Copper ions are essential for life but posses a redox-activity which makes them potentially toxic, and their cellular availability is highly regulated by an intricate network of intracellular chaperones, transcription factors and membrane transporters. Copper homeostatic imbalance is connected to several major neurological diseases. The detailed mechanisms of copper movement across membranes remain unknown due to the difficulty to characterize at atomic level the different proteins involved, which are mainly integral membrane systems. In humans, high-affinity copper uptake is modulated by hCTR1, a trimeric membrane transporter which has so far fled from high-resolution x-ray or cryo-EM investigations and is extremely challenging to produce and recover in workable amounts for structural studies. The central objective of the present project is to develop and apply a solid-state Magic-Angle Spinning (MAS) NMR approach to allow complete characterization of the structure and mechanism of lipid-bound hCTR1. Building on a decade of continuous advances of the NMR community, the recent development of very fast (up to 100 kHz) MAS probes has revolutionised this field, with developments that speed up the analysis of proteins of considerable size and open the way to complex biological solids available in limited amounts. We propose to leverage the unique expertise and equipment available in the consortium, and achieve the objectives above through a combination of innovative strategies for isotopic sample preparation, advanced spectroscopic tools to obtain NMR signatures of the structure and dynamics, and new instrumentation capable of even faster MAS rates. The project will provide breakthrough data for understanding structure-activity relationships in a challenging integral membrane protein, and will allow the addition of solid-state NMR to the method portfolio for the characterization of medically relevant targets.

Pseudomonas aeruginosa (PA) is a major human opportunistic pathogen. It causes infections in immune-compromised patients, cystic fibrosis patients and is responsible for many nosocomial infections worldwide. PA provokes acute and chronic infections. The choice between these two modes of infection depends on the ability of PA to switch from a planktonic (free-swimming) to a sedentary (living in a community called a biofilm) lifestyle. In PA, the transition between these two lifestyles is finely regulated by complex regulatory networks involving several two-component regulatory systems. This proposal focuses on the GacS signal transduction network that controls biofilm formation and virulence gene expression. In this network, two trans-membrane sensor kinases, GacS and RetS, interact extensively and function antagonistically via a series of biochemical mechanisms involving multi-modular interactions on both sides of the membrane. This project aims to perform functional and molecular characterization of this regulatory system and identify small molecules to interfere with this network. Our goals will be pursued using interdisciplinary approaches, including genetics, microbiology, biochemistry, structural biology and cellular microscopy. Unravelling the molecular determinants responsible for the GacS-RetS signalling pathway, and identify inhibitors molecules, will contribute to the development of new antibacterial molecules to control PA pathogenicity.

G Protein-Coupled Receptors (GPCRs) are central players in the complex relationship of inter-cellular signaling. They act in concert by activating and influencing each other according to the context or the pathology in a complex web whose figures and meanings remain, even today, often indecipherable. Advances in the study of these phenomena is made difficult by the lack of molecular tools, in particular specific antibodies (Abs), allowing to point and modulate very precisely one of these proteins among many similar ones. Although highly anticipated, the development of such tools is hampered by the difficulty to identify among a plethora of inactive candidates the few that possess a high affinity and selectivity for one single receptor of interest. In this context, we propose an approach taking advantage of new methods for Ab production against these elusive targets and then to measure one by one, on millions, the affinity of the Abs thus obtained for the native protein in its membrane environment. This precise measurement is made possible via microfluidic technologies which, combined with a biocompatible chemistry, allows the formation of millions of microcapsules whose surface serves as sensors. These sensors allow to measure finely the protein-Ab interaction at a speed of a thousand events per second, to sort the best ones and to recover the producing clones alive. The methodology will be applied to the development of Abs specifically targeting the ß1 and ß2 adrenergic receptors, key GPCRs involved in the heart physiology. In a second step, a measure of selectivity between several targets will be directly integrated into the sorting criterion in order to reach the very rare specific antibodies. These candidates will be produced on a large scale and validated by biophysical measurements. They will be then used to decipher the precise involvement of ß1 and ß2 adrenergic receptors in heart cardiomyocytes and their relative contribution in heart failure phenomena.

The efficient treatment of chronic and neuropathic pain is a yet unsolved medical, economic and societal problem. It affects an increasing number of patients and severely impairs their daily life (weekly symptoms over ca. 7 years on average, 60% of patients obliged to work at home, 13% of unemployed patients). Current treatment of chronic pain is purely symptomatic (NSAIDs, weak opiates, antiepileptics, antidepressants, anxiolytics) and inefficient in 75% of the cases. Among the many signaling pathways that are currently investigated to better define molecular mechanisms involved in the appearance and maintenance of chronic pain, our consortium is particularly interested in a cytokine (FL) and its receptor tyrosine kinase (FLT3) whose expression on primary sensory neurons has been shown to mediate mechanical as well as thermal hyperalgesia through the activation of different TRP channels. Administration of the FL cytokine in mice induces a FLT3-specific hyperalgesia that can be reversed by inhibiting the FLT3 receptor (FLT3 ko mice, anti-FLT3 siRNAs). Importantly, reversal of FL-induced hyperlgesia with anti-FLT3 agents is devoid of addiction and tolerance, as classically observed with opiates. FLT3 ko mouse displays normal response to acute pain stimuli, but almost failed to develop neuropathic pain syndrome after peripheral nerve injury. Intrathecal administration of anti-FLT3 siRNA suppressed both the development and the maintenance of tactile allodynia after sciatic nerve ligature. Taken together, our results indicate a previously unknown role for FLT3 expressed by sensory neurons in maintaining sensitization that has been implicated in persistent neuropathic pain. Thus blocking FLT3 signaling in somato-sensory neurons might be a new strategy for the therapy of chronic neuropathic pain induced by nerve injury. Since mutations of the FLT3 gene is the most common genetic lesion in acute myeloid leukaemia (AML), many FLT3 inhibitors have been developed for the treatment of AML. However, they suffer from a lack of selectivity for the receptor tyrosine kinase FLT3 since they all target the conserved kinase catalytic ATP-binding site. Severe side effects associated with the therapeutic use of FLT3 inhibitors are tolerated in oncology but not in the perspective of a long-lasting treatment of chronic and neuropathic pains. Combining virtual screening of compound libraries and experimental binding/functional assays, our consortium has identified the first extracellular FLT3 receptor antagonist able to inhibit, at a low micromolar range, the binding of the cytokine FL to its FLT3receptor and further block FL-induced FLT3 autophosphorylation. When administered to rats, the inhibitor completely reverses neuropathic pain induced by a chronic constriction injury (CCI) of the sciatic nerve. This compound is however not directly usable in humans because of a still moderate affinity for its primary target, the existence of two off-targets that may lead to undesired effects, and a low oral bioavailability. Having made the proof-of-concept that extracellular FLT3 inhibition by a low molecular weight compound is feasible and really reverses neuropathic pain in rodents, the BIODOL project is aimed at developing convergent approaches towards the identification of a potent and orally available selective FLT3 inhibitor along 3 main axes: (i) medicinal chemistry optimization of pharmacodynamic and pharmacokinetic properties of the existing initial lead; (ii) identifying novel hits by experimentally screening the French National Library on an already developed in vitro HTRF assay, (iii) evaluate the ability of peptides featuring the transmembrane segment of FLT3 and related receptor tyrosine kinases (e.g. PDGFR, c-kit, Fms) to block the receptor dimerization necessary for downstream signaling. The final deliverable of the project will be the development of a potential clinical candidate for treating neuropathic pain in humans.

The project nano-WAC aims to develop a new miniaturized affinity chromatography-based technology (nano-WAC) that will enable the innovative ligand-screening against native membrane proteins (MPs), notably G protein–coupled receptors (GPCRs), a large family of MPs with a tremendous clinical potential. To date, no robust biophysical technique is available for the discovery of small ligands (fragments) targeting MPs that are available in minute amounts. Our strategy, based on the combination of the separation/detection powers of affinity chromatography and mass spectrometry (nano-WAC-MS), will open the way to the screening of fragment library for the identification of weak affinity fragments in the context of the Fragment Based Drug Discovery approach. After development with one prototypical GPCR, this assay will be extended to the ghrelin receptor GHSR that is a prominent target in biomedicine.