Marie Trovaslet

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.

Progress in the development of enzyme-based nerve agent bioscavengers

Acetylcholinesterase is the physiological target for acute toxicity of nerve agents. Attempts to protect acetylcholinesterase from phosphylation by nerve agents, is currently achieved by reversible inhibitors that transiently mask the enzyme active site. This approach either protects only peripheral acetylcholinesterase or may cause side effects. Thus, an alternative strategy consists in scavenging nerve agents in the bloodstream before they can reach acetylcholinesterase. Pre- or post-exposure administration of bioscavengers, enzymes that neutralize and detoxify organophosphorus molecules, is one of the major developments of new medical counter-measures. These enzymes act either as stoichiometric or catalytic bioscavengers. Human butyrylcholinesterase is the leading stoichiometric bioscavenger. Current efforts are devoted to its mass production with care to pharmacokinetic properties of the final product for extended lifetime. Development of specific reactivators of phosphylated butyrylcholinesterase, or variants with spontaneous reactivation activity is also envisioned for rapid in situ regeneration of the scavenger. Human paraoxonase 1 is the leading catalytic bioscavenger under development. Research efforts focus on improving its catalytic efficiency toward the most toxic isomers of nerve agents, by means of directed evolution-based strategies. Human prolidase appears to be another promising human enzyme. Other non-human efficient enzymes like bacterial phosphotriesterases or squid diisopropylfluorophosphatase are also considered though their intrinsic immunogenic properties remain challenging for use in humans. Encapsulation, PEGylation and other modifications are possible solutions to address this problem as well as that of their limited lifetime. Finally, gene therapy for in situ generation and delivery of bioscavengers is for the far future, but its proof of concept has been established.

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.

Activity and molecular dynamics relationship within the family of human cholinesterases.

The temperature dependence of the dynamics of recombinant human acetylcholinesterase (hAChE) and plasma human butyrylcholinesterase (hBChE) is examined using elastic incoherent neutron scattering. These two enzymes belong to the same family and present 50% amino acid sequence identity. However, significantly higher flexibility and catalytic activity of hAChE when compared to the ones of hBChE are measured. At the same time, the average height of the potential barrier to the motions is increased in the hBChE, e.g. more thermal energy is needed to cross it in the latter case, which might be the origin of the increase in activation energy and the reduction in the catalytic rate of hBChE observed experimentally. These results suggest that the motions on the picosecond timescale may act as a lubricant for those associated with activity occurring on a slower millisecond timescale.

Energy landscapes of human acetylcholinesterase and its huperzine A-inhibited counterpart

Enzymes are animated by a hierarchy of motions occurring on time scales that span more than 15 orders of magnitude from femtoseconds (10(-15) s) to several minutes. As a consequence, an enzyme is characterized by a large number of conformations, so-called conformational substates that interconvert via molecular motions. The energy landscapes of these macromolecules are very complex, and many conformations are separated by only small energy barriers. Movements at this level are fast thermal atomic motions occurring on a time scale between 10(-7) and 10(-12) s, which are experimentally accessible by incoherent neutron scattering techniques. They correspond to local fluctuations within the molecule and are believed to act as coupling links for larger, conformational changes. Several questions related to this hierarchy of motions are a matter of very active research: which of the motions are involved in the biological functions of the macromolecule and are motions of different energy (and thus time) scale correlated? How does the distribution of motions change when an enzyme is inhibited? We report here on investigations of the enzyme human acetylcholinesterase, unliganded and in complex with the noncovalent inhibitor Huperzine A, by incoherent neutron scattering. Different time scales are explored to shed light on the interplay of enzyme activity, dynamics, and inhibition. Surprisingly the average molecular dynamics do not seem to be altered by the presence of the inhibitor used in this study within the considered time scales. The activation energy for the free and the inhibited form of the enzyme is moreover found to be almost identical despite changes of interactions inside the gorge, which leads to the active site of the enzyme.

Interaction of prion protein with acetylcholinesterase: potential pathobiological implications in prion diseases

IntroductionThe prion protein (PrP) binds to various molecular partners, but little is known about their potential impact on the pathogenesis of prion diseasesResultsHere, 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-infected cell cultures. Furthermore, reduction of AChE levels in prion-infected heterozygous AChE knock-out mice leads to slightly but significantly prolonged incubation time. Finally, we found that AChE levels were altered in prion-infected cells and tissues, suggesting that AChE might be directly associated with abnormal PrP.ConclusionOur results indicate that AChE deserves consideration as a new actor in expanding pathologically relevant PrP morphotypes and as a therapeutic target.

Correlation of the dynamics of native human acetylcholinesterase and its inhibited huperzine A counterpart from sub-picoseconds to nanoseconds

It is a long debated question whether catalytic activities of enzymes, which lie on the millisecond timescale, are possibly already reflected in variations in atomic thermal fluctuations on the pico- to nanosecond timescale. To shed light on this puzzle, the enzyme human acetylcholinesterase in its wild-type form and complexed with the inhibitor huperzine A were investigated by various neutron scattering techniques and molecular dynamics simulations. Previous results on elastic neutron scattering at various timescales and simulations suggest that dynamical processes are not affected on average by the presence of the ligand within the considered time ranges between 10 ps and 1 ns. In the work presented here, the focus was laid on quasi-elastic (QENS) and inelastic neutron scattering (INS). These techniques give access to different kinds of individual diffusive motions and to the density of states of collective motions at the sub-picoseconds timescale. Hence, they permit going beyond the first approach of looking at mean square displacements. For both samples, the autocorrelation function was well described by a stretched-exponential function indicating a linkage between the timescales of fast and slow functional relaxation dynamics. The findings of the QENS and INS investigation are discussed in relation to the results of our earlier elastic incoherent neutron scattering and molecular dynamics simulations.