A method for the efficient evaluation of substrate-based cholinesterase imaging probes for Alzheimer's disease.

Cholinesterase (ChE) enzymes have been identified as diagnostic markers for Alzheimer disease (AD). Substrate-based probes have been synthesised to detect ChEs but they have not detected changes in ChE distribution associated with AD pathology. Probes are typically screened using spectrophotometric methods with pure enzyme for specificity and kinetics. However, the biochemical properties of ChEs associated with AD pathology are altered. The present work was undertaken to determine whether the Karnovsky-Roots (KR) histochemical method could be used to evaluate probes at the site of pathology. Thirty thioesters and esters were synthesised and evaluated using enzyme kinetic and KR methods. Spectrophotometric methods demonstrated all thioesters were ChE substrates, yet only a few provided staining in the brain with the KR method. Esters were ChE substrates with interactions with brain ChEs. These results suggest that the KR method may provide an efficient means to screen compounds as probes for imaging AD-associated ChEs.

Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

Introduction: Alzheimer's disease (AD) is characterized by neurotoxic immuno-inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aß) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses. Methods: We performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic-molecular mechanisms of cytokine-mediated and Aß-mediated neurotoxicities in AD.  Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small-molecule therapeutics for AD. Results: In response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aß is released as an early responder immunopeptide triggering an innate immunity cascade in which Aß exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon "self" neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane-penetrating attack by antimicrobial peptides (AMPs) such as Aß. After this self-attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aß, leading to a chronic self-perpetuating autoimmune cycle.  AD thus emerges as a brain-centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti-AD molecules capable of chemical modification into multi-site therapeutic modulators targeting AD's complex immunopathic-proteopathic pathogenesis. Discussion:  Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug-like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

Synthesis and Preliminary Evaluation of Phenyl 4-123I-Iodophenylcarbamate for Visualization of Cholinesterases Associated with Alzheimer Disease Pathology.

UNLABELLED: Acetylcholinesterase and butyrylcholinesterase accumulate with brain ß-amyloid (Aß) plaques in Alzheimer disease (AD). The overall activity of acetylcholinesterase is found to decline in AD, whereas butyrylcholinesterase has been found to either increase or remain the same. Although some cognitively normal older adults also have Aß plaques within the brain, cholinesterase-associated plaques are generally less abundant in such individuals. Thus, brain imaging of cholinesterase activity associated with Aß plaques has the potential to distinguish AD from cognitively normal older adults, with or without Aß accumulation, during life. Current Aß imaging agents are not able to provide this distinction. To address this unmet need, synthesis and evaluation of a cholinesterase-binding ligand, phenyl 4-(123)I-iodophenylcarbamate ((123)I-PIP), is described. METHODS: Phenyl 4-iodophenylcarbamate was synthesized and evaluated for binding potency toward acetylcholinesterase and butyrylcholinesterase using enzyme kinetic analysis. This compound was subsequently rapidly radiolabeled with (123)I and purified by high-performance liquid chromatography. Autoradiographic analyses were performed with (123)I-PIP using postmortem orbitofrontal cortex from cognitively normal and AD human brains. Comparisons were made with an Aß imaging agent, 2-(4'-dimethylaminophenyl)-6-(123)I-iodo-imidazo[1,2-a]pyridine ((123)I-IMPY), in adjacent brain sections. Tissues were also stained for Aß and cholinesterase activity to visualize Aß plaque load for comparison with radioligand uptake. RESULTS: Synthesized and purified PIP exhibited binding to cholinesterases. (123)I was successfully incorporated into this ligand. (123)I-PIP autoradiography with human tissue revealed accumulation of radioactivity only in AD brain tissues in which Aß plaques had cholinesterase activity. (123)I-IMPY accumulated in brain tissues with Aß plaques from both AD and cognitively normal individuals. CONCLUSION: Radiolabeled ligands specific for cholinesterases have potential for use in neuroimaging AD plaques during life. The compound herein described, (123)I-PIP, can detect cholinesterases associated with Aß plaques and can distinguish AD brain tissues from those of cognitively normal older adults with Aß plaques. Imaging cholinesterase activity associated with Aß plaques in the living brain may contribute to the definitive diagnosis of AD during life.

Butyrylcholinesterase-knockout reduces brain deposition of fibrillar ß-amyloid in an Alzheimer mouse model.

In Alzheimer's disease (AD), numerous ß-amyloid (Aß) plaques are associated with butyrylcholinesterase (BChE) activity, the significance of which is unclear. A mouse model, containing five human familial AD genes (5XFAD), also develops Aß plaques with BChE activity. Knock-out of BChE in this model showed diminished fibrillar Aß plaque deposition, more so in males than females. This suggests that lack of BChE reduces deposition of fibrillar Aß in AD and this effect may be influenced by sex.

Thioesters for the in vitro evaluation of agents to image brain cholinesterases.

Cholinesterases are associated with pathology characteristic of conditions such as Alzheimer's disease and are therefore, considered targets for neuroimaging. Ester derivatives of N-methylpiperidinol are promising potential imaging agents; however, methodology is lacking for evaluating these compounds in vitro. Here, we report the synthesis and evaluation of a series of N-methylpiperidinyl thioesters, possessing comparable properties to their corresponding esters, which can be directly evaluated for cholinesterase kinetics and histochemical distribution in human brain tissue. N-methylpiperidinyl esters and thioesters were synthesized and they demonstrated comparable cholinesterase kinetics. Furthermore, thioesters were capable, using histochemical method, to visualize cholinesterase activity in human brain tissue. N-methylpiperidinyl thioesters can be rapidly evaluated for cholinesterase kinetics and visualization of enzyme distribution in brain tissue which may facilitate development of cholinesterase imaging agents for application to conditions such as Alzheimer's disease.

Synthesis and preliminary evaluation of piperidinyl and pyrrolidinyl iodobenzoates as imaging agents for butyrylcholinesterase.

PURPOSE: The purpose of this study is to synthesize and evaluate specific agents for molecular imaging of butyrylcholinesterase (BuChE), known to be associated with neuritic plaques and neurofibrillary tangles in Alzheimer's disease (AD). In this study, these agents were tested in a normal rat model. The distribution of radiolabel was compared with known BuChE histochemical distribution in the rat brain. PROCEDURES: Iodobenzoate esters were synthesized and tested, through spectrophotometric analysis, as specific substrates for BuChE. These compounds were converted to the corresponding (123)I esters from tributyltin intermediates and purified for studies in the rat model. Whole body dynamic scintigraphic images were obtained for biodistribution studies. Autoradiograms of brain sections were obtained and compared to histochemical distribution of the enzyme in this model system. RESULTS: The three iodobenzoate esters studied were specific substrates for BuChE. Whole body biodistribution studies with (123)I-labeled compounds showed rapid disappearance from the body while radioactivity was retained in the head region. Brain section autoradiography of animals injected with these labeled compounds indicated that most areas known to contain BuChE corresponded to areas of radioactivity accumulation. CONCLUSION: BuChE-specific radiolabeled iodobenzoates enter the brain and, in general, label areas known to exhibit BuChE activity in histochemical studies. Such molecules may represent a new direction for the development of agents for the molecular imaging of BuChE in the living brain, especially in regions where BuChE-containing neuropathological structures appear in AD.

A significant reduction of the diaphragm in mdx:MyoD-/-(9th) embryos suggests a role for MyoD in the diaphragm development.

To further investigate the role of MyoD during skeletal myogenesis, we backcrossed mdx mutant mice (lacking dystrophin) with MyoD knock-out mice to obtain viable mice with MyoD allele on a pure mdx background. However, after nine generations of backcrossing, it was not possible to obtain a viable mdx:MyoD-/- phenotype (designated as: mdx:MyoD-/-(9th)). The compound-mutant embryos were examined just before birth. Essentially normal Myf5-dependent and most of the MyoD-dependent musculature was observed. By contrast, the skeletal muscle compartment of the diaphragm was significantly reduced. The mesenchymal compartment of the diaphragm was intact and no herniations were observed. Other examined organs (e.g., liver, kidney, brain, etc.) showed no histological abnormalities. Pulmonary hypoplasia was determined as the cause of neonatal death. Therefore, using a different approach, our new data supplement our previous findings and suggest an essential role for MyoD in development of skeletal muscle of the diaphragm. The failure of mdx:MyoD-/-(9th) diaphragm to develop normally is not caused by a reduced number of satellite cells, but from the inability of stem cells to progress through the myogenic program. Our data also suggest that functions of MyoD and Myf5 (and the respective muscle precursor cell sub-populations) are not entirely redundant by term, as previously suggested, since Myf5 is not capable of fully substituting for MyoD in the diaphragm development.