Ludovic Jean

Crystal structures of human cholinesterases in complex with huprine W and tacrine: elements of specificity for anti-Alzheimer's drugs targeting acetyl- and butyryl-cholinesterase

The multifunctional nature of Alzheimers disease calls for MTDLs (multitarget-directed ligands) to act on different components of the pathology, like the cholinergic dysfunction and amyloid aggregation. Such MTDLs are usually on the basis of cholinesterase inhibitors (e.g. tacrine or huprine) coupled with another active molecule aimed at a different target. To aid in the design of these MTDLs, we report the crystal structures of hAChE (human acetylcholinesterase) in complex with FAS-2 (fasciculin 2) and a hydroxylated derivative of huprine (huprine W), and of hBChE (human butyrylcholinesterase) in complex with tacrine. Huprine W in hAChE and tacrine in hBChE reside in strikingly similar positions highlighting the conservation of key interactions, namely, π-π/cation-π interactions with Trp86 (Trp82), and hydrogen bonding with the main chain carbonyl of the catalytic histidine residue. Huprine W forms additional interactions with hAChE, which explains its superior affinity: the isoquinoline moiety is associated with a group of aromatic residues (Tyr337, Phe338 and Phe295 not present in hBChE) in addition to Trp86; the hydroxyl group is hydrogen bonded to both the catalytic serine residue and residues in the oxyanion hole; and the chlorine substituent is nested in a hydrophobic pocket interacting strongly with Trp439. There is no pocket in hBChE that is able to accommodate the chlorine substituent.

Human butyrylcholinesterase produced in insect cells: Huprine-based affinity purification and crystal structure

Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low‐glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ∼ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine‐based affinity chromatography, which is superior to the classical procainamide‐based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low‐glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 Å. The overall monomer structure is similar to the low‐glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low‐glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.

Synthesis and structure–activity relationship of Huprine derivatives as human acetylcholinesterase inhibitors

New series of Huprine (12-amino-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinolines) derivatives have been synthesized and their inhibiting activities toward recombinant human acetylcholinesterase (rh-AChE) are reported. We have synthesized two series of Huprine analogues; in the first one, the benzene ring of the quinoline moiety has been replaced by different heterocycles or electron-withdrawing or electron-donating substituted phenyl group. The second one has been designed in order to evaluate the influence of modification at position 12 where different short linkers have been introduced on the Huprine X, Y skeletons. All these molecules have been prepared from ethyl- or methyl-bicyclo[3.3.1]non-6-en-3-one via Friedländer reaction involving selected o-aminocyano aromatic compounds. The synthesis of two heterodimers based on these Huprines has been also reported. Activities from moderate to same range than the most active Huprines X and Y taken as references have been obtained, the most potent analogue being about three times less active than parent Huprines X and Y. Topologic data have been inferred from molecular dockings and variations of activity between the different linkers suggest future structural modifications for activity improvement.

Phenyltetrahydroisoquinoline-pyridinaldoxime conjugates as efficient uncharged reactivators for the dephosphylation of inhibited human acetylcholinesterase.

Pyridinium and bis-pyridinium aldoximes are used as antidotes to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus nerve agents. Herein, we described a series of nine nonquaternary phenyltetrahydroisoquinoline-pyridinaldoxime conjugates more efficient than or as efficient as pyridinium oximes to reactivate VX-, tabun- and ethyl paraoxon-inhibited human AChE. This study explores the structure-activity relationships of this new family of reactivators and shows that 1b-d are uncharged hAChE reactivators with a broad spectrum.

Syntheses and in vitro evaluations of uncharged reactivators for human acetylcholinesterase inhibited by organophosphorus nerve agents

Organophosphorus nerve agents (OPNAs) are highly toxic compounds that represent a threat to both military and civilian populations. They cause an irreversible inhibition of acetylcholinesterase (AChE), by the formation of a covalent P-O bond with the catalytic serine. Among the present treatment of nerve agents poisoning, pyridinium and bis-pyridinium aldoximes are used to reactivate this inhibited enzyme but these compounds do not readily cross the blood brain barrier (BBB) due to their permanent cationic charge and thus cannot efficiently reactivate cholinesterases in the central nervous system (CNS). In this study, a series of seven new uncharged oximes reactivators have been synthesized and their in vitro ability to reactivate VX and tabun-inhibited human acetylcholinesterase (hAChE) has been evaluated. The dissociation constant K(D) of inhibited enzyme-oxime complex, the reactivity rate constant kr and the second order reactivation rate constant k(r2) have been determined and have been compared to reference oximes HI-6, Obidoxime and 2-Pralidoxime (2-PAM). Regarding the reactivation of VX-inhibited hAChE, all compounds show a better reactivation potency than those of 2-PAM, nevertheless they are less efficient than obidoxime and HI-6. Moreover, one of seven described compounds presents an ability to reactivate tabun-inhibited hAChE equivalent to those of 2-PAM.

Huprine derivatives as sub-nanomolar human acetylcholinesterase inhibitors: from rational design to validation by X-ray crystallography.

Alzheimer’s disease (AD) is the most common cause of senile dementia. Because of its dramatic human and economic impact, it has become one of the major public health issues of the 21st Century, with millions affected worldwide. A decrease in the levels of the neurotransmitter acetylcholine (ACh) is invariably observed in the brains of AD victims, resulting in a progressive decrease in cholinergic neurotransmission that correlates with cognitive impairment. 3] It was therefore proposed that restoring ACh levels in the brain may slow the progress of AD. Because acetylcholinesterase (AChE, EC 3.1.1.7) is the enzyme responsible for the breakdown of ACh at cholinergic synapses, currently approved drugs for treating the symptoms of AD are AChE inhibitors, with the notable exception of the NMDA receptor antagonist, memantine. While the causative agent for AD remains unclear, an amyloid hypothesis was put forward based on the observation that the amyloid-b peptide (Ab) is the main constituent of the proteinaceous deposits observed in the brain tissue of AD victims. Ab can take on a variety of oligomeric and fibrous forms that display different levels of neurotoxicity. Current therapeutic approaches are accordingly directed at decreasing Ab production (secretase inhibitors) and aggregation (anti-Ab aggregation agents such as tramiprosate), or increasing Ab clearance (immunotherapy). The investigation of AChE ligands, however, continues to receive experimental scrutiny, as it was discovered that the enzyme peripheral site accelerates Ab aggregation and deposition. The current strategy for designing new drug candidates focuses on the design of molecules that are able to exert dual action, that is, interacting simultaneously with the AChE active site (inhibition of cholinesterase activity) and with the peripheral site (inhibition of AChE-mediated Ab deposition). Over the past few years, many such dual inhibitors have been designed in which both functionalities are combined; however, a detailed understanding of the molecular basis for their binding to the human enzyme is still lacking. In particular, the influence of the interactions between the human AChE (hAChE) active site gorge residues and the linker connecting the two moieties of the dual inhibitors has been overlooked. Huprines are the best AChE active site ligands reported to date, with binding affinities in the low nanomolar range. Previous work by our research group has suggested that ligation to a peripheral site binder could be performed at position 9 of

A convenient route to 1-benzyl 3-aminopyrrolidine and 3-aminopiperidine

Abstract 1-Benzyl 3-aminopyrrolidine 1 and 1-benzyl 3-aminopiperidine 2 were prepared rapidly mainly in aqueous conditions in 55 and 75% yields, respectively, on a multi-gram scale starting from inexpensive and commercially available starting materials. The key step involved the Curtius rearrangement mediated by sodium nitrite and trifluoroacetic acid of the appropriate acylhydrazides. All the reactions (except LAH reductions) were performed in water.

New huprine derivatives functionalized at position 9 as highly potent acetylcholinesterase inhibitors.

A series of 24 huprine derivatives diversely functionalized at position 9 have been synthesized and evaluated for their inhibitory activity against human recombinant acetylcholinesterase (AChE). These derivatives were prepared in one to five steps from huprine 1 bearing an ester function at position 9. Ten analogues (1, 2, 6–9, 13–15, and 23) are active in the low nanomolar range (IC50 <5 nM), very close to the parent compound huprine X. Compounds 2, 6, and 7 show a very good selectivity for AChE, with AChE inhibitory activities 700–1160‐fold higher than those for butyrylcholinesterase (BChE). The inhibitory potency of these compounds decreases with the steric bulk of the substituents at position 9. According to docking simulations, small substituents fit into the acyl‐binding pocket, whereas the larger ones stick out of the active site gorge of AChE. Determination of the kinetic parameters of three of the most potent huprines (2, 6, and 7) showed that most of the difference in KD is accounted by a decrease in kon, which is correlated to the increase of the substituent size. A first in vivo evaluation has been performed in mice for the most active compound 2 (IC50=1.1 nM) and showed a rather weak toxicity (LD50=40 mg kg−1) and an ability to cross the blood–brain barrier with doses above 15 mg kg−1.

Synthesis and luminescence properties of new red-shifted absorption lanthanide(III) chelates suitable for peptide and protein labelling

The synthesis and photo-physical properties of an original bis-pyridinylpyrazine chromophore efficiently sensitising europium(III) and samarium(III) are described. The corresponding lanthanide(III) complexes display in aqueous solutions a maximum excitation wavelength which is significantly red-shifted compared to the usual terpyridine-based chelates, and a valuable luminescence brightness above 2,000 dm(3) mol(-1) cm(-1) at 345 nm was obtained with a europium(III) derivative. Further functionalisation with three different bioconjugatable handles was also investigated and their ability to efficiently label a model hexapeptide was evaluated and compared. Finally, the best bioconjugatable europium(III) chelate was used in representative labelling experiments involving monoclonal antibodies and the luminescence features of the corresponding bioconjugates remained satisfactory.

Synthesis and in vitro evaluation of donepezil-based reactivators and analogues for nerve agent-inhibited human acetylcholinesterase

Poisoning by organophosphorus nerve agents and pesticides is a serious public and military health issue with over 200 000 fatalities annually worldwide. Conventional emergency treatment consists of rapid administration of atropine and pyridinium oxime as an antidote. The reactivation of acetylcholinesterase (AChE) in the central nervous system (CNS) by the oxime is inefficient due to the fact that positively charged pyridiniums do not readily cross the blood brain barrier (BBB). Herein, we described the synthesis and in vitro evaluation of four donepezil-based non quaternary reactivators. The compounds 1–4 have been prepared in 7–8 linear steps in 1–9% overall yields and oximes 1–3 show better ability (8 fold higher) than pralidoxime to reactivate VX-inhibited human AChE (VX-hAChE). Besides, oxime 2 is 5 to 11 fold more efficient than pralidoxime and HI-6 respectively for the reactivation of VX-inhibited human butyrylcholinesterase (VX-hBChE).