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.

A phenothiazine urea derivative broadly inhibits coronavirus replication via viral protease inhibition.

Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (Mpro) and papain-like protease (PLpro), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 Mpro and PLpro protease activity was identified. In silico docking studies also predicted the binding of the phenothiazine urea to the active sites of structurally similar Mpro and PLpro proteases from distantly related alphacoronavirus, HCoV-229 E (229 E), and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cellulo. It was further demonstrated that the compound inhibited 229 E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles, and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative readily inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses.

Solvents and detergents compatible with enzyme kinetic studies of cholinesterases.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes that serve a wide range of physiological functions including the hydrolysis of the neurotransmitter acetylcholine and several other xenobiotics. The development of inhibitors for these enzymes has been the focus for the treatment of several conditions, such as Alzheimer's disease. Novel chemical entities are evaluated as potential inhibitors of AChE and BChE using enzyme kinetics. A common issue encountered in these studies is low aqueous solubility of the possible inhibitor. Additives such as cosolvents or detergents can be included in these studies improve the aqueous solubility. Typical cosolvents include acetonitrile or dimethyl sulfoxide while typical detergents include Polysorbate 20 (Tween 20) or 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS). When solubility is not improved, these molecules are often not evaluated further. To address this issue eleven cosolvents and six detergents that could facilitate aqueous solubility were evaluated to understand how they would affect cholinesterase enzymes using Ellman's assay. These studies show that propylene glycol, acetonitrile, methanol, Tween 20, Polysorbate 80 (Tween 80), polyoxyethylene 23 lauryl ether (Brij 35) and polyoxyethylene 10 oleoyl ether (Brij 96v) have the least inhibitory effects towards cholinesterase activity. It is concluded that these cosolvents and detergents should be considered as solubilizing agents for evaluation of potential cholinesterase inhibitors with low aqueous solubility.

2-Pyridone natural products as inhibitors of SARS-CoV-2 main protease.

The disease, COVID-19, is caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2) for which there is currently no treatment. The SARS-CoV-2 main protease (Mpro) is an important enzyme for viral replication. Small molecules that inhibit this protease could lead to an effective COVID-19 treatment. The 2-pyridone scaffold was previously identified as a possible key pharmacophore to inhibit SARS-CoV-2 Mpro. A search for natural, antimicrobial products with the 2-pyridone moiety was undertaken herein, and their calculated potency as inhibitors of SARS-CoV-2 Mpro was investigated. Thirty-three natural products containing the 2-pyridone scaffold were identified from the literature. An in silico methodology using AutoDock was employed to predict the binding energies and inhibition constants (Ki values) for each 2-pyridone-containing compound with SARS-CoV-2 Mpro. This consisted of molecular optimization of the 2-pyridone compound, docking of the compound with a crystal structure of SARS-CoV-2 Mpro, and evaluation of the predicted interactions and ligand-enzyme conformations. All compounds investigated bound to the active site of SARS-CoV-2 Mpro, close to the catalytic dyad (His-41 and Cys-145). Thirteen molecules had predicted Ki values <1 µM. Glu-166 formed a key hydrogen bond in the majority of the predicted complexes, while Met-165 had some involvement in the complex binding as a close contact to the ligand. Prominent 2-pyridone compounds were further evaluated for their ADMET properties. This work has identified 2-pyridone natural products with calculated potent inhibitory activity against SARS-CoV-2 Mpro and with desirable drug-like properties, which may lead to the rapid discovery of a treatment for COVID-19.

1-(3-Tert-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM/MM Studies.

Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with Ki = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with Ki* = 0.53 nM. Reversible binding of TFK provides a long residence time, τ = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the "isomerization" step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called "transition state analog", followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer's disease and protection of central AChE against organophosphorus compounds.

Targeting butyrylcholinesterase for preclinical single photon emission computed tomography (SPECT) imaging of Alzheimer's disease.

INTRODUCTION: Diagnosis of Alzheimer's disease (AD) in vivo, by molecular imaging of amyloid or tau, is constrained because similar changes can be found in brains of cognitively normal individuals. Butyrylcholinesterase (BChE), which becomes associated with these structures in AD, could elevate the accuracy of AD diagnosis by focusing on BChE pathology in the cerebral cortex, a region of scant BChE activity in healthy brain. METHODS: N-methylpiperidin-4-yl 4-[123I]iodobenzoate, a BChE radiotracer, was injected intravenously into B6SJL-Tg(APPSwFlLon, PSEN1∗M146 L∗L286 V) 6799Vas/Mmjax (5XFAD) mice and their wild-type (WT) counterparts for comparative single photon emission computed tomography (SPECT) studies. SPECT, computed tomography (CT), and magnetic resonance imaging (MRI) enabled comparison of whole brain and regional retention of the BChE radiotracer in both mouse strains. RESULTS: Retention of the BChE radiotracer was consistently higher in the 5XFAD mouse than in WT, and differences were particularly evident in the cerebral cortex. DISCUSSION: Cerebral cortical BChE imaging with SPECT can distinguish 5XFAD mouse model from the WT counterpart.

Development of acetophenone ligands as potential neuroimaging agents for cholinesterases.

Association of cholinesterase with ß-amyloid plaques and tau neurofibrillary tangles in Alzheimer's disease offers an opportunity to detect disease pathology during life. Achieving this requires development of radiolabelled cholinesterase ligands with high enzyme affinity. Various fluorinated acetophenone derivatives bind to acetylcholinesterase with high affinity, including 2,2,2-trifluoro-1-(3-dimethylaminophenyl)ethanone (1) and 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (2). Such compounds also offer potential for incorporation of radioactive fluorine (18F) for Positron Emission Tomography (PET) imaging of cholinesterases in association with Alzheimer's disease pathology in the living brain. Here we describe the synthesis of two meta-substituted chlorodifluoroacetophenones using a Weinreb amide strategy and their rapid conversion to the corresponding trifluoro derivatives through nucleophilic substitution by fluoride ion, in a reaction amenable to incorporating 18F for PET imaging. In vitro kinetic analysis indicates tight binding of the trifluoro derivatives to cholinesterases. Compound 1 has a Ki value of 7nM for acetylcholinesterase and 1300nM for butyrylcholinesterase while for compound 2 these values are 0.4nM and 26nM, respectively. Tight binding of these compounds to cholinesterase encourages their development for PET imaging detection of cholinesterase associated with Alzheimer's disease pathology.

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.

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 of 6H-dibenzo[b,d]pyran-6-ones using the inverse electron demand Diels-Alder reaction.

A set of coumarin-fused electron-deficient 1,3-dienes was synthesized, which differ in the nature of the electron-withdrawing group (EWG) at the terminus of the diene unit and (when EWG = CO(2)Me) the nature and position of substituents. These dienes reacted with the enamine derived from cyclopentanone and pyrrolidine to afford the corresponding cyclopenteno-fused 6H-dibenzo[b,d]pyran-6-ones, most likely via a domino inverse electron demand Diels-Alder (IEDDA)/elimination/transfer hydrogenation sequence. The parent diene (EWG = CO(2)Me, no substituents) was reacted with a range of electron-rich dienophiles (mostly enamines) to afford the corresponding 6H-dibenzo[b,d]pyran-6-ones or their nondehydrogenated precursors, which were aromatized upon treatment with a suitable oxidant. The enamines could either be synthesized prior to the reaction or generated in situ. The syntheses of 30 dibenzopyranones are reported.

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.

Cysteine thioesters as myelin proteolipid protein analogues to examine the role of butyrylcholinesterase in myelin decompaction.

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disorder involving demyelination, axonal transection, and neuronal loss in the brain. Recent studies have indicated that active MS lesions express elevated levels of butyrylcholinesterase (BuChE). BuChE can hydrolyze a wide variety of esters, including fatty acid esters of protein. Proteolipid protein (PLP), an important transmembrane protein component of myelin, has six cysteine residues acylated, via thioester linkages, with fatty acids, usually palmitic, that contribute to the stability of myelin. Experimental chemical deacylation of PLP has been shown to lead to decompaction of myelin. Because of elevated levels of BuChE in active MS lesions and its propensity to catalyze the hydrolysis of acylated protein, we hypothesized that this enzyme may contribute to deacylation of PLP in MS, leading to decompaction of myelin and contributing to demyelination. To test this hypothesis, a series of increasing chain length (C2-C16) acyl thioester derivatives of N-acetyl-l-cysteine methyl ester were synthesized and examined for hydrolysis by human cholinesterases. All N-acetyl-l-cysteine fatty acyl thioester derivatives were hydrolyzed by BuChE but not by the related enzyme acetylcholinesterase. In addition, it was observed that the affinity of BuChE for the compound increased the longer the fatty acid chain, with the highest affinity for cysteine bound to palmitic acid. This suggests that the elevated levels of BuChE observed in active MS lesions could be related to the decompaction of myelin characteristic of the disorder.

Differential binding of phenothiazine urea derivatives to wild-type human cholinesterases and butyrylcholinesterase mutants.

A series of N-10 urea derivatives of phenothiazine was synthesized and each compound was evaluated for its ability to inhibit human cholinesterases. Most were specific inhibitors of BuChE. However, the potent inhibitory effects on both cholinesterases of one sub-class, the cationic aminoureas, provide an additional binding mechanism to cholinesterases for these compounds. The comparative effects of aminoureas on wild-type BuChE and several BuChE mutants indicate a binding process involving salt linkage with the aspartate of the cholinesterase peripheral anionic site. The effect of such compounds on cholinesterase activity at high substrate concentration supports ionic interaction of aminoureas at the peripheral anionic site.