Small molecules antiviral drugs have demonstrated their efficiency against a number of viruses. They are available for the treatment of infections with herpes viruses, HIV, HBV, HCV as well as with influenza viruses. Yet for many other emerging and/or neglected viruses causing life-threatening infections there are no drugs available. Ideally, potent and broad-spectrum (i.e. pan-genus or pan family virus activity) antiviral drugs should be developed whereby one drug could be used for the treatment of a number of such viral infections. This proposal will address this goal taking advantage of two recent breakthroughs: 1. The viral cap methyltransferase (MTase) is a newly identified therapeutic target for antiviral treatments, 2. We have developed a new strategy to screen inhibitors of its activity. This new concept in antiviral discovery will be the first step in organizing a task force in emerging viruses management. Partner 2 is the French leader in chemical libraries management and a recognized expert in medicinal chemistry. The development of chemical libraries with added value against identified viral target is the missing key tool for the identification of antivirals targeting the viral MTase. Partner 1 combines an expertise in enzymology of viral enzymes and in developing new approaches in screening. Partner 3 and 4 are internationally recognized for their clinical isolates viruses collection and potential in the evaluation of inhibitors in BSL3/4 environment. This consortium responds to the dual aspect of the ASTRID call, military and civilian purposes and to the DGA POS (Politique et Objectifs Scientifiques). Our strategy is based on the selection of clinically relevant viruses: 1) which represent a threat to exposed humans, 2) from two different Flaviviruses, and Filovirus virus families. Furthermore, the MTase target has never been challenged to that extent, as there was not the possibility to screen against it. This target represents a new opportunity because not only its inhibition will hamper the replication potential of the virus but it will allow the immune system to fight the infection as the viral genome will be unmasked. Then, based on the results of the screening against the enzymes and the clinical isolates, the consortium will demonstrated the proof of concept that targeting MTases have applications in the antiviral fields. The second aim of the proposal is to validate hits and to propose first structure-activity relationship. Our expertise in antiviral development (partner 1 and 3) prompt us in establishing an iterative development with 3 key skills: A-enzyme kinetics/ B-clinical isolates/C-medicinal chemistry, which concern in our consortium A-partner 1/B-partners 3 and 4/C-partner 2. The results arising from these studies are expected to contribute to the development of anti-Flaviviruses, and anti-Filovirus drugs that could prevent and/or treat infection in exposed populations, such as people living nearby an infected livestock or members of the French army and emergency teams that should be present in highly endemic regions for strategic (i.e. military, economical, emergency etc..) reasons.

Find an efficient solution for neutralization of organophosphorus nerve agents in vivo, in order to protect or cure people is a concern of public health. Such organophosphorus-based compounds are used as chemical weapons or pest control agents. The need of a biocompatible mean for their neutralization takes into account both their potential use within wartime or terrorist actions, and acute or chronic intoxications by insecticides. This project aims at unifying the efforts of two research groups in order to develop catalytic biocompatible scavengers. Depending on their biocompatibility, these agents will be used either in vivo as prophylactic or curative means, or will be incorporated into creams or foams for skin, mucosae and wound decontamination. Organophosphorus compounds (OP) are among the most toxic compounds synthesized due to their irreversible inhibition of acetylcholinesterase. A catalytic bioscavenger present in the bloodstream can neutralize the neurotoxics before they reach their biological target at the synapse or neuromuscular junction. Injected bioscavenger will be an important pretreatment tool in case of severe intoxication risk, for example for military personnel going in a conflict area where neurotoxics are potentially used, or for civilian emergency personnel going to the site of a chemical terrorist attack. For intoxicated people, a massive dose of bioscavenger can be injected to rapidly eliminate the toxic from the body, thus leading to a faster recovery. Human butyrylcholinesterase (BChE) is an enzyme for which modifying its catalytic activity has been an ongoing subject of interest. Considerable interest has been shown in BChE because it hydrolyses a wide range of toxic esters, including heroin and cocaine, and because it scavenges organophosphorus pesticides and nerve agents. Redesign of its active site has improved its cocaine hydrolase activity 2000-fold, turning it into a very efficient cocaine-detoxifying tool. This BChE variant is effective at detoxifying cocain in vivo, and we propose a similar approach for neurotoxics in the present project. IRBA teams and others have explored the development of catalytic scavenger for OP molecules during the last decades. Nowadays, the BChE G117H variant and human paraoxonase (PON1) have a catalytic activity against OP, but this activity is not efficient enough for practical use. In 2008, Baker succeeded in modifying the activity of a protein by adding an appropriate chemical function with the help of molecular modeling and bioinformatics tools. The aim here is to use these tools to modify BChE in order to introduce a performant OP hydrolase activity. Since 2002, our institute develops a softwaresoftware called SuMo whichSuMo, which will be included in the general methodology published by Baker. The use of this software will unlock the limitation of the choice of scaffold protein. Each protein constructed and validated in silico will be synthesized by the IRBA team and tested for hydrolase activity on nerve agents and pesticide. The lead hit will constitute the active ingredient of new medical countermeasure against the intoxication by organophosphorus neurotoxics.

Since the use of chemical weapons in Syria in 2013 and 2017, a potential chemical threat using organophosphorus nerve Agents (OPNA) renewed the scientific interest in developing new antidotes against OPNA poisoning. In case of OPNA poisoning, on the battlefield or during a terrorist attack, conventional emergency medical treatment consists in the injection of antidotes composed of pyridinium aldoxime (pralidoxime, HI-6 or obidoxime depending on the country concerned) for reactivation of human acetylcholinesterase (hAChE) and substances countering the effects of excess acetylcholine (ACh) in the central nervous system (diazepam and atropine). Currently, the french army uses a two-compartment auto-injector containing a mixture of pralidoxime methylsulfate, atropine sulfate (blocking muscarinic receptors (mAChRs) responsible for adverse effects: e.g. bronchial hypersecretions, myosis, etc. ), and avizafone (anticonvulsant). However, the current treatment does not address the nicotinic effects responsible for the death of the person intoxicated through respiratory muscles paralysis. Based on a first “hit” developed in the frame or Multidote ANR ASTRID project ANR-17-ASTR-0012, this project aims at developing original multi-target antidotes able to reactivate hAChE, block ACh nicotinic receptors (nAChRs), thus limiting the peripheral nicotinic effects and reactivate the circulating endogenous enzyme Butyrylcholinesterase (hBChE), which will be used as a pseudo-catalytic scavenger to hydrolyze OPNA in the blood. This hit combines 3-hydroxypyridinaldoxime and a quiniclidinium, nAChR orthosteric antagonist, and presents unmatched capabilities to reactivate both esterases. It will be the starting point of this multidisciplinary project combining organic synthesis, medicinal chemistry, electrophysiology, enzymology and in vivo toxicology using the real OPNA.

Organophosphates (OPs) constitute a class of synthetic molecules still widely used as an insecticide but also as chemical weapons. This class has been the subject of numerous investigations by the chemical industry in search of pesticides that are more effective and potentially less harmful to the environment. In addition, the military authorities are interested in these molecules to counteract their effects. Indeed, a number of OPs pose serious environmental toxicity problems as well as the civilian or military populations targeted by attacks perpetrated with such compounds. Globally, there are around 250,000 fatal cases of OP poisoning per year. OPs interfere with the esterase activity essential for the degradation of certain active molecules in organisms. The most studied is acetylcholinesterase (AChE) which hydrolyzes acetylcholine. This neurotransmitter is essential in the cholinergic transmission of nerve impulses in the central (CNS) and peripheral (PNS) nervous systems, the failure of which leads to a major cholinergic syndrome which combines peripheral manifestations and epileptic seizures. In the event of accidental poisoning by an OP or during terrorist attacks, conventional emergency medical treatment consists of the injection of antidotes composed of pyridinium oxime for the reactivation of AChE and substances countering the effects of excess acetylcholine. However, pyridinium oxime remains ineffective in reactivating CNS AChE due to its poor passage through the blood brain barrier (BBB). The BHE-OP-Antidotes project aims to discover transient opening treatments for BBB or vector drug delivery systems to be used to facilitate the passage of BBB through the AChE reactivating molecules of the CNS. The project uses two complementary and validated in vivo models for this type of work, the zebrafish larva and the mouse. These two models are predictive for applications in humans given the very high evolutionary conservation of the biological processes studied. The zebrafish opens up the possibility of carrying out many combinations of experiments that will make it possible to amend the mouse model and whose results will be validated with the mammalian model. These models will be used to test the conditions for opening the BBB according to a genetic or pharmacological approach in association with a decrease in neurotoxicity induced by conventional pyridinium oximes following intoxication by OPs. The ability of vectorized biomimetic nanoparticles (NPs) loaded with conventional pyridinium oximes or new antidotes to prevent and treat the effects on the CNS of poisoning by OPs will be evaluated. For the two animal models used, the combination of biochemical, molecular and cellular studies, functional imaging, electroencephalographic studies and locomotor tests will be implemented to assess the restoration of the functional integrity of the CNS. The project mobilizes is carried out in collaboration between four teams which have internationally recognized expertise in chemistry, biochemistry, molecular biology, neurosciences, toxicology, pharmacology and the use of zebrafish and mouse animal models including the effects of OPs on the CNS and PNS, study of AChE reactivators, and study of BBB permeability: Pr. P. Babin, project coordinat or (Partner n ° 1, University of Bordeaux, INSERM U1211) (zebrafish model, OPs and antidotes, cholinergic and neuropathic effects), Dr. N. Soussi-Yanicostas (Partner n ° 2, NeuroDiderot INSERM U1141, Hôpital Robert Debré) (OPs and models of epilepsy in zebrafish), Dr. A.-G . Calas (Partner n ° 3, Toxicology & Chemical Risks Department, IRBA, Brétigny) (mouse model, OPs and antidotes, effects on the CNS and SNP) and Dr. C. Chapouly (Partner n ° 4, INSERM U1034, University de Bordeaux) (mouse model, in vitro and in vivo studies of BBB and its permeability).