Discovery Of A Novel Muscarinic M1Agonist And Its Evaluation In Alzheimer’s Disease Animal Models

Nowadays, the significant increase in human lifespan promotes the rapid growth of the aging population. The prevalence of age-related neurodegenerative diseases, such as Alzheimer’s Disease (AD), also grows at an alarming rate. AD is the most common dementia among the elderly. It has been over a century since the first description by Dr. Alzheimer, but the mechanism underlies AD remains enigma, and there is still no cure or effective treatment for this disease.Based upon the cholinergic hypothesis, currently most FDA-approved drugs for the treatment of AD are acetylcholinesterase inhibitors (AChE I), which prevent the breakdown of acetylcholine (ACh), thus, restoring brain ACh level in AD patients. However, their effectiveness diminishes over time due to the progressive loss of cholinergic neurons. On the other hand, levels of Ml muscarinic acetylcholine receptors (mAChRs) are relatively well preserved in the brains of AD patients. There is increasing evidence that M1receptor plays a central role in modulating AD pathology and serves as an appealing AD target. M1agonists target core features of AD, such as:(1) the formation and accumulation of Ap, caused by the abnormal metabolism of Amyloid precursor protein (APP);(2) hyperphosphorylation of the tau protein;(3) cholinergic deficiency, caused by degeneration of cholinergic neurons. Some Ml agonists have been tested in clinical trials, such as Xanomeline. It has shown some therapeutic effect in the treatment of the memory impairment of AD in Phase Ⅲ, but unwanted side effects precluded further development.Accordingly, attempts were made to optimize Xanomeline in pursuit of novel M1agonists with higher affinity, potency, and tolerability. After systematic structural modifications, a series of Xanomeline derivatives were designed and synthesized, which was where the present study started from. There are four chapters in this thesis:(1) in chapter I, the screening of these derivatives, which led to the discovery of EUK1001, will be described in detail, as well as the effects of EUK1001on APP processing;(2) in chapter2, the therapeutic efficacy of EUK1001on a AD model, the forebrain-specific presenilin1/presenilin2conditional double knockout (PS cDKO) mice, will be presented;(3) in chapter3, a preliminary discussion will be made on the possible function of klotho, a famous anti-aging factor, in the AD-like PS cDKO mice;(4) in the last chapter, a review focused on the relationship between M1agonist and AD, as well as the background and progress in klotho research, will be provided.1. Discovery of EUK1001, a novel Ml agonist and Xanomeline derivativeA cell-based reporter gene assay was utilized to screen a collection of172Xanomeline derivatives. Eighteen compounds were found to activate M1receptor, including the two best hits, EUK1001l and EUK1002. Compared with Xanomeline, EUK1001demonstrated markedly lower EC50and higher potency. The M1agonist activity of EUK1001and EUK1002was then confirmed in Chinese hamster ovary (CHO) cells transiently co-transfected with muscarinic M1receptor and reporter gene. In an AD cell model overexpressing the human APP Swedish double mutant (APPsw), EUK1001and EUK1002effectively promoted the secretion of neuroprotective sAPPa via M1receptor. Based on these data, it can be inferred that EUK1001and EUK1002may possess much better capability in activating Ml receptor, and they may also function as a-cut enhancers for APP, which could be a definite help to improve the Aβ pathology of AD.2. EUK1001attenuates neurodegenerative phenotypes in AD-like presenilin1/presenilin2conditional double knockout miceDuring the in vivo pharmacological screening and pharmacokinetics analysis, EUK1001, but not EUK1002, demonstrated a better side effect profile than Xanomeline. In the present study, we studied the effects of3-month chronic administration of EUK1001and Xanomeline (0.5mg/kg/day) in AD-like presenilin1/presenilin2conditional double knockout (PS cDKO) mice. Only EUK1001was found to significantly ameliorate the deficit in recognition memory. Histological analysis demonstrated partial attenuation of the brain atrophy in EUK1001-treated PS cDKO mice and minimal effect in the Xanomeline-treated mice. Both compounds effectively suppressed the elevation of brain tau phosphorylation in the PS cDKO mice, but neither inhibited the increased inflammatory responses. These results indicate that EUK1001showed superiority to Xanomeline with regard to attenuation of several AD-like neurodegenerative phenotypes in PS cDKO mice. These results suggest further investigation of the development of EUK1001for the treatment of AD is indicated.3. Decreased klotho expression and elevated blood phosphate level in AD-like presenilin1/presenilin2conditional double knockout miceThe deficiency of klotho, a famous anti-aging factor, in mice mimics human premature aging. Klotho is predominantly expressed in the kidney and brain, but acts as a circulating factor and has several distinct functions in the whole body. Klotho protein plays essential roles in the regulation of phosphate and calcium homeostasis and acts as an anti-oxidative and anti-inflammation factor. The expression level of klotho declines during aging process and in many diseases, but whether klotho participates in the syndromes of AD has so far not been elucidated. In the present study, using presenilin1/presenilin2conditional double knockout (PS cDKO) mice as an AD model, we have investigated the expression of klotho and its possible involvement in premature aging phenotypes. Our results showed that renal klotho level was significantly decreased in12-month old PS cDKO mice, compared with control mice. Meanwhile, significant elevations of plasma phosphorus, slight reduction of calcium, and almost no sign of renal dysfunction were observed. While at3-month, the renal klotho level, plasma phosphorus, as well as the plasma calcium were not altered, but the brain klotho expression was found to be reduced. These preliminary findings indicate a possible role of klotho in the disorders of PS cDKO mice. The declined renal klotho expression may be responsible for the excess blood phosphate, but it seems that such a moderate decline in renal klotho level and impaired phosphate metabolism were not sufficient to elicit renal dysfunction in PS cDKO mice. On the other hand, as klotho is an anti-oxidative and anti-inflammation factor, klotho decline may contribute to the increased oxidative stress and inflammation in PS cDKO mice, but more evidence is indicated.