Microglia-Based Phenotypic Screening Identifies a Novel Inhibitor of

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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

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ACS Chemical Neuroscience

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Microglia-based phenotypic screening identifies a novel inhibitor of

2

neuroinflammation effective in Alzheimer’s disease models

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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, *

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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

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3

11

Republic of China

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4

13

People's Republic of China

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5

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

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China

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#

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]).

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ACS Paragon Plus Environment


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ABSTRACT

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

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

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

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

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

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

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

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proinflammatory cytokines. The identified novel pharmacological inhibitor of

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

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

31

vivo efficacy of cognitive impairment relief to donepezil and memantine

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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

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constitutes a unique chemical probe for pathogenesis research and drug development

35

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

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

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agents.

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KEYWORDS

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

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INTRODUCTION

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

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

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memory and cognition functions1. However, insufficient understanding of the

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pathophysiology limits available therapies to symptomatic relief, rather than

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

46

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

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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

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stop the progression of this neurodegenerative disorder2.

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

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

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

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

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

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

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

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conventional approaches based on phenotypic screenings should not be marginalized

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



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

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that employs phenotypic screenings to identify small molecules with in vivo

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therapeutic potential against AD.

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

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

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

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

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

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

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

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hyperphosphorylation kinases to promote the formation of neurofibrillary tangles.

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

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transport and thus lead to synaptic dysfunction and neurodegeneration10. Neuronal

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

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

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

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

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

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

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

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novel compounds that alter the phenotypes of neuroinflammation and bear favorable

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

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

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

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

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

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

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

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

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through microglia-based phenotypic screenings. We also characterized its

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

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RESULTS AND DISCUSSION

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

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Lipopolysaccharides (LPS) stimulated microglia was adopted as the phenotypic

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

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

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

98

used to assess anti-neuroinflammation potency of compounds.

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With this model, we developed an anti-neuroinflammatory molecule

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

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



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different bioisosteres to replace the original pharmacophore the pyridazine of

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

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

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for further structural modification, while retaining its anti-neuroinflammatory

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

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

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

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

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

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

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

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

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

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

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

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

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

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an acceptable half-life.

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Figure 1

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LPS-stimulated microglia possess the phenotypes of cytotoxicity and

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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might

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neuroinflammation cycle at early stages, which were mediated by activated

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microglia in AD brains.

alleviate

neurotoxic

oxidative

stress and

prevent

the

19

.

deleterious

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Figure 2

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

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

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



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microglia activation that selectively inhibits M1 polarization of microglia22. The

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

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

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

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

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

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

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activation amplification by IL-1β itself24.

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

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

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

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

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

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

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

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

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

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

162

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

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

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


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Table 1

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

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

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proinflammatory factors and neuroinflammation amplification, both of which are

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associated with neuronal degeneration25,

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

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AD progression by attenuating the microglial overproduction of various neurotoxic

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and proinflammatory factors.

26

. AD is characterized by progressive

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

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

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

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

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

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

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

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group. Behavioural changes in each group were comprehensively examined using

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

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

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

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respectively (Fig. 3a).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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donepezil-treated

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

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

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

and

memantine-treated

groups,

but

also

in

all

three

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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

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

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performance. These results demonstrated that GIBH-130 treatment relatively

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

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

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

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

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respectively. These results exhibited that donepezil and memantine symptomatically

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

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

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

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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

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anti-neuroinflammatory compound 115.

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Figure 3

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

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

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

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

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



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

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

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

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

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

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

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

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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.

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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


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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).

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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.

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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



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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

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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


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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



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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

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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



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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

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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



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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

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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



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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

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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



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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

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634

[Displays and footnotes]

635

Figure 1

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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.

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Figure 2

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

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

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

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

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

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

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

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

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used for statistical analyses.

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Figure 3

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

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

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

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

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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

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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

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

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

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

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

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

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

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

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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

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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

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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.

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For Table of Contents Use Only

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Microglia-based phenotypic screening identifies a novel inhibitor of

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neuroinflammation effective in Alzheimer’s disease models

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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, *

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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)


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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


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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)


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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



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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)


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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 Novel Inhibitor of

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