Eugénie Carletti

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.

Structure, Activities and Biomedical Applications of Human Butyrylcholinesterase

Human butyrylcholinesterase (BuChE) is a serine enzyme present in most organs and plasma. No clear physiological function has yet been assigned to BuChE, but it is a pharmacologically and toxicologically important enzyme that plays a role in degradation of numerous ester-containing drugs and poisonous esters. Thus, BuChE-based bioscavengers are an alternative for prophylaxis and treatments of intoxications by these compounds. Also, BuChE has been integrated in biosensors for detection of organophosphorus compounds and other cholinesterase inhibitors.

Reactibodies Generated by Kinetic Selection Couple Chemical Reactivity with Favorable Protein Dynamics.

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (VL) and variable heavy (VH) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.

X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger

OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water.

Structural Study of the Complex Stereoselectivity of Human Butyrylcholinesterase for the Neurotoxic V-Agents.

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the PS adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VXR-(+) and VXS-(−) solutions lead to the formation of the PS and PR adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VXR-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VXS-(−). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with PR enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the PS enantiomer and ages.

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

Inhibition Pathways of the Potent Organophosphate Cbdp with Cholinesterases Revealed by X-Ray Crystallographic Snapshots and Mass Spectrometry

Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aerotoxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bioscavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 Å snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H(2)(16)O or H(2)(18)O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP.

Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase

Phosphotriesterase from Pseudomonas diminuta (PTE; EC 3.1.8.1) hydrolyzes organophosphate insecticides and chemical warfare agents. The two zinc cations in the active center can be substituted. Co2+-containing PTE is the most efficient but least stable isoform. Gel filtration showed that PTE is monomeric at the submicromolar concentrations used in kinetic assays. The analysis of the recombinant enzyme by X-ray fluorescence spectrometry and CCT-ICP-MS, confirms that recombinant Zn-PTE contains only Zn2+ whereas Co-PTE has Zn2+ and Co2+ in equimolar amount, with Co2+ most likely in the reported labile β-site. We noted that recombinant PTE is unstable at low concentrations and must be stabilized by a protein environment. We tested the effect of excess of various metal cofactors on PTE-catalyzed hydrolysis of paraoxon. We notably observed that ZnCl2 induces a non-competitive partial inhibition of Zn2+- and Co2+-PTE at pH 8.5 (apparent Ki=155 μM and 52 μM, respectively). Inhibition results from interactions with colloidal Zn(OH)2 formed in alkaline buffer that alters the catalytic machinery. NiCl2 caused a similar effect at higher concentrations (apparent Ki=3 mM). We observed that mutating His123, a surface residue close to an alleged allosteric site, dramatically altered the bacterial expression yield of Co2+-PTE, Ki for Zn(OH)2 inhibition, kcat (up to 60 fold) for paraoxon hydrolysis, but not KM. Issues addressed in this work are important for future biotechnological developments of PTE as a detoxifying enzyme.