Ischemic stroke is a sudden neurological disorder which constitutes the third leading cause of death and the leading cause of acquired disability in adults in industrialized countries. The current treatment for the acute phase of ischemic stroke is to remove the blood clot obstructing the cerebral bloodflow by enzymatic degradation (thrombolysis) or by removing it mechanically through catheterization (thrombectomy). To guide clinical practitioners in their choice of treatment, magnetic resonance imaging (MRI) is essential. But MRI has its limits and does not allow the diagnosis of microthrombi, which are however partly responsible for post stroke sequelae. This project aims to (i) study the physiopathology of microthrombi in ischemic stroke, (ii) develop a contrast agent to reveal microthrombi on MRI and (iii) establish a treatment for micrthrombi. 1. Study of microvascular thrombosis in ischemic stroke The presence and impact of distal microthrombi will be studied in 2 models of ischemic stroke in mice. The first model we will use is thromboembolic, it is obtained by injection of thrombin into the middle cerebral artery (MCA). In the second model, cerebral ischemia is induced by occlusion of the MCA with a filament; removing the filament will allow us to reproduce the abrupt recanalization encountered in patients who benefited from thrombectomy. We will study precisely on histological sections the quantity, the stability over time and the composition of microthrombi in these 2 models. 2. Synthesis of a novel MRI contrast agent to reveal microthrombi Previous work in the PhIND laboratory has demonstrated the great potential of the molecular magnetic resonance imaging (MRI) strategy with microparticles of iron oxide (MPIO). Despite the promises of this strategy, the MPIOs used in these studies are composed of inert and toxic materials. The development of biodegradable and non-toxic MPIOs is therefore necessary to make this technology applicable to humans. In this project we propose to synthesize MPIOs from iron oxide nanoparticles assembled in a biodegradable matrix. We will use an emulsion coupled to a crosslinking process to cluster the iron oxide nanoparticles in the biodegradable matrix. We refer to these particles by the acronym PHysIOMIC. To optimize this synthesis and characterize the particles obtained, we will work with the organic chemistry department of the pharmaceutical research laboratory of Caen (CERMN). We will then use this novel MRI contrast agent to reveal the microthrombi present in ithe schemic stroke models using the molecular MRI technique. Preliminary results confirm that this method is effective in revealing the occlusive microthrombi present in the ischemic thromboembolic stroke model. In order to increase the specificity of our system, we will work on functionalizing the PHysIOMIC with antibodies specific to the platelets contained in the microthrombi. 3. Preclinical study of a thrombolytic therapy for the treatment of microthrombi We will test 3 different thrombolytic treatments that are known to be effective in degrading platelet and von Willebrand factor rich clots. We will test N-acetylcystein, the powerful thrombolytic effect of which has recently been demonstrated in the PhIND laboratory, and 2 treatments whose fibrinolytic activity is triggered by the presence of thrombin, which is generated in very large quantities by the activated platelets present in microthrombi.

TREATABLE project focuses on tuberculosis that remains a major global-health threat, also causing a quarter of the global antimicrobial resistance emergency, and is further exacerbated by the COVID-19 pandemic. The ability of Mycobacterium tuberculosis to persist despite the pressure exerted by the host and antimicrobials is associated with its intrinsic phenotypic variation and its enhanced diversification potential under stressful conditions. Here we propose to target persistence-associated biomarkers, formerly identified at the subpopulation level, aiming to counter this phenomenon, and to potentiate and accelerate the anti-tubercular therapy. To achieve this goal, we established an interdisciplinary consortium, where both academia and private sector will work in synergy. Together we will implement a state-of-the-art research program, merging complementary expertise: bulk- and single-cell microbiology; molecular and cell biology; advanced microfluidic culture-systems engineering; control of microflow dynamics; computer-aided drug design and drug discovery. In particular, we plan to: 1) develop a robust and automated microfluidic system for single-cell imaging, based on an existing prototype; 2) carry out in silico drug discovery using a chemically diverse compound library and focusing on a shortlist of persistence biomarkers; 3) characterize the single-cell activity of our putative anti-persistence compounds, pre-selected at the population level; 4) validate our compound hits and look for synergies with standard anti-tubercular drugs. With TREATABLE, we will increase our fundamental knowledge of the single-cell bases of M. tuberculosis persistence, contribute to a technological breakthrough, which will prove useful to probe single-cell biology in different fields, we will generate new weapons for healthcare intervention and improve tuberculosis therapeutics.

Cryptophanes (Crs) are cage-like host molecules composed of two molecular crowns, cyclotriveratrylene (CTV) units usually connected by three ether linkers. These molecular hosts are known since decades to be able to encapsulate many different entities, in particular they are well suited for xenon. Then, the development of Cr-based biosensors has been tremendously studied for applications in hyperpolarized xenon magnetic resonance imaging (MRI) in biological systems. Several works have demonstrated their applicability and relevance, in particular in cells assays, but to date, no application of such a bioprobe has been reported in vivo. Indeed, the complex preparation of Cr-monosubstituted derivatives and the poor solubility of these molecules in biocompatible media precludes the obtention of an efficient xenon based bioprobe. Nevertheless, over the past four decades, considerable progress has been made in the characterization of the cryptophane’s structural aspects and exploration of their unique host–guest chemistry, in both organic and aqueous solutions. However, the synthetic schemes to produce such derivatives have not evolved so much, leading to minor modifications of the whole backbone of cryptophanes, then narrowing their potential others applications. The purpose of Neo-Crypto project is to extend the cryptophane chemical space by using and improving a newly described synthetic strategy. The obtained new molecular hosts will be evaluated as hyperpolarized Xe-sensors for MRI. Other physicochemical properties will also be evaluated through collaborations: radon complexation and chiroptical properties.

Malaria is still a major public health problem in 2018, since this infection due to the Plasmodium parasite, present in 91 countries, kills more than 440,000 people every year, with a majority of African children. As observed in most of infectious diseases, the emergence of resistant strains of P. falciparum toward antimalarials is nowadays responsible for a major concern, especially regarding the increasing resistant rate observed toward artemisinin derivatives (ref. treatments for P. falciparum malaria) in Asia, and the subsequent development of multi-resistant strains that could spread worldwide without any therapeutic option. Then, to bypass parasitic resistance, new antimalarials are expected to be active on artemisinin-resistant strains and to possess a novel mechanism of action. It is also crucial to develop new molecules targeting both the asexual (hepatic and erythrocytic) and sexual (gametocytes) stages of the parasite, in a view to block malaria transmission. We previously identified a multi-stage acting lead compound in the thienopyrimidinone series, called Gamhepathiopine (or M1), which fulfills these criteria by acting on erythrocytic, hepatic and sexual stages of P. falciparum. This original molecule displays excellent in vitro activities even on the artemisinin-resistant parasites. In addition, our lead molecule does not exert its antiplasmodial activity by a mechanism of action already described for marketed antimalarials. Such promising results are however tempered by a rapid hepatic metabolization and a poor aqueous solubility, which limit gamhepathiopine activity in vivo. In this context, we thus propose to pharmacomodulate gamhepathiopine to optimize its physico-chemical and pharmacokinetic properties and to potentiate its in vivo activity. To this aim, the metabolic stability issue will be addressed by modifying the two identified sites of metabolization of gamhepathiopine. In addition, the aqueous solubility will be improved by introduction of polar groups and/or synthesis of hydrophilic prodrugs. Additional pharmacomodulation work is also proposed in order to identify new antiplasmodial leads and to modulate the purine-analog character of the scaffold, in the hypothesis of a mode of action related to plasmodial kinase inhibition. The new molecules will be evaluated in vitro on the erythrocytic stages (sensible and resistant strains) and for their cytotoxicity. The most active compounds will be studied 1) for their mutagenicity 2) in vitro against hepatic stages ( (P. yoelii, P. falciparum and P. vivax and for their action on gametocytes and their capacity to block parasite transmission to Anopheles (vector of malaria)) then 3) in vivo, after potential preparation of lipidic nanoemulsions, on a murine model either infected by P. yoelii or humanized and infected by P. falciparum or P. vivax to validate the benefit of the chemical modifications toward PK properties and guarantee the preservation of the multi-stage acting properties. The last part of the project concerns the identification of the gamhepathiopine plasmodial target, in a view to elucidate its novel mechanism of action. A first hypothesis based on M1 chemical structure (purine analogue) and on recent literature data consists in postulating the possible involvement of a plasmodial kinase to explain its antiplasmodial activity. A phospho-proteomic study will thus be conducted to answer this question. In parallel and to extent the study of the mechanism of action of the lead molecule to potential non-kinase targets, an affinity chromatography procedure will be applied to Gamhepathiopine (immobilized on a solid support via a spacer) to try to isolate its target from a plasmodial lysate and to proceed to its identification by MALDI-TOF.

This project aims at designing novel pleiotropic prodrugs, under a mucoadhesive liposomal formulation for intranasal administration, with potential therapeutic interest in Down Syndrome (DS) and Alzheimer's disease (AD). The prodrugs will be activated through the covalent inhibition of butyrylcholinesterase (BuChE), able to counteract the cholinergic neurodegeneration in AD. The released drugs will then target either the beta2-A or 5-HT4 receptors or the pro-neuroinflammatory HMGB1 protein, in order to display a potential additional neuroprotective effect. The liposomal formulation and the intranasal administration will enhance the CNS distribution of the prodrugs for a selective activation by the cerebral BuChE, in order to improve the central effects of the released drugs and potentially avoid their peripheral side effects.