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

Viruses evolved strategies to disseminate by hiding into immune cells and enhance their migrating properties. Viruses induce drastic subcellular rearrangements that may lead to particular types of migratory behavior, but the underlying mechanisms remain poorly understood. We aim at unravelling the molecular determinants induced by Zika virus (ZIKV) responsible for the increased migrating properties of monocytes and dendritic cells. We recently showed that ZIKV promotes transmigration of monocytes and we are now proposing to investigate the impact of ZIKV on cell migration in a broader range of assays, including microchannels, cerebral organoids and mouse lymphatic vessels. The molecular partners promoting ZIKV-induced cell migration will be identified by proteomics, CRISPR and pharmacological approaches. Our work should offer seminal bases for the development of antiviral and/or anti-inflammatory therapeutic strategies through the specific prevention/induction of immune cell migration.

Efforts to combat dengue require a better understanding of the pathophysiological mechanisms involved in the transition to severe forms of dengue and the identification of new targets for therapeutic intervention. The hypothesis of the METABODEN project is based on the assumption that lipid metabolism plays a major role in dengue pathogenesis. METABODEN is a multidisciplinary project that aims (i) to investigate the crosstalk between blood metabolic imprinting and disease severity using longitudinal pediatric clinical samples and metabolomics approaches. (ii) to decrypt the molecular events accounting for DENV-induced lipidome remodeling (iii) to evaluate the pathogenic consequences of host lipidome remodeling on the innate immune response and vascular leakage. Besides increasing fundamental knowledge on the role of lipid metabolism in DENV pathogenesis, the METABODEN project will identify reliable biomarkers to predict diseases severity and potential targets for antiviral intervention. Our consortium is composed of molecular virologists specialized in the field of DENV research, immunologists, and clinicians providing access to blood samples from DENV-infected children. Previous joint publications and preliminary results guarantee cohesion, feasibility, and progress of the project.

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.

Antibiotic-resistant bacteria become a major threat for public health worldwide. Tuberculosis is responsible for ~1,5 million of deaths every year and the number of multi-drug resistant cases increases. Nosocomial infections are responsible for ~ 25 000 deaths a year in the Europe. Development of new therapeutics requires deep understanding of the molecular mechanisms conferring resistance. Bacterial RNA polymerase is an essential enzyme of gene expression and the proven target for clinical drugs. This project aims to uncover molecular mechanism by which RNA polymerase modify potency of the clinical drug fidaxomicin and to design novel, fidaxomicin-derived, antibacterial molecules.