Discovery of Selective Butyrylcholinesterase (BChE) Inhibitors through a Combination of Computational Studies and Biological Evaluations.
ABSTRACT: As there are increased levels and activity of butyrylcholiesterase (BChE) in the late stage of Alzheimer's disease (AD), development of selective BChE inhibitors is of vital importance. In this study, a workflow combining computational technologies and biological assays were implemented to identify selective BChE inhibitors with new chemical scaffolds. In particular, a pharmacophore model served as a 3D search query to screen three compound collections containing 3.0 million compounds. Molecular docking and cluster analysis were performed to increase the efficiency and accuracy of virtual screening. Finally, 15 compounds were retained for biological investigation. Results revealed that compounds 8 and 18 could potently and highly selectively inhibit BChE activities (IC50 values < 10 ?M on human BChE, selectivity index BChE > 30). These active compounds with novel scaffolds provided us with a good starting point to further design potent and selective BChE inhibitors, which may be beneficial for the treatment of AD.
Project description:Cholinesterase inhibitors have long been used in the treatment of Alzheimer's Disease (AD) via the protection of acetylcholine levels. However, recent research has shown that the specific inhibition of butyrylcholinesterase (BChE) could better ameliorate symptoms within patients. In addition, it has recently been shown that selective inhibition of BChE can also significantly attenuate the toxicity and physiological effects of heroin. Currently, there are no specific and potent inhibitors of BChE approved for use in AD or heroin abuse. Through a combined use of in silico and in vitro screening, we have found three compounds with sub-50?nM IC50 values that specifically target BChE. These newly discovered BChE inhibitors can act as the lead scaffolds for future development of the desirably potent and selective BChE inhibitors.
Project description:AIM: To identify the critical chemical features, with reliable geometric constraints, that contributes to the inhibition of butyrylcholinesterase (BChE) function. METHODS: Ligand-based pharmacophore modeling was used to identify the critical chemical features of BChE inhibitors. The generated pharmacophore model was validated using various techniques, such as Fischer's randomization method, test set, and decoy set. The best pharmacophore model was used as a query in virtual screening to identify novel scaffolds that inhibit BChE. Compounds selected by the best hypothesis in the virtual screening were tested for drug-like properties, and molecular docking study was applied to determine the optimal orientation of the hit compounds in the BChE active site. To find the reactivity of the hit compounds, frontier orbital analysis was carried out using density functional theory. RESULTS: Based on its correlation coefficient (0.96), root mean square (RMS) deviation (1.01), and total cost (105.72), the quantitative hypothesis Hypo1 consisting of 2 HBA, 1 Hy-Ali, and 1 Hy-Ar was selected as the best hypothesis. Thus, Hypo1 was used as a 3D query in virtual screening of the Maybridge and Chembridge databases. The hit compounds were filtered using ADMET, Lipinski's Rule of Five, and molecular docking to reduce the number of false positive results. Finally, 33 compounds were selected based on their critical interactions with the significant amino acids in BChE's active site. To confirm the inhibitors' potencies, the orbital energies, such as HOMO and LUMO, of the hit compounds and 7 training set compounds were calculated. Among the 33 hit compounds, 10 compounds with the highest HOMO values were selected, and this set was further culled to 5 compounds based on their energy gaps important for stability and energy transfer. From the overall results, 5 hit compounds were confirmed to be potential BChE inhibitors that satisfied all the pharmacophoric features in Hypo1. CONCLUSION: This study pinpoints important chemical features with geometric constraints that contribute to the inhibition of BChE activity. Five compounds are selected as the best hit BchE-inhibitory compounds.
Project description:According to recent research advance, it is interesting to identify new, potent and selective inhibitors of human butyrylcholinesterase (BChE) for therapeutic treatment of both the Alzheimer's disease (AD) and heroin abuse. In this study, we carried out a structure-based virtual screening followed by in vitro activity assays, with the goal to identify new inhibitors that are selective for BChE over acetylcholinesterase (AChE). As a result, a set of new, selective inhibitors of human BChE were identified from natural products with solanaceous alkaloid scaffolds. The most active one of the natural products (compound 1) identified has an IC50 of 16.8?nM against BChE. It has been demonstrated that the desirable selectivity of these inhibitors for BChE over AChE is mainly controlled by three key residues in the active site cavity, i.e. residues Q119, A277, and A328 in BChE versus the respective residues Y124, W286, and Y337 in AChE. Based on this structural insight, future rational design of new, potent and selective BChE inhibitors may focus on these key structural differences in the active site cavity.
Project description:In this study, a series of selective butyrylcholinesterase (BChE) inhibitors was designed and synthesized from the structural optimization of hit 1, a 4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid derivative identified by virtual screening our compound library. The in vitro enzyme assay results showed that compounds 9 ((4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)(pyrrolidin-1-yl)methanone) and 23 (N-(2-bromophenyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide) displayed improved BChE inhibitory activity and good selectivity towards BChE versus AChE. Their binding modes were probed by molecular docking and further validated by molecular dynamics simulation. Kinetic analysis together with molecular modeling studies suggested that these derivatives could target both the catalytic active site (CAS) and peripheral anionic site (PAS) of BChE. In addition, the selected compounds 9 and 23 displayed anti-A?1-42 aggregation activity in a dose-dependent manner, and they did not show obvious cytotoxicity towards SH-SY5Y neuroblastoma cells. Also, both compounds showed significantly protective activity against A?1-42-induced toxicity in a SH-SY5Y cell model. The present results provided a new valuable chemical template for the development of selective BChE inhibitors.
Project description:Butyrylcholinesterase (BChE) is a nonspecific cholinesterase enzyme that hydrolyzes choline-based esters. BChE plays a critical role in maintaining normal cholinergic function like acetylcholinesterase (AChE) through hydrolyzing acetylcholine (ACh). Selective BChE inhibition has been regarded as a viable therapeutic approach in Alzheimer's disease. As of now, a limited number of selective BChE inhibitors are available. To identify BChE inhibitors rapidly and efficiently, we have screened 8998 compounds from several annotated libraries against an enzyme-based BChE inhibition assay in a quantitative high-throughput screening (qHTS) format. From the primary screening, we identified a group of 125 compounds that were further confirmed to inhibit BChE activity, including previously reported BChE inhibitors (e.g., bambuterol and rivastigmine) and potential novel BChE inhibitors (e.g., pancuronium bromide and NNC 756), representing diverse structural classes. These BChE inhibitors were also tested for their selectivity by comparing their IC<sub>50</sub> values in BChE and AChE inhibition assays. The binding modes of these compounds were further studied using molecular docking analyses to identify the differences between the interactions of these BChE inhibitors within the active sites of AChE and BChE. Our qHTS approach allowed us to establish a robust and reliable process to screen large compound collections for potential BChE inhibitors.
Project description:: Late-onset Alzheimer's disease (AD) is clinically characterized by a progressive decline of memory and other cognitive functions leading to the loss of the ability to perform everyday activities. Only a few drugs have been approved to treat AD dementia over the past century since the first AD patient was diagnosed. Drugs increasing the availability of neurotransmitters at synapses in the brain are used clinically in the treatment of AD dementia, and cholinesterase inhibitors (ChEIs) are the mainstay of the therapy. A detrimental effect on cognitive function has been reported in patients with pharmacological inhibition of acetylcholinesterase (AChE) by ChEIs and reduced butyrylcholinesterase (BChE) activity due to the single nucleotide polymorphisms. The BChE K-variant (rs1803274), the most common genetic variant of the BCHE gene, was thought to reduce enzyme activity reflecting the lower clinical response to rivastigmine in AD patients. During ChEIs therapy, patients carrying reduced-activity BChE do not present such improved attention like patients with the wild-type enzyme. On the other hand, alterations in the BCHE gene causing enzyme activity reduction may delay AD onset in patients at risk by preserving the level of cortical acetylcholine (ACh). Based on our previous results, we conclude that SNPs localized outside of the coding sequence, in 5'UTR (rs1126680) and/or intron 2 (rs55781031) of the BCHE gene, but not solely K-variant alteration (p.A539T) itself, are responsible for reduced enzyme activity. Therefore, we suspect that not BChE-K itself, but these coexisting SNPs (rs1126680 and rs55781031), could be associated with deleterious changes in cognitive decline in patients treated with ChEIs. Based on the results, we suggest that SNPs (rs1126680) and/or (rs55781031) genotyping should be performed to identify subjects at risk for lowered efficacy ChEIs therapy, and such patients should be treated with a lower rivastigmine dosage. Finally, our sequence analysis of the N-terminal end of N-BChE revealed evolutionarily conserved amino acid residues that can be involved in disulfide bond formation and anchoring of N-BChE in the cell membrane.
Project description:Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer's disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.
Project description:Cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors have been attracted as candidate treatments for Alzheimer's disease (AD). Fifteen khellactone-type coumarins from the roots of Peucedanum japonicum Thunberg were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and MAO inhibitory activities. Compound 3'-angeloyl-4'-(2-methylbutyryl)khellactone (PJ13) most potently inhibited AChE (IC<sub>50</sub>?=?9.28 µM), followed by 3'-isovaleryl-4'-(2-methylbutyroyl)khellactone (PJ15) (IC<sub>50</sub>?=?10.0 ?M). Compound senecioyl-4'-angeloyl-khellactone (PJ5) most potently inhibited BChE (IC<sub>50</sub>?=?7.22 ?M) and had the highest selectivity index (>?5.54), followed by 3'-senecioyl-4'-(2-methylbutyryl)khellactone (PJ10) and 3',4'-disenecioylkhellactone (PJ4) (IC<sub>50</sub>?=?10.2 and 10.7 ?M, respectively). Compounds PJ13, PJ15, and PJ5 showed reversible and mixed-types of inhibition with K<sub>i</sub> values of 5.98, 10.4 (for AChE), and 4.16 µM (for BChE), respectively. However, all 15 compounds weakly inhibited MAO-A and MAO-B. Molecular docking simulation revealed that PJ13 had a higher binding affinity (-?9.3 kcal/mol) with AChE than PJ15 (-?7.8 kcal/mol) or PJ5 (- 5.4 kcal/mol), due to the formation of a hydrogen bond with Tyr121 (distance: 2.52 Å). On the other hand, the binding affinity of PJ5 (-?10.0 kcal/mol) with BChE was higher than for PJ13 (-?7.7 kcal/mol) or PJ15 (-?8.1 kcal/mol), due to the formation of a hydrogen bond with Ser198 (distance: 2.05 Å). These results suggest that PJ13 and PJ5 are potential reversible selective inhibitors of AChE and BChE, respectively, for the treatment of AD.
Project description:Alzheimer's disease (AD) is a neurodegenerative disorder representing the leading cause of dementia and is affecting nearly 44 million people worldwide. AD is characterized by a progressive decline in acetylcholine levels in the cholinergic systems, which results in severe memory loss and cognitive impairments. Expression levels and activity of butyrylcholinesterase (BChE) enzyme has been noted to increase significantly in the late stages of AD, thus making it a viable drug target. A series of hydroxylated 2-phenylbenzofurans compounds were designed, synthesized and their inhibitory activities toward acetylcholinesterase (AChE) and BChE enzymes were evaluated. Two compounds (15 and 17) displayed higher inhibitory activity towards BChE with IC50 values of 6.23??M and 3.57??M, and a good antioxidant activity with EC50 values 14.9??M and 16.7??M, respectively. The same compounds further exhibited selective inhibitory activity against BChE over AChE. Computational studies were used to compare protein-binding pockets and evaluate the interaction fingerprints of the compound. Molecular simulations showed a conserved protein residue interaction network between the compounds, resulting in similar interaction energy values. Thus, combination of biochemical and computational approaches could represent rational guidelines for further structural modification of these hydroxy-benzofuran derivatives as future drugs for treatment of AD.
Project description:Alzheimer's disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.