Immobilized butyrylcholinesterase in the characterization of new inhibitors that could ease Alzheimer's disease.

Focus of this work was the development and characterization of a new immobilized enzyme reactor (IMER) containing human recombinant butyrylcholinesterase (rBChE) for the on-line kinetic characterization of specific, pseudo-irreversible and brain-targeted BChE inhibitors as potential drug candidates for Alzheimer's disease (AD). Specifically, a rBChE-IMER containing 0.99 U of covalently bound target enzyme was purposely developed and inserted into a HPLC system connected to a UV-vis detector. Selected reversible cholinesterase inhibitors, (-)-phenserine and (-)-cymserine analogues, were then kinetically characterized by rBChE-IMER, and by classical in solution assays and their carbamoylation and decarbamoylation constants were determined. The results support the elucidation of the potency, inhibition duration, mode of action and specific structure/activity relations of these agents and allow cross-validation of the two assay techniques.

Tetrahydrofurobenzofuran cymserine, a potent butyrylcholinesterase inhibitor and experimental Alzheimer drug candidate, enzyme kinetic analysis.

Synaptic loss, particularly related to the forebrain cholinergic system, is considered to be an early event that leads to Alzheimer's disease (AD) and has led to the development of acetylcholinesterase inhibitors (AChE-Is) as the mainstay of treatment for several degenerative disorders that culminate in dementia. The primary dose-limiting toxicities of all clinically available AChE-Is are, similar to useful actions on cognition, cholinergically mediated and they ultimately limit the value of this drug class in achieving anything but symptomatic improvements. In addition, AChE levels in brain areas associated with AD decline with disease progression, which likely ultimately limits the therapeutic utility of this drug class. New research indicates that selective inhibition of butyrylcholinesterase (BuChE), a closely related enzyme that is markedly elevated in AD brain, increases acetylcholine (ACh) and augments cognition in rodents free of the characteristic undesirable actions of AChE-Is. BuChE inhibition hence represents an innovative treatment approach for AD, and agents are currently being synthesized to optimally achieve this. The novel compound, tetrahydrofurobenzofuran cymserine (THFBFC), is derived from our effort to produce a potent and BuChE-selective inhibitor as a candidate to test the hypothesis that BuChE-Is would be efficacious and better tolerated than AChE-Is in AD. Herein, we applied innovative enzyme kinetic analyses to characterize the quantitative interaction of THFBFC with human BuChE. These provided values for the agent's IC(50), together with specific new kinetic constants, such as K (T50), K (T1/2), R (I), (o)K (RT), (o)P(max), K(PT) and PT(1/2), to aid define target concentrations for clinical translation. Additional classical kinetic parameters, including K(i), K(m)or K(s), k(cat) or V(max) and V (mi) were also determined. THFBFC proved to be a potent competitive inhibitor of human BuChE and, like its isomer dihydrobenzodioxepine cymserine, is a potentially interesting AD drug candidate.

Identification of novel small molecule inhibitors of amyloid precursor protein synthesis as a route to lower Alzheimer's disease amyloid-beta peptide.

A wealth of independent research with transgenic mice, antibodies, and vaccines has pointed to a causative role of the amyloid-beta peptide (A beta) in Alzheimer's disease (AD). Based on these and earlier associative studies, A beta represents a promising target for development of therapeutics focused on AD disease progression. Interestingly, a cholinesterase inhibitor currently in clinical trials, phenserine, has been shown to inhibit production of both amyloid precursor protein (APP) and A beta. We have shown that this inhibition occurs at the post-transcriptional level with a specific blocking of the synthesis of APP relative to total protein synthesis (Shaw et al., 2001). However, the dose of phenserine necessary to block APP production is far higher than that needed to elicit its anticholinesterase activity, and it is these latter actions that are dose limiting in vivo. The focus of this study was to screen 144 analogs of phenserine to identify additional small molecules that inhibit APP protein synthesis, and thereby A beta production, without possessing potent acetylcholinesterase (AChE) inhibitory activity. An enzyme-linked immunosorbent assay was used to identify analogs capable of suppressing APP production following treatment of human neuroblastoma cells with 20 muM of compound. Eight analogs were capable of dose dependently reducing APP and A beta production without causing cell toxicity in further studies. Several of these analogs had little to no AChE activities. Translation of APP and A beta actions to mice was demonstrated with one agent. They thus represent interesting lead molecules for assessment in animal models, to define their tolerance and utility as potential AD therapeutics.