold and replacement the thiophene ring with a phenyl group enhanced the half-life of compound 2 in rat. The insertion of a carbonyl group between the scaffold and the pharmacophore greatly improved its in vitro efficacy (compound 3). Further optimization of compound 3 eventually led to the identification of compound 4 (GIBH-130) with improved IC50 and acceptable half-life in rat.

28 / 37


Page 29 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


650

Figure 2

651

Figure 2 GIBH-130 inhibits the production of proinflammatory and neurotoxic

652

mediators from microglia. N9 microglial cells were pre-treated with minocycline (20 µM) or

653

various concentrations of GIBH-130 for 18 h. Subsequently microglia cells were stimulated

654

with LPS (1 µg/ml). The NO levels were determined in the culture medium using an NO

655

detection assay 24 h later (a). After stimulation with LPS for 24 h or 48 h, levels of tumour

656

necrosis factor (TNF-α) (b) and interleukin-1β (IL-1β) (c) were measured in the culture

657

medium using ELISA, respectively. Each column and vertical bar represented means ± s.e.m.

658

(n = 6 per group). *P < 0.05, **P < 0.01. One-way ANOVA followed by Dunnett's t-test was

659

used for statistical analyses.

29 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

660

Page 30 of 42

Figure 3

661 662

Figure 3 Oral administration of GIBH-130 attenuates memory impairments and

663

cognitive decline in rats with bilateral hippocampal injection of Aβ β25-35. (a) Schematic

664

diagram of the experimental schedule. Rats were injected with the Aβ25-35 peptide or

665

physiological saline (sham-operated group) in two sides of the hippocampal CA1 region on

666

day 0. The Aβ25-35-injected rats were orally administered GIBH-130 (a daily dose of 0.18

667

mg/kg, 0.02 mg/kg or 0.0022 mg/kg in three different groups), donepezil hydrochloride (a

668

daily dose of 0.9 mg/kg), memantine hydrochloride (a daily dose of 1.8 mg/kg) or distilled

669

water (model group). Cognitive function was assessed in all groups through a Y-maze test

670

(YMT) on day 8, a novel object recognition test (NORT) on days 9–12, a Morris water maze

671

test (MWMT) on days 14–18 and a step-through test (STT) on days 19-20. The results of

672

YMT and STT are presented in Supporting Information. Discrimination index (b) represented

673

the percentage of time that rat spent more on exploring a novel object than on a familiar

30 / 37


Page 31 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


674

object 24 h after the training session in NORT. And the preference index (c) represented the

675

percentage of time that rats spent on exploring a novel object. In the reference memory task

676

of MWMT, distance that rats in each group swam before they reached a hidden safety

677

platform were recorded (d). In the following 90-s probe task, the hidden platform was

678

removed. The percentage of time that rats swam in the target quadrant where the platform

679

was previously located were presented (e). The results are expressed as the means ± s.e.m.

680

(n = 9 or 10 per group, half male and half female) and analyzed using y a one-way or

681

two-way ANOVA followed by the LSD post hoc test for multiple comparisons. *P < 0.05, **P 100 µM

Acute toxicity

MTD > 2000mg/kg

Safety

721

Abbreviation: MLM, mouse liver microsome; hERG, the human Ether-à-go-go-Related Gene;

722

BBB, blood brain barrier; AUC, area under the curve; MTD, maximum tolerance dose.

36 / 37


Page 37 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


723

For Table of Contents Use Only

724

Microglia-based phenotypic screening identifies a novel inhibitor of

725

neuroinflammation effective in Alzheimer’s disease models

726 727 728 729 730

Wei Zhou1,5,#, Guifa Zhong1,# , Sihai Fu2, Hui Xie3, Tianyan Chi4, Luyi Li4, Xiurong Rao1, Shaogao Zeng1, Dengfeng Xu1, Hao Wang1, Guoqing Sheng1, Xing Ji2, Xiaorong Liu1, Xuefei Ji4, Donghai Wu1, Libo Zou4, Micky Tortorella1, Kejian Zhang2 & Wenhui Hu1, *

731

37 / 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1 GIBH-130 is identified among compounds structurally derived from compound 1 using phenotypic screening assays. LPS-stimulated N9 microglial line was adopted as the phenotypic screening assays to search for small molecules that can suppress proinflammatory cytokine production, with IL-1β as the phenotypic marker to assess the anti-neuroinflammation potency of compounds. Structural optimization started from compound 1, which was an effective inhibitor of microglia activation. Introduction of a quinazoline scaffold and replacement the thiophene ring with a phenyl group enhanced the half-life of compound 2 in rat. The insertion of a carbonyl group between the scaffold and the pharmacophore greatly improved its in vitro efficacy (compound 3). Further optimization of compound 3 eventually led to the identification of compound 4 (GIBH-130) with improved IC50 and acceptable half-life in rat. 197x111mm (120 x 120 DPI)


Page 38 of 42

Page 39 of 42


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3 Oral administration of GIBH-130 attenuates memory impairments and cognitive decline in rats with bilateral hippocampal injection of Aβ25-35. (a) Schematic diagram of the experimental schedule. Rats were injected with the Aβ25-35 peptide or physiological saline (sham-operated group) in two sides of the hippocampal CA1 region on day 0. The Aβ25-35-injected rats were orally administered GIBH-130 (a daily dose of 0.18 mg/kg, 0.02 mg/kg or 0.0022 mg/kg in three different groups), donepezil hydrochloride (a daily dose of 0.9 mg/kg), memantine hydrochloride (a daily dose of 1.8 mg/kg) or distilled water (model group). Cognitive function was assessed in all groups through a Y-maze test (YMT) on day 8, a novel object recognition test (NORT) on days 9–12, a Morris water maze test (MWMT) on days 14–18 and a step-through test (STT) on days 19-20. The results of YMT and STT are presented in supplementary information. Discrimination index (b) represented the percentage of time that rat spent more on exploring a novel object than on a familiar object 24 h after the training session in NORT. And the preference index (c) represented the percentage of time that rats spent on exploring a novel object. In the reference memory task of MWMT, distance that rats in each group swam before they reached a hidden safety platform were recorded (d). In the following 90-s probe task, the hidden platform was removed. The percentage of time that rats swam in the target quadrant where the platform was previously located were presented (e). The results are expressed as the means ± s.e.m. (n = 9 or 10 per group, half male and half female) and analyzed using y a one-way or two-way ANOVA followed by the LSD post hoc test for multiple comparisons. *P < 0.05, **P < 0.01. 415x257mm (120 x 120 DPI)


Page 40 of 42

Page 41 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4 GIBH-130-treated APP/PS1 transgenic mice show memory and cognition recovery. (a) Schematic diagram of the experimental schedule. APP/PS1 transgenic mice were orally administered with GIBH-130 (at a daily dose of 0.25, 0.028 or 0.0031 mg/kg), donepezil hydrochloride (at a daily dose of 1.3 mg/kg), memantine hydrochloride (at a daily dose of 2.6 mg/kg) or distilled water (model group) at 15 weeks of age and continuously for 25 weeks. C57BL/6J mice were given distilled water as a control group (n = 10). Memory performances of the control, treated and untreated groups were assessed using a Y-maze test (YMT) at week 32, a novel object recognition test (NORT) at weeks 32-36, a Morris water maze test (MWMT) at weeks 37-39 and a step-through test (STT) at week 40. Results of NORT, YMT and STT are presented in the supplementary information.(b) The swimming distance that mice swam until they reached the hidden safety platform in the reference memory task of the Morris water maze test. (c) In the following 90-s probe trail task of the Morris water maze test, the time that mice spent on exploring the target quadrant where the hidden safety platform previously located were recorded. (d) In the repeated acquisition task, escape latency represented the time that mice of each group used to find the randomly-located safety platform in



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the Morris water maze. The results are expressed as the mean ± s.e.m. (n = 9 or 10 per group, half male and half female) and analysed using a one-way or two-way ANOVA followed by the LSD post hoc test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001 155x190mm (300 x 300 DPI)


Page 42 of 42

Page 43 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5 Oral administration of GIBH-130 alters in vivo phenotypes of Aβ-injected AD mice. The Aβ1-42injected mice were orally administered GIBH-130 (a daily dose of 0.0025 mg/kg, 0.025 mg/kg or 0.25 mg/kg in three experimental groups), donepezil hydrochloride (a daily dose of 1.3 mg/kg) and distilled water (model group) for 8 days. (a-b) Concentrations of IL-4 (a) and IL-6 (b) in the cortex and hippocampus were measured using ELISA. (c) Frozen sections of mouse brains (5 µm thick) in each group were stained with anti-CD11b mAb. Integral optical density (IOD) of images of sections in each group were displayed in columns. (d) The ultrastructure of the hippocampus CA1 neurons in the 7 groups of mice was observed using transmission electron microscopy. Scale bars: 2 µm. Each column and vertical bar represented are means ± s.e.m. (n = 8 per group, half male and half female). *P < 0.05, **P < 0.01, ***P < 0.001. Oneway ANOVA followed by Dunnett's t-test was used for statistical analyses. 191x110mm (300 x 300 DPI)


Microglia-Based Phenotypic Screening Identifies a ... - ACS Publications

Recommend Documents