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Organophosphorus compounds (OPs) interfere with the catalytic mechanism of acetylcholinesterase (AChE) by rapidly phosphorylating the catalytic serine residue. The inhibited enzyme can at least partly be reactivated with nucleophilic reactivators such as oximes. The covalently attached OP conjugate may undergo further intramolecular dealkylation or deamidation reactions, a process termed "aging" that results in an enzyme considered completely resistant to reactivation. Of particular interest is the inhibition and aging reaction of the OP compound tabun since tabun conjugates display an extraordinary resistance toward most reactivators of today. To investigate the structural basis for this resistance, we determined the crystal structures of Mus musculus AChE (mAChE) inhibited by tabun prior to and after the aging reaction. The nonaged tabun conjugate induces a structural change of the side chain of His447 that uncouples the catalytic triad and positions the imidazole ring of His447 in a conformation where it may form a hydrogen bond to a water molecule. Moreover, an unexpected displacement of the side chain of Phe338 narrows the active site gorge. In the crystal structure of the aged tabun conjugate, the side chains of His447 and Phe338 are reversed to the conformation found in the apo structure of mAChE. A hydrogen bond between the imidazole ring of His447 and the ethoxy oxygen of the aged tabun conjugate stabilizes the side chain of His447. The displacement of the side chain of Phe338 into the active site gorge of the nonaged tabun conjugate may interfere with the accessibility of reactivators and thereby contribute to the high resistance of tabun conjugates toward reactivation.

Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.

Organophosphonates such as isopropyl metylphosphonofluoridate (sarin) are extremely toxic as they phosphonylate the catalytic serine residue of acetylcholinesterase (AChE), an enzyme essential to humans and other species. Design of effective AChE reactivators as antidotes to various organophosphonates requires information on how the reactivators interact with the phosphonylated AChEs. However, such information has not been available hitherto because of three main challenges. First, reactivators are generally flexible in order to change from the ground state to the transition state for reactivation; this flexibility discourages determination of crystal structures of AChE in complex with effective reactivators that are intrinsically disordered. Second, reactivation occurs upon binding of a reactivator to the phosphonylated AChE. Third, the phosphorous conjugate can develop resistance to reactivation. We have identified crystallographic conditions that led to the determination of a crystal structure of the sarinnonaged-conjugated mouse AChE in complex with [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]-oxoazanium dichloride (HI-6) at a resolution of 2.2 Å. In this structure, the carboxyamino-pyridinium ring of HI-6 is sandwiched by Tyr124 and Trp286, however, the oxime-pyridinium ring is disordered. By combining crystallography with microsecond molecular dynamics simulation, we determined the oxime-pyridinium ring structure, which shows that the oxime group of HI-6 can form a hydrogen-bond network to the sarin isopropyl ether oxygen, and a water molecule is able to form a hydrogen bond to the catalytic histidine residue and subsequently deprotonates the oxime for reactivation. These results offer insights into the reactivation mechanism of HI-6 and design of better reactivators.

Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.

Organophosphorus compounds (OPs), such as nerve agents and a group of insecticides, irreversibly inhibit the enzyme acetylcholinesterase (AChE) by a rapid phosphorylation of the catalytic Ser203 residue. The formed AChE-OP conjugate subsequently undergoes an elimination reaction, termed aging, that results in an enzyme completely resistant to oxime-mediated reactivation by medical antidotes. In this study, we present crystal structures of the non-aged and aged complexes between Mus musculus AChE (mAChE) and the nerve agents sarin, VX, and diisopropyl fluorophosphate (DFP) and the OP-based insecticides methamidophos (MeP) and fenamiphos (FeP). Non-aged conjugates of MeP, sarin, and FeP and aged conjugates of MeP, sarin, and VX are very similar to the noninhibited apo conformation of AChE. A minor structural change in the side chain of His447 is observed in the non-aged conjugate of VX. In contrast, an extensive rearrangement of the acyl loop region (residues 287-299) is observed in the non-aged structure of DFP and in the aged structures of DFP and FeP. In the case of FeP, the relatively large substituents of the phosphorus atom are reorganized during aging, providing a structural support of an aging reaction that proceeds through a nucleophilic attack on the phosphorus atom. The FeP aging rate constant is 14 times lower than the corresponding constant for the structurally related OP insecticide MeP, suggesting that tight steric constraints of the acyl pocket loop preclude the formation of a trigonal bipyramidal intermediate.

Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.acetylcholinesterase | density functional theory | crystallography | nerve agent | reactivation

Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer's disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators.