al Journal of Neuropsychopharmacology(2009), 12, 1409-1419. Copyright G CINP 2009 ARTICLE /S1461145709000455 Novel piperazine derivative pms1339 exhibits O CINP tri-functional properties and cognitive improvement in mice Jean Marc Miezan Ezoulin*, Bi-yun Shao*, Zheng Xia*, Qiong Xie, Juan Li, Yong-yao Cui, Hao Wang, Chang-zhi Dong, Yan-xing Zhao, France Massicot, Zhui-bai Qiu, Francoise Heymans and Hong-zhuan Chen' i Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong LIniversity School of Medicine, Shanghai, P.R. China Unite de Pharmacochimie Moleculare et Systemes Membranaires(EA2381), Laboratoire de plarmacochimie Moleculare Universite Paris 7-Denis diderot, Batiment Lavoisier, Paris, france Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, PR China 4 UNiversite Paris Descartes, Faculte des Sciences Pharmaceutiques et Biologiques, Laboratoire de Toxicologie, Paris, France Abstract Amyloid-B-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons nd cognitive decline in Alzheimers disease. The acetylcholinesterase(AChE)inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, AB aggregation cantly inhibit both mice brain AChE (ICs0=4.41+0. 63 uM) and sera butyrylcholinesterase(BuChE, ICso=1.09+0.20 uM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (Co=17.95+2.31 uM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and ChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site(PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Ap aggregation. In-vivo study indicated PMS1339(1 mg/kg i.p. reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement Received 17 February 2009; Reviewed 12 March 2009; Revised 30 March 2009; Accepted 9 April 2009 First published online 22 May 2009 Key words: AChE inhibitor, Alzheimer's disease, amyloid B, PAF antagonist, PMS1339 Introduction Clinically, this neuropathology is characterized by an Alzheimers disease(AD) is a progressive neuro- sidious loss of memory and cognitive abilities in close connection with an impairment of brain cholin- degenerative disorder considered to be the leading ergic neurotransmission(Walsh Selkoe, 2004) cause of dementia affecting the elderly population. Despite enormous research efforts devoted to AD so Author for correspondence: Professor H-Z. Chen, Department of far, an effective treatment seems to be out of reach. The Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong use of acetylcholinesterase(AChE) inhibitors to en- University School of Medicine, 280 South Chongqing Road, hance surviving cholinergic activity is still the main Shanghai 200025, P.R. China. Tel.:+862164674721Fax:+862164674721 Email:hongzhuan_chen@hotmail.com(H-Z.Chen Email: heymans@ccr jussieu. fr(Professor F Heymans) improving cognition as well as behavioural and func- a These authors contributed equally to this work. tional daily activities but do not stop the diseasesNovel piperazine derivative PMS1339 exhibits tri-functional properties and cognitive improvement in mice Jean Marc Miezan Ezoulin1,2 *, Bi-yun Shao1 *, Zheng Xia1 *, Qiong Xie3 , Juan Li1 , Yong-yao Cui1 , Hao Wang1 , Chang-zhi Dong2 , Yan-xing Zhao1 , France Massicot4 , Zhui-bai Qiu3 , Franc¸oise Heymans2 and Hong-zhuan Chen1 1 Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, P.R. China 2 Unite´ de Pharmacochimie Mole´culaire et Syste`mes Membranaires (EA2381), Laboratoire de Pharmacochimie Mole´culaire, Universite´ Paris 7-Denis Diderot, Baˆtiment Lavoisier, Paris, France 3 Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, P.R. China 4 Universite´ Paris Descartes, Faculte´ des Sciences Pharmaceutiques et Biologiques, Laboratoire de Toxicologie, Paris, France Abstract Amyloid-b-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons and cognitive decline in Alzheimer’s disease. The acetylcholinesterase (AChE) inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, Ab aggregation inhibition and cognitive improvement. PMS1339 could signifi- cantly inhibit both mice brain AChE (IC50=4.41¡0.63 mM) and sera butyrylcholinesterase (BuChE, IC50=1.09¡0.20 mM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (IC50=17.95¡2.31 mM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor. Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and AChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site (PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Ab aggregation. In-vivo study indicated PMS1339 (1 mg/kg i.p.) reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement in vivo, and revealed the emergence of a multi-target-directed ligand to tackle the determinants of Alzheimer’s disease. Received 17 February 2009; Reviewed 12 March 2009; Revised 30 March 2009; Accepted 9 April 2009; First published online 22 May 2009 Key words : AChE inhibitor, Alzheimer’s disease, amyloid b, PAF antagonist, PMS1339. Introduction Alzheimer’s disease (AD) is a progressive neuro￾degenerative disorder considered to be the leading cause of dementia affecting the elderly population. Clinically, this neuropathology is characterized by an insidious loss of memory and cognitive abilities in close connection with an impairment of brain cholin￾ergic neurotransmission (Walsh & Selkoe, 2004). Despite enormous research efforts devoted to AD so far, an effective treatment seems to be out of reach. The use of acetylcholinesterase (AChE) inhibitors to en￾hance surviving cholinergic activity is still the main therapy for Alzheimer’s patients. These compounds temporally aim to alleviate their disabilities in terms of improving cognition as well as behavioural and func￾tional daily activities but do not stop the disease’s Author for correspondence : Professor H.-Z. Chen, Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, P.R. China. Tel. : +86 21 6467 4721 Fax : +86 21 6467 4721 Email : hongzhuan_chen@hotmail.com (H.-Z. Chen) Email : heymans@ccr.jussieu.fr (Professor F. Heymans). * These authors contributed equally to this work. International Journal of Neuropsychopharmacology (2009), 12, 1409–1419. Copyright f CINP 2009 doi:10.1017/S1461145709000455 ARTICLE