Molecular movies of nerve agents reacting with their biological targets and antidotes by time-resolved serial synchrotron crystallography. – SerialX-OP

The use of organophosphorus chemical warfare nerve agents (NOPs) has renewed great concerns worldwide since their recent use in Syria (sarin), and in England and Russia where new NOPs were applied (Skripal and Navalny cases). The chemical threat has also regained scientific interest to develop new antidotes against NOP poisoning. The lethal action of NOPs is based on the irreversible inhibition of acetylcholinesterase (AChE), an essential enzyme in the central nervous system (CNS) that hydrolyzes the neurotransmitter acetylcholine. In the event of NOP poisoning, during military operations or a terrorist attack, the conventional emergency medical treatment consists of the injection of antidotes. These are made up of active compounds that counter the effects of excess acetylcholine, including damaging neuronal excitation in the CNS, and molecules capable of reactivating AChE (oxime-based reactivators). These reactivators have the disadvantage of having an efficacy that largely depends on the used NOP, and they are particularly ineffective on phosphoramidates (tabun, novichoks). Moreover, the reactivators do not cross the blood-brain barrier and therefore cannot access and reactivate inhibited AChE in the CNS. New generations of oxime-based reactivators have therefore been developed over the last decade, some showing in vitro efficacy on a broad spectrum of NOP. Additionally, biocompatible scavengers of NOP have also been developed. These are proteins capable of rapidly binding to NOP and neutralizing them in the body of poisoned patients. Upon inhibition of AChE, or reaction with an esterase-type bioscavenger, the NOP covalently binds to the catalytic serine of the enzyme which then becomes inactive. The oximes are then able to carry out a nucleophilic attack on the organophosphorus group derived from the NOP and displace it from the catalytic serine, thus restoring the enzyme’s activity. While this general chemical mechanism has been known for decades, the precise course of these reactions at the atomic scale remains largely hypothetical. Molecular simulations to reproduce them only gave partial information, that could not be verified experimentally. Structural studies by X-ray crystallography have also been carried out but only provided static images before or after the reaction. Recent advances in X-ray crystallography now make it possible to study time-resolved reactions (“time-resolved serial crystallography”) and thus to fill these important knowledge gaps. The increased power of the European synchrotron (ESRF) in Grenoble gives access to this technique and makes it possible to visualize the progress of a reaction between a protein and a ligand at atomic resolution and on the relevant time scales (ms, s). Our goal is to implement this new technique to: i) study the reaction mechanism of NOPs with small thermophilic esterases that are easy to produce and can be optimized by artificial intelligence, with the goal to increase their ability to rapidly capture NOPs and use them as bioscavengers (objective 1); and ii) to study the mechanism of AChE inhibition by NOPs and subsequent reactivation by oxime-based reactivators, in order to better understand these processes and acquire the structural information to optimize the oximes and make them more effective on NOPs resistant to reactivation (objective 2). The multidisciplinary nature of the teams involved in this partnership (biochemistry, molecular biology, enzymology, structural biology, and molecular modeling) will be the determining factor for the success of this project.