Original Contribution Discovery of a benzofuran derivative (MBPTA) as a novel ROCK inhibitor that protects against MPP þ -induced oxidative stress and cell death in SH-SY5Y cells Cheong-Meng Chong a , Mingyun Shen b , Zhong-Yan Zhou a , Peichen Pan b , Pui-Man Hoi a , Shang Li a , Wang Liang a , Nana Ai a , Lun-Qing Zhang a , Cheuk-Wing Li a , Huidong Yu c , Tingjun Hou b,d , Simon Ming-Yuen Lee a,n a State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China b Institute of Functional Nano & Soft Materials and Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou, Jiangsu 215123, China c Rongene Pharma Co., Ltd., International Business Incubator, Guangzhou Science Town, Guangdong 510663, China d College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China article info Article history: Received 17 January 2014 Received in revised form 16 June 2014 Accepted 18 June 2014 Available online 26 June 2014 Keywords: Parkinson disease MPP þ Benzofuran ROCK ROS NO Free radicals abstract Parkinson disease (PD) is a neurodegenerative disease with multifactorial etiopathogenesis. The discovery of drug candidates that act on new targets of PD is required to address the varied pathological aspects and modify the disease process. In this study, a small compound, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl- 1,3,4-thiadiazol-2-yl) acetamide (MBPTA) was identified as a novel Rho-associated protein kinase inhibitor with significant protective effects against 1-methyl-4-phenylpyridinium ion (MPP þ )-induced damage in SH- SY5Y neuroblastoma cells. Further investigation showed that pretreatment of SH-SY5Y cells with MBPTA significantly suppressed MPP þ -induced cell death by restoring abnormal changes in nuclear morphology, mitochondrial membrane potential, and numerous apoptotic regulators. MBPTA was able to inhibit MPP þ - induced reactive oxygen species (ROS)/NO generation, overexpression of inducible NO synthase, and activation of NF-κB, indicating the critical role of MBPTA in regulating ROS/NO-mediated cell death. Furthermore, MBPTA was shown to activate PI3K/Akt survival signaling, and its cytoprotective effect was abolished by PI3K and Akt inhibitors. The structural comparison of a series of MBPTA analogs revealed that the benzofuran moiety probably plays a crucial role in the anti-oxidative stress action. Taken together, these results suggest that MBPTA protects against MPP þ -induced apoptosis in a neuronal cell line through inhibition of ROS/NO generation and activation of PI3K/Akt signaling. & 2014 Published by Elsevier Inc. Parkinson disease (PD) 1 is a common progressive degenerative movement disorder that is mainly caused by the death of dopa- minergic neurons in the substantia nigra of the central nervous system. The primary symptoms of PD patients are characterized by tremor, slowness, stiffness, and balance problems. The incidence of PD is becoming a global health concern as the population ages [1,2]. Two monoamine oxidase type B (MAO-B) inhibitors, selegi- line and rasagiline, are both used to treat early-stage PD, but do not effectively prevent, slow, or halt the progression of this disease [3]. Although the brain neurochemical defects and pathological behavioral characteristics of PD have been well defined, the etiology and pathogenesis mechanisms are not fully elucidated. There is increasing evidence to indicate the critical roles of mitochondrial dysfunction, oxidative stress, neuroinflammation, and apoptosis in the pathogenesis of PD [4]. Numerous agents targeting these pathways have been identified for symptomatic control. Owing to the multifactorial etiopathogenesis of PD, the identification of new drug candidates acting on new targets of PD is urgently needed to tackle the multiple pathological aspects of PD. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine http://dx.doi.org/10.1016/j.freeradbiomed.2014.06.014 0891-5849/& 2014 Published by Elsevier Inc. Abbreviations: AD, Alzheimer disease; CM-H 2 DCFDA, 2 0 ,7 0 -dichlorofluorescein diacetate; DAF-FM, 4-amino-5-methylamino-2 0 ,7 0 -difluorofluorescein; FBS, fetal bovine serum; iNOS, inducible nitric oxide synthase; JC-1, 5,5 0 ,6,6 0 -tetrachloro- 1,1 0 ,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide; LDH, lactate dehydrogenase; MBPTA, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl-1,3,4-thiadiazol-2-yl) acetamide; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; MAO-B, monoamine oxidase type B; MPP þ , 1-methyl-4-phenylpyridinium ion; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PS, penicillin–streptomycin; ROCK, Rho-associated protein kinase; ROS, reactive oxygen species; PARP, poly(ADP- ribose) polymerase; PD, Parkinson disease; PMSF, phenylmethanesulfonyl fluoride; Δψ m , mitochondrial membrane potential n Corresponding author. E-mail addresses: tingjunhou@zju.edu.cn, tingjunhou@hotmail.com (T. Hou), simonlee@umac.mo (S.-Y. Lee). Free Radical Biology and Medicine 74 (2014) 283–293