The aim of NMR4mAbs project is to develop innovative Nuclear Magnetic Resonance (NMR) tools to better characterize the impact of chemical modifications on structure of monoclonal Antibodies (mAbs) clinical batches. So far, 2D NMR spectra of methyl groups of therapeutic antibodies at natural abundance are used as "fingerprints" to ensure the structural compliance of mAbs batches. Although this fingerprinting approach is a powerful and effective tool for quality monitoring, it does not grant access to in-depth structural information, as it does not identify precisely the structural modifications involved on mAbs. As a result, it is challenging to explain the structural origin of any potency change after stress tests and in turn to understand precisely biotherapeutic molecules degradation pathways. A precise characterization of mAbs is limited by the inability to assign each observed NMR signal to the corresponding atom in the target mAb. To solve this bottleneck, the partners already set up new in vivo and in vitro isotopic labelling methods optimized for mAbs, enabling to initiate the first assignment of a full mAb. Based on this first assignment of a mAb, and exploiting sequence and structure similarities, our goal is to develop fast and efficient NMR-based and labelling-based methods to speed up frequencies assignment to any other mAbs. These new approaches will enable to decipher the whole structural information encoded in 2D CH3-NMR spectra, so far unexploited. While advanced assignment approaches will need to be implemented at early stages of therapeutical mAb development, the frequencies identification will be used in all the following phases of the development pipeline. In this project, we will demonstrate how previous knowledge of mAb NMR frequencies, will enable an in-depth characterization of any structural modification using natural abundance 2D CH3-NMR spectra, acquired directly on the mAb clinical batches.

The incidence of sepsis is continually increasing, with 300 cases per 100,000 per year in the US. Mortality is still in the order of 30-50% resulting in 800,000 deaths per year in the US. In most cases the pathogens involved are sensitive to commonly used antibiotics but treatment is insufficient due to host factors including massive inflammation, microvasculature dysfunction and vascular leak, among others. A central assumption of this project is that there is a necessity for drugs targeting these host responses to complement antibiotic treatment. We reason that once an infection has reached a certain threshold, bacterial killing by antibiotics is restricted by biofilm-like resistance mechanisms and that furthermore, bacterial killing is not sufficient to prevent the adverse effects of infection on blood vessels. The strategy of this project is to perform a pharmacological screen to find molecular entities targeting the host pathways/receptors that block the pathogenic interaction of bacteria with the endothelium: bacterial colonization, junction destabilization and inflammation processes. We will use Neisseria meningitidis as the model of a sepsis causing pathogen as it recapitulates many key features of sepsis pathology and because of recent progress in understanding the mechanisms underlying the disease caused by this bacterium. Identified compounds will then be tested with two other sepsis causing pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. We expect to generate a selection of compounds that can then be developed into drugs to treat deadly bacterial infections.

Malaria is the most important human parasitic disease and a major public health concern. Since 2007, an ambitious goal of malaria elimination and eventually eradication has been set, control interventions have been scaled up and malaria cases have declined. Yet, there were still 220 million malaria cases in 2009. Innovative funding initiatives have been launched to subsidise drugs to the populations most at need. The market of antimalarials has been estimated at about 500 M€ for approx. 400 million treatments per year. Control now faces the threatening obstacle of drug parasite resistance, including resistance to recently introduced artemisinin-based combination therapies. To sustain control efficiency and to meet the challenge of elimination, new drugs are required. Development of novel antimalarials is long and expensive. Focus so far has been on a few drug targets of Plasmodium intraerythrocytic stages. This project aims at replenishing the drug pipeline with antimalarials active on new targets, expressed at different stages of both P.falciparum and P.vivax. The project builds on the validation by the Institut Pasteur (IP) team of novel promising drug targets, i.e. the SUB1 and SUB2 subtilisin proteases that orchestrate a cascade of events culminating in the egress of the parasite from and invasion into new host cells. Importantly, available evidence indicates that these subtilisins are essential in blood stages as well as in pre-erythrocytic stages. This R&D proposal will capitalise on recent drug discovery efforts by the IP team to execute in collaboration with Sanofi Aventis (SA) the translational research activities needed for the critical hit/early lead to lead generation. We will combine the expertise from the IP teams in parasitology, structural biology and epidemiology with the expertise of SA teams of Therapeutic Strategic Unit dedicated to Infectious diseases (TSU-ID), LG-CR (Lead Generation and Compound Realisation) and DSAR (Disposition Sécurité et Recherche Animale) to develop a new generation of anti-malarial lead compound qualified to enter into pre-clinical phases. The overall strategy will be to use iterative process of optimisation and testing of compounds targeting the Plasmodium SUBs proteases starting from the existing hits. The products will be evaluated for ADMET and for a set of biological activity criteria (inhibition of Pv/Pf/PbSUB1 recombinant enzymes, of SUB2 enzymes, of P.falciparum growth in vitro and P.berghei in vivo. Product design and optimisation will articulate medicinal chemistry with crystallographic and NMR structural studies, in silico docking, activity against the enzymes and inhibition assays on parasites, including multidrug-resistant field isolates of P.falciparum and P. vivax from Cambodia. The objective is to obtain a molecule fulfilling the specifications of being non-toxic, potent and soluble for further development of oral therapy against P.falciparum and P. vivax malaria. Three main approaches will be pursued: a) lead generation starting from the early lead CD3; b) validation of SUB1 inhibitor A11H6, and hit to lead development; c) a fragment-based approach (SA). The main emphasis will be on the CD3-based strategy, with the aim to increase CD3 anti-parasite potency by ten to twenty-fold and to increase its solubility, while the two alternate approaches will be explored as backup strategies in case CD3 fails to meet the desired product specification. The project has two main partners, an academic (IP) and an industrial (SA). Each partner will engage several teams and significant ressources. The objective of the project and its workplan is fully in line with the stated objectives of the call. We bridge the gap between academic, product-oriented research and industrial research to build upon and valorise the patented outcome of academic research and jointly execute a critical forward step towards development of new products needed by the market.

Since almost a century electrical activities of hearth and brain have been visualized by electrocardiography (ECG) or electroencephalogry (EEG). Since three decades, these electrical activities of hearth and brain could also be visualized by fluorescent dyes. The general idea is to change the electrical signal of a cell into an optical signal. For that purpose fluorescent molecules are introduced in the plasma membrane of cells. These fluorescent dyes are lipophilic and localized in the lipidic bilayers of the plasma membrane of the cells. The fluorescent properties of the dyes will be modified with the changes of membrane voltage. These fluorescent dyes sensitive to potential are known under the name of “Voltage Sensitive Dyes” or VSDs. The goal of the present proposal is to develop new VSDs and to use them for visualization of hearth and brain electrical activities in several animal models including non-human primates. The final goal is to develop new non-toxic VSDs which could be use in humans. The first application would be done in the neurosurgery of epileptic foyer with the aim to precisely localize the pathological tissue. VSDs have already been used with great success in hearth and brain imaging. Electrical maps of the complete intact tissue are obtained. VSDs could also been used to monitor electrical activity of a single neuron and even to follow the action potential in a single axon. Electrophysiological techniques and the optical techniques using VSDs have the same high temporal resolution in the millisecond. The spatial resolution of VSDs is much more superior to classical EEG or ECG recordings. The cellular resolution is achieved with VSDs. The recent progresses of functional imaging are amazing. VSDs are widely used in animal models by cardiologists and neuroscientists. On the other hand, VSDs are not widely used in the clinics as they are not exempted from toxic effect. One of the first goal of our project is the preparation of VSDs devoid of toxic effect. The fluorescent excitation emission spectra of the present available VSDs are in the visible. Visible light is absorbed in the tissues and to get enough fluorescent signals, illumination has to be high enough. Strong illumination causes phototoxicity. Preparation of infrared fluorescent compounds should decrease tissue absorption and phototoxicity. One of the main tasks of this proposal is centred on the chemical synthesis of new VSDs with fluorescent properties in the infrared. Already three classes of new VSDs with fluorescent properties in the near infrared have already been synthetized. Several of the fluorescent molecules developed are excited at over 700 nm with emission over 800 nm. This choice of excitation-emission is optimal for use in living blood perfused tissue. The maximum absorption of haemoglobin is between 433 nm and 577 nm. Over 850 nm, water becomes a problem. The final goal of this proposal is to prepare new VSDs for in vivo functional imaging of electrical activity in humans for cardiology and neurology. Already in the last two years more than ten fluorescent dyes have been synthetized. They belong to three different families. They will be tested in vitro on neuronal cultures in order to select the dyes giving a good electrochromicity i.e. the relation between changes in cellular potential and fluorescent signals. The selected dyes will then be tested in vivo. In cardiology they will be tested in Langendorff-perfused rat and pig hearths and compared with dyes currently used. The selected dyes will also be tested for imaging neocortical activities in response to sensory stimulation in mice and also in non-human primates. If one the selected dyes has passed toxicity and regulatory tests, it will be used in neurosurgical treatment of epileptic seizures. It is expected that the topical application of the VSD on the exposed cortex could help the neurosurgeon in delineating the dysplasic tissue from the healthy one.