This year, the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to an unprecedented pandemic termed Covid-19 (Coronavirus disease 2019). Even as human trials of candidate vaccines are already ongoing, there is still a doubt that people can develop protective immunity against SARS-CoV-2. There is therefore an urgent need to identify safe and effective drugs for treatment. The goal of this project is to implement an innovative assay to rapidly detect SARS-CoV-2 experimental infections to allow for the high-throughput screening (HTS) of antivirals to tackle Covid-19. We have established an assay, termed Alpha-Centauri, that allows the quantification, using a Nanoluciferase-based Protein Complementation Assay (PCA), of the nuclear translocation of innate immunity transcription factors by HTS. Since all viral infections trigger signalling pathways downstream of PRR to varying degrees, which all converge on the phosphorylation and nuclear translocation of IRF3, our system should provide a fast and reliable read-out for experimental SARS-CoV-2 infections. Our project falls within axis 2 of the ANR Flash COVID-19 call: Development of animal and cell models & Therapeutic targets and models to assess candidate drugs. Our team has already started on the project, and has secured a proof-of-concept with Sendai Virus, a RNA virus that causes severe respiratory disease in mice For this project, we aim to (i) translate our system to lung adenocarcinoma A549 cells, which are an appropriate cellular model for SARS-CoV-2; we will use both overexpressing cell lines and generate cell lines in which endogenous IRF3 is modified by CRISPR/Cas9-mediated genome editing to allow nuclear PCA of endogenous protein (Aims 1 and 2); and (ii) validate our system for HTS by miniaturisation of the protocol and by testing reported positives and negatives on SARS-CoV-2 infection (Aim 3). The project will be carried out as a partnership between two teams from the "Institut de Recherche en Infectiologie de Montpellier" (IRIM): Team "Viral Trafficking, Restriction and Innate Signaling" (Sébastien Nisole) who will develop the cellular models, and Team "Membrane Dynamics & Viruses" (Raphaël Gaudin) who will perform all infections with SARS-CoV-2 in the biosafety level 3 (BSL3) laboratories at the "Centre d’études des Maladies Infectieuses et Pharmacologie Anti-Infectieuse" (CEMIPAI, Montpellier). The deliverables of the project will be the tools and the engineered cell lines that will be made accessible to the academic and pharmaceutical scientific community to screen compound libraries for antivirals active against SARS-CoV-2. Our technology will allow to screen chemical libraries for antiviral molecules, validate candidate molecules, or optimise validated antivirals. Our assay will enable to identify antivirals that target SARS-CoV-2 directly, and also molecules that boost the host cell interferon response. The latter will therefore constitute a precious arsenal to target other viral infections, which like SARS-CoV-2, are highly sensitive to interferon.

The recent SARS-CoV-2 pandemic has highlighted the need to develop new antivirals against RNA viruses that are responsible for more than a third of new emerging or re-emerging infections. As many RNA viruses use cellular helicases during their infectious cycle, these proteins have become promising therapeutic targets. VIR2RHA is a collaborative research project aimed at developing RHA inhibitors to effectively fight against a broad spectrum of RNA viruses. Thus, using drug design approaches, we have developed new original molecules which bind specifically to RHA, which are non-toxic and which demonstrate broad-spectrum antiviral properties in vitro against Chikungunya virus (CHIKV), the virus Dengue fever (DENV), influenza virus (IAV), human immunodeficiency virus type -1 (HIV-1), T-cell leukemia virus type 1 (HTLV-1) and also the SARS-CoV-2 responsible for the COVID-19 pandemic. In the VIR2RHA project, our objectives are to study the mechanisms of action of our inhibitors in order to continue their preclinical development. This program is also designed to elucidate the proviral functions of RHA in the infectious cycle of these RNA viruses. Our program is part of the pandemic preparness action and adapts to the axes "Life, Health and Well-being" and "Medical innovation, Nanotechnologies, Regenerative medicine, Innovative therapies and vaccines" of the ANR 2022 call

The goal of this interdisciplinary project is to combine virology to the powerful nano/microfluidic strategies (we called "viro-fluidics") to study, for the first time, the viral egress dynamics at the scales of living single-cells and single-viral nanoparticles. Viro-fluidics will be adapted to study retroviruses that reproduce by multiplying within host cells and propagate into the extracellular medium to complete the cycle of infection. The crucial step of the virus egress remains incompletely deciphered because conventional approaches are conducted on cell populations with highly heterogenous virus production kinetics. Key issues will be addressed such as how optimal viral production rates vary with time during infection and could adapt to cells with different lifespans. More globally, the viro-fluidic tools developed here for the pandemic HIV virus, both for single-cell trapping and for single-nanoparticle detection in flow will be easily adaptable to other pandemic viruses such as Coronavirus (SARS-CoV-2) and Hepatitis E virus which are both global public health threats, as well as for many host-parasite systems. Indeed, the viro-fluidics will allow the study of virus production and spreading in bodily fluids (saliva, blood, ...). In addition, the EV field will also gain from the viro-fluidics with aspects of EVs biogenesis, release pathways and propagation.

The recent SARS CoV-2 outbreak raised awareness on the global burden of RNA viral infections and pointed out the weakness of public health systems. Inhibitors developed to target viral enzymes globally collide with the emergence of resistances that defeat these antiviral strategies. In this context, cellular factors required for optimal viral infections such as cellular RNA helicases represent valuable targets for antiviral applications. Helicases separate double helix made of DNA or RNA and allow cellular proteins to access, read, or rearrange genetic information. Brai et al. recently developed specific inhibitors of the DDX3 helicase and they demonstrated the antiviral properties of these molecules. Above all, by establishing the excellent toxicity profile of these compounds, they validated the possibility to target cellular helicases without significant adverse effects. Nevertheless, inhibitors against other cellular helicases remain to be developed. Based on these observations, and on the pleiotropic role of DHX9 in the replication of various RNA viruses, we consider DHX9 as a potential therapeutic target for the development of anti-SARS-CoV-2 molecules. In collaboration with two other teams (Dr. Briant and Dr. Chaloin) of the Institute of research in infectious diseases of Montpellier (IRIM), we have developed small molecules able to specifically bind a hydrophobic pocket in the Helicase domain of DHX9 and to inhibit its PCE activity. We also demonstrated the efficiency of our compounds on RNA viruses (CHIKV) et retroviruses (HTLV-1). These molecules are active in the micromolar range of concentrations. ADME/Tox studies showed that our inhibitors have no significant cytotoxic effect on non-infected cells. Last March at the beginning of the epidemy in France with the help of SATT AxLR and in collaboration with 3 other teams from IRIM including Dr. Briant, we set-up a model of SARS-CoV-2 infection using Vero E6 and A549-ACE2 cells. In our preliminary study, we showed that DHX9 is interacting with several proteins of the SARS-CoV-2 virus and that our DHX9 inhibitors demonstrated strong anti-SARS CoV-2 antiviral properties without any major cytotoxic effect in the A549-ACE2 model. In this project, our objectives are to further identify the molecular basis of DHX9 interactions with viral SARS-CoV-2 proteins to increase the specificity of our compounds; to improve the stability and solubility of the lead molecules developed in our proof of concept study, and to measure the antiviral activity spectrum of the optimized compounds in vitro and in vivo. The NAT_DHX9 project ultimately aims to contribute to satisfying urgent and still unmet medical needs regarding the SARS-CoV-2 virus by bringing efficient disease-modifying treatments to the clinical stage and finally to the market in case of success. It will increase the targeted-therapeutic arsenal to treat RNA viruses’ infection.

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), is one of the deadliest human pathogen. Despite existing chemotherapy, Mtb has been responsible for the death of 1.4 million people and about 10 million new infections in 2015 (WHO report on TB, 2016). This concerns not only developing countries since 5000 new cases are reported yearly in France. The current problems in TB eradication are: lengthy treatments, co-infection with HIV and emergence of multidrug-resistant strains of Mtb. During the last 50 years, very few antitubercular drugs have been discovered and put on the market. Therefore, identifying new molecules targeting mycobacteria is urgent although highly challenging. Most mycobacteria are naturally resistant to antibiotics for which the highly hydrophobic cell wall represents an impermeable barrier. Mycolic acids (MA) are very long lipids made of 90 carbon atoms that are essential components of the mycomembrane and contribute to the high hydrophobicity of the cell wall. MA are synthesized in the cytoplasm and then transported to the periplasm by a specific transporter, MmpL3, which belongs to the superfamily of Resistance-Nodulation-Division permeases. To date, our knowledge about the mechanism by which MA are transported by MmpL3 remains very limited, due to the lack of both in vitro characterization and structural information. The fact that MmpL3 is essential for mycobacterial growth makes it an extremely attractive drug target for future translational applications. Recent whole-cell-based screening conducted by several independent teams, including ours, led to the identification of various chemical entities exhibiting potent antitubercular activity. The mode of action of all these chemotypes involves the inhibition of MA transport to the bacterial surface. In most studies, MmpL3 was designated as the primary target based on the presence of mutations occurring in mmpL3 in spontaneous resistant strains. Among them, some have already reached phases II or III of clinical trials and/or have shown to exhibit synergetic effects with existing antitubercular drugs. Despite these exciting promises, concerns have recently been raised regarding the real implication of MmpL3 as the target of many of these compounds as well as their mechanism of action. Therefore, describing, at a molecular and structural level, the MmpL3-mediated transport mechanism might help to understand how MA are translocated to the cell surface and to validate the importance of MmpL3 in cell wall assembly. This may also greatly help to describe the mode of action of some of the recently identified MmpL3 inhibitors. The objectives of MyTraM consists of the 1) expression and purification of large amounts of recombinant MmpL3; 2) implementation of hybrid structural biology approaches including X-ray crystallography and cryo-electron microscopy to determine the three-dimensional structure(s) of MmpL3; 3) development of an innovative biochemical in vitro assay to assess MA transport and to investigate the mode of action of several MmpL3 inhibitors; and 4) determination of the binding constants of MmpL3 substrates and inhibitors in solution using microscale thermophoresis. We anticipate that this 3-year project should add important breakthroughs in our understanding of MmpL structure-function relationships and lead to a more precise description of the mode of inhibition of a family of promising anti-TB compounds. On a longer term, these studies should also aid in the future improvement of already existing MmpL3 inhibitors and in the conception of new generations of MmpL3-based drugs for the treatment of TB and other mycobacterial infections.