Dissociation Between the Potent B-Amyloid Protein Pathway Inhibition and Cholinergic Actions of the Alzheimer Drug Candidates Phenserine and Cymserine
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Chapter 47 Dissociation Between the Potent b-Amyloid Protein Pathway Inhibition and Cholinergic Actions of the Alzheimer Drug Candidates Phenserine and Cymserine Nigel H. Greig, Tada Utsuki, Qian-sheng Yu, Harold W. Holloway, Tracyann Perry, David Tweedie, Tony Giordano, George M. Alley, De-Mao Chen, Mohammad A. Kamal, Jack T. Rogers, Kumar Sambamurti, and Debomoy K. Lahiri Introduction Alzheimer’s disease (AD) is characterized by selective neuronal loss, wide- spread deposition of amyloid fibrils that are comprised of a 39-43 amino acid amyloid-b peptide (Ab), and the formation of neurofibrillary tangles in the brain of afflicted individuals [1–4]. Currently, cholinesterase inhibitors (ChEIs) are the primary strategy for treating mild to moderate AD subjects in the United States [5–11], to which the recently approved N-methyl-D-aspartate (NMDA) antagonist memantine is sometimes added [8,12]. However, conjoint with the increasing understanding of the complex pathology of AD over recent years, there is an emergence of more novel targets for therapy [3,4,7,9,10,13]. Such targets are based on the actions of Ab, the inflammatory cascade, and the role of tau proteins in the formation of neurofibrillary tangles, oxidative neu- ronal damage, and neurotransmitter depletion. Because both Ab precursor protein (APP) processing and cholinesterase activity are affected in the AD brain [2–4,7], we examined the effects of several clinically relevant drugs on the APP pathway to elucidate whether common molecular mechanisms linked the two targets. Current research indicates that Ab plays a key role in the progressive neurodegeneration observed in AD [1–4,7]. The smaller Ab peptide [molecular weight (MW) 4.1 kDa] is generated proteo- lytically from a larger integral membrane protein called APP (about 110-130 kDa). Specifically, APP is cleaved by three enzymes—a-, b-, and g-secretases—to different protein fragments, including toxic Ab and several carboxyl-terminal fragments that, additionally, may be involved in the pathogenesis of AD [1–4]. Our goal has been to investigate, design, and synthesize various classes of agents that can potentially lower APP expression and levels, as APP is the N.H. Greig Drug Design & Development Section, Laboratory of Neurosciences, Intramural Research Program, National Institute on Aging, Baltimore, MD 21224, USA A. Fisher et al. (eds.), Advances in Alzheimer’s and Parkinson’s Disease, 445 Ó Springer 2008 446 N.H. Greig et al. originator to all the toxic fragments. Over the last several years, we have examined the effects of a dozen clinically useful drugs/compounds on the amyloid pathway [2,3,7,14–20]. In the present study, our evaluation of the drugs’ effects was focused at four levels: (1) APP itself; (2) the toxic cleaved products of APP, explicitly Ab; (3) anticholinesterase activity; and (4) the chemical structure of the ChEI. We have developed a family of unique cholinesterase inhibitors, phenserine and analogs, that utilize the optically active tricyclic backbone of the natural product and the classic ChEI, physostigmine (also known as eserine) [14,16] (Fig. 1). They differ from physostigmine, however, on the basis of a common phenylcarbamate structure that, for phenserine, is unsubstituted and for its close analog, cymserine, has an isopropyl in the para (40) position [21–25]. In contrast to physostigmine, which is short acting and has a brain/plasma con- centration ratio of unity [14,15], phenserine and cymserine are reversible and long-acting anticholinesterases that have a brain/plasma concentration ratio of 10:1 and 40:1 and selectivity for acetylcholinesterase (AChE) and butyrylcho- linesterase (BuChE), respectively [14,16,22]. Consequently, phenserine has been demonstrated to improve cognitive performance dramatically in rodents over an unusually wide dose range [14,16]. In addition, phenserine administration reduced brain APP production in naive animals and prevented a rise in APP brain levels, which are associated with forebrain cholinergic lesions in rats, a model that mimics the cholinergic loss found in the AD brain [26]. Phenserine is currently being assessed in clinical trials for mild to moderate AD potentially to perturb the cognitive and behavioral symptoms associated with AD and lower brain levels of Ab [16]. Cymserine analogs are in preclinical development for the potential treatment of severe AD, when BuChE levels are dramatically elevated H CH3 N O C C 1 A B N CH3 CH O N 8 3 CH3 Physostigmine 11.6 Å H H CH3 CH3 N O O C N C N CH CH 4' CH O N 3 3 O N 3 CH3 CH3 CH3 Phenserine 14.6 Å Cymserine 18.1 Å Fig. 1 Chemical structures of physostigmine (top), phenserine (bottom left), and cymserine (bottom right). Hexahydropyrroloindole marked as tricyclic A, B, C rings; N1 and N8 nitrogens; and 40 (para) position are shown 47 Phenserine/ Cymserine Effect on Ab 447 and mismatched to dwindling levels of acetylcholine and AChE [27,28]. In contrast, AD clinical trials with physostigmine, available as a slow-release oral formulation (Synapton; Forest Laboratories, New York, NY, USA) were suspended based on its poor toxicity profile [29]. Consequent to the fact that both APP processing and cholinesterase activity are affected in the AD brain, a focus for our current studies is the molecular changes induced by ChEIs [17–20,30]. In this regard, our present work is a continuation of earlier research on tacrine, a first-generation aminoacridine ChEI with beneficial cognitive action in the treatment of AD that, additionally, showed activity on the APP pathway [19,20]. Specifically, tacrine treatment reduced the levels of secreted APP and Ab of neural cells in culture; but whether this effect was related to tacrine’s chemical structure or anticholinesterase action remained unclear. We therefore have studied the mechanism through which other structurally divergent ChEI drugs, such as phenserine, interact with the cellular processing of APP [17,18,30–33]. Herein, we report that phenserine and cymserine, close structural analogs of physostigmine, reduced levels of APP and Ab, whereas physostigmine had no effect on neuroblastoma cells. Taken together, the prior tacrine action and the differential effects of three structurally related compounds (phenserine, cymserine, physostigmine) on APP pathways contrast with their similar potency to inhibit either AChE or BuChE, thereby dissociating these two pharmacological actions. Elucidating the mechanism(s) via which selected ChEIs alter APP synthesis/processing and hence lower Ab deposition may allow optimization of their use and, additionally, aid in the design of better drugs for AD treatment. Methods Chemicals Phenserine ([3aS]- or [–] -phenserine) and cymserine ([3aS]- or [–]-cymserine) were synthesized as their water-soluble tartrate salts, as described previously [21–25] and were of high (> 99.9%) chemical and optical purity. Physostigmine ([3aS]– or [–]-physostigmine) was purchased as eserine salicylate from Sigma (St. Louis, MO, USA), and all other chemicals were of the highest purity and were obtained from Sigma unless stated otherwise. Lipophilicity Determination Octanol-water partition coefficients were determined both experimentally and by computation. Octanol solutions (0.5 mM), 5 ml, of phenserine, cymserine, and physostigmine were prepared, and their ultraviolet (UV) absorbences, A1, were determined by spectrophotometer at 254 nm wavelength. The octanol 448 N.H. Greig et al. solutions then were vigorously mixed with an equal volume of 0.1 M phosphate buffer (pH 7.4) for 15 minutes. Following separation by centrifugation and appropriate drying, the absorbence of the octanol was again measured, A2. An octanol-water partition coefficient, P, was calculated from the formula: P = A2/A1 À A2 Cell Culture Human neuroblastoma (SK-N-SH) cells were obtained from the American Type Culture Collection (ATCC) and cultured in minimal essential medium (MEM) containing 10% fetal bovine serum (FBS) to 70% confluence, as described elsewhere [18–20]. To initiate the experiment, cells were first fed with fresh medium with low FBS (0.5%) and treated separately with vehicle, phenserine, cymserine, or physostigmine at defined concentrations for specific periods of time (16 and 24 hours). LDH and MTT Assays Following drug treatment, cells were allowed to grow, and conditioned medium samples were collected at 16- to 24-hour intervals. The released lactate dehydrogenase (LDH) was measured in the conditioned medium samples. Cells were harvested at the end of the experiment. The cells were resuspended and immediately assayed for MTT [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenylte- trazolium bromide]. Both LDH and MTT measurements were undertaken using sensitive and quantitative methods within their linear ranges, as described previously [18,19]. APP Measurement Levels of secretory APP were assayed in the conditioned medium by Western immunoblotting using 22C11 antibody (Roche), as described previously [18,19]. Ab Assay Levels of total Abx-40 peptide levels were determined in the conditioned medium samples using a sandwich enzyme-linked immunosorbent assay (ELISA) method after the modification of IBL reagents [18–20]. Abx-40 is the most abundant species among all Ab isoforms secreted and can be detected in this cell line. 47 Phenserine/ Cymserine Effect on Ab 449 Cholinesterase Measurements The inhibition of freshly prepared human AChE and BuChE by phenserine and physostigmine was independently measured between the concentrations of 0.3 nM and 10 mM to obtain both an IC50 value (concentration required to inhibit 50% enzyme activity) and the level of enzyme inhibition obtained in the described cell culture studies, as described previously [21–25]. Results Phenserine, Cymserine, and Physostigmine Assessed at Nontoxic Doses in Human Neuronal (SK-N-SH) Cell Cultures Human neuroblastoma cells were incubated with vehicle, phenserine, cymser- ine, or physostigmine as described in the Methods section. When monitoring cells morphologically under a phase contrast microscope, we observed no difference between control and drug-treated cells. This was confirmed by sub- jecting the cell culture samples to various biochemical viability assays.