Histoenzymatic methods and in situ hybridization were used to follow AChE expression in rabbit em... more Histoenzymatic methods and in situ hybridization were used to follow AChE expression in rabbit embryos from 10 to 15 days. Transcripts of AChE are detected at the same developmental stages in all structures where enzymatic activity is found, except in neuronal extension and the ventral part of mesonephros. AChE and BChE expression were compared. BChE transcripts are detected before BChE activity can be revealed in blood cells and mesonephros.
Organophosphates (OPs) are either found in nature or synthetized for use as pesticides, flame ret... more Organophosphates (OPs) are either found in nature or synthetized for use as pesticides, flame retardants, neurotoxic warfare agents or drugs (cholinergic enhancers in Alzheimer's disease and myasthenia gravis, or inhibitors of lipases in metabolic diseases). Because of the central role of acetylcholinesterase cholinergic neurotransmission in humans, one of the main purposes for using OPs is inactivation of the enzyme by phosphorylation of the nucleophilic serine residue in the active center. However, hundreds of serine hydrolases are expressed in the human proteome, and many of them are potential targets for OP adduction. In this review, we first situate the α/β hydrolase fold proteins among the distinctively folded proteins known to interact with OPs, in particular the different lipases, peptidases, and enzymes hydrolyzing OPs. Second, we compile the human α/β hydrolases and review those that have been experimentally shown to interact with OPs. Among the 120 human α/β hydrolase fold proteins, 102 have a serine in the consensus GXSXG pentapeptide compatible with an active site, 6 have an aspartate or a cysteine as the active site nucleophile residue, and 12 evidently lack an active site. 76 of the 120 have been experimentally shown to bind an OP.
The enzyme butyrylcholinesterase (BChE) and the human cannabinoid receptor 2 (hCBR) represent pro... more The enzyme butyrylcholinesterase (BChE) and the human cannabinoid receptor 2 (hCBR) represent promising targets for pharmacotherapy in the later stages of Alzheimer's disease. We merged pharmacophores for both targets into small benzimidazole-based molecules, investigated SARs, and identified several dual-acting ligands with a balanced affinity/inhibitory activity and an excellent selectivity over both hCBR and hAChE. A homology model for the hCBR was developed based on the hCBR crystal structure and used for molecular dynamics studies to investigate binding modes. In vitro studies proved hCBR agonism. Unwanted μ-opioid receptor affinity could be designed out. One well-balanced dual-acting and selective hBChE inhibitor/hCBR agonist showed superior in vivo activity over the lead CBagonist with regards to cognition improvement. The data shows the possibility to combine a small molecule with selective and balanced GPCR-activity/enzyme inhibition and in vivo activity for the therapy...
The prion protein (PrP) binds to various molecular partners, but little is known about their pote... more The prion protein (PrP) binds to various molecular partners, but little is known about their potential impact on the pathogenesis of prion diseases RESULTS: Here, we show that PrP can interact in vitro with acetylcholinesterase (AChE), a key protein of the cholinergic system in neural and non-neural tissues. This heterologous association induced aggregation of monomeric PrP and modified the structural properties of PrP amyloid fibrils. Following its recruitment into PrP fibrils, AChE loses its enzymatic activity and enhances PrP-mediated cytotoxicity. Using several truncated PrP variants and specific tight-binding AChE inhibitors (AChEis), we then demonstrate that the PrP-AChE interaction requires two mutually exclusive sub-sites in PrP N-terminal domain and an aromatic-rich region at the entrance of AChE active center gorge. We show that AChEis that target this site impair PrP-AChE complex formation and also limit the accumulation of pathological prion protein (PrPSc) in prion-infe...
Structure and Function of Cholinesterases and Related Proteins, 1998
Numerous observations suggest a “non classical” role for acetylcholinesterase during development ... more Numerous observations suggest a “non classical” role for acetylcholinesterase during development (1). We are interested in testing this hypothesis during early development of the teleost Danio rerio (zebrafish).
The peptidasic site of highly purified human plasma cholinesterase was investigated using active‐... more The peptidasic site of highly purified human plasma cholinesterase was investigated using active‐site‐directed inhibitors. Peptidase activity was assayed taking substance P as substrate. Inhibition by organophosphates indicated that the peptidasic site contained an active serine. The presence of essential histidine residues associated with serine was revealed by histidine modifications. Carboxyl group reagents showed that the active centre contained carboxyl groups in a non‐polar environment. The removal of sialic acids did not alter peptidase activity. The peptidasic site of cholinesterase shared many properties with serine proteases sites and esteratic sites of cholinesterases. In addition, with the peptidasic site, as well as the esteratic site, there was always the possibility of ‘aging’ when inhibited by DFP or soman.
Human isolated pulmonary vessels were treated with cholinesterase (ChE) inhibitors to determine t... more Human isolated pulmonary vessels were treated with cholinesterase (ChE) inhibitors to determine the role of these enzymes in regulating vascular muscle tone. In addition, kinetic parameters were determined for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in human pulmonary vessel homogenates. Carbachol (CCh) and acetylcholine (ACh) were equipotent contractile agonists in human pulmonary arteries (pD2 values, 5.28±0.05 and 5.65±0.16; Emax, 0.91±0.26 and 0.98±0.30 g wt. for CCh and ACh, respectively; n=7). In venous preparations, ACh was ineffective and CCh induced small contractions (Emax, 0.08±0.04 g wt.; n=13). In human pulmonary arteries following pretreatment with tetraisopropylpyrophosphoramide (iso‐OMPA, 100 μM), an increased sensitivity to the contractile agonist ACh was observed (pD2 values, 5.80±0.13 and 6.37±0.19 for control and treated preparations, respectively; n=5). This pretreatment had no effect on the CCh concentration response curve. In contrast, hum...
Histoenzymatic methods and in situ hybridization were used to follow AChE expression in rabbit em... more Histoenzymatic methods and in situ hybridization were used to follow AChE expression in rabbit embryos from 10 to 15 days. Transcripts of AChE are detected at the same developmental stages in all structures where enzymatic activity is found, except in neuronal extension and the ventral part of mesonephros. AChE and BChE expression were compared. BChE transcripts are detected before BChE activity can be revealed in blood cells and mesonephros.
Organophosphates (OPs) are either found in nature or synthetized for use as pesticides, flame ret... more Organophosphates (OPs) are either found in nature or synthetized for use as pesticides, flame retardants, neurotoxic warfare agents or drugs (cholinergic enhancers in Alzheimer's disease and myasthenia gravis, or inhibitors of lipases in metabolic diseases). Because of the central role of acetylcholinesterase cholinergic neurotransmission in humans, one of the main purposes for using OPs is inactivation of the enzyme by phosphorylation of the nucleophilic serine residue in the active center. However, hundreds of serine hydrolases are expressed in the human proteome, and many of them are potential targets for OP adduction. In this review, we first situate the α/β hydrolase fold proteins among the distinctively folded proteins known to interact with OPs, in particular the different lipases, peptidases, and enzymes hydrolyzing OPs. Second, we compile the human α/β hydrolases and review those that have been experimentally shown to interact with OPs. Among the 120 human α/β hydrolase fold proteins, 102 have a serine in the consensus GXSXG pentapeptide compatible with an active site, 6 have an aspartate or a cysteine as the active site nucleophile residue, and 12 evidently lack an active site. 76 of the 120 have been experimentally shown to bind an OP.
The enzyme butyrylcholinesterase (BChE) and the human cannabinoid receptor 2 (hCBR) represent pro... more The enzyme butyrylcholinesterase (BChE) and the human cannabinoid receptor 2 (hCBR) represent promising targets for pharmacotherapy in the later stages of Alzheimer's disease. We merged pharmacophores for both targets into small benzimidazole-based molecules, investigated SARs, and identified several dual-acting ligands with a balanced affinity/inhibitory activity and an excellent selectivity over both hCBR and hAChE. A homology model for the hCBR was developed based on the hCBR crystal structure and used for molecular dynamics studies to investigate binding modes. In vitro studies proved hCBR agonism. Unwanted μ-opioid receptor affinity could be designed out. One well-balanced dual-acting and selective hBChE inhibitor/hCBR agonist showed superior in vivo activity over the lead CBagonist with regards to cognition improvement. The data shows the possibility to combine a small molecule with selective and balanced GPCR-activity/enzyme inhibition and in vivo activity for the therapy...
The prion protein (PrP) binds to various molecular partners, but little is known about their pote... more The prion protein (PrP) binds to various molecular partners, but little is known about their potential impact on the pathogenesis of prion diseases RESULTS: Here, we show that PrP can interact in vitro with acetylcholinesterase (AChE), a key protein of the cholinergic system in neural and non-neural tissues. This heterologous association induced aggregation of monomeric PrP and modified the structural properties of PrP amyloid fibrils. Following its recruitment into PrP fibrils, AChE loses its enzymatic activity and enhances PrP-mediated cytotoxicity. Using several truncated PrP variants and specific tight-binding AChE inhibitors (AChEis), we then demonstrate that the PrP-AChE interaction requires two mutually exclusive sub-sites in PrP N-terminal domain and an aromatic-rich region at the entrance of AChE active center gorge. We show that AChEis that target this site impair PrP-AChE complex formation and also limit the accumulation of pathological prion protein (PrPSc) in prion-infe...
Structure and Function of Cholinesterases and Related Proteins, 1998
Numerous observations suggest a “non classical” role for acetylcholinesterase during development ... more Numerous observations suggest a “non classical” role for acetylcholinesterase during development (1). We are interested in testing this hypothesis during early development of the teleost Danio rerio (zebrafish).
The peptidasic site of highly purified human plasma cholinesterase was investigated using active‐... more The peptidasic site of highly purified human plasma cholinesterase was investigated using active‐site‐directed inhibitors. Peptidase activity was assayed taking substance P as substrate. Inhibition by organophosphates indicated that the peptidasic site contained an active serine. The presence of essential histidine residues associated with serine was revealed by histidine modifications. Carboxyl group reagents showed that the active centre contained carboxyl groups in a non‐polar environment. The removal of sialic acids did not alter peptidase activity. The peptidasic site of cholinesterase shared many properties with serine proteases sites and esteratic sites of cholinesterases. In addition, with the peptidasic site, as well as the esteratic site, there was always the possibility of ‘aging’ when inhibited by DFP or soman.
Human isolated pulmonary vessels were treated with cholinesterase (ChE) inhibitors to determine t... more Human isolated pulmonary vessels were treated with cholinesterase (ChE) inhibitors to determine the role of these enzymes in regulating vascular muscle tone. In addition, kinetic parameters were determined for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in human pulmonary vessel homogenates. Carbachol (CCh) and acetylcholine (ACh) were equipotent contractile agonists in human pulmonary arteries (pD2 values, 5.28±0.05 and 5.65±0.16; Emax, 0.91±0.26 and 0.98±0.30 g wt. for CCh and ACh, respectively; n=7). In venous preparations, ACh was ineffective and CCh induced small contractions (Emax, 0.08±0.04 g wt.; n=13). In human pulmonary arteries following pretreatment with tetraisopropylpyrophosphoramide (iso‐OMPA, 100 μM), an increased sensitivity to the contractile agonist ACh was observed (pD2 values, 5.80±0.13 and 6.37±0.19 for control and treated preparations, respectively; n=5). This pretreatment had no effect on the CCh concentration response curve. In contrast, hum...
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