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.

Protein-carbohydrate interactions play a key role in the first step of numerous biological processes, e.g. fertilization and tissue homing of immune cells, but also in infection, inflammation, migration of tumor cells and other pathologies. Pathogenic microorganisms (viruses, bacteria, fungi and parasites) have developed strategies for utilizing glycan epitopes on human tissues for specific recognition, for adhesion and sometimes for cellular internalization. The proteins involved can be viral capsid domains, adhesins on top of pili, soluble lectins or carbohydrate binding domains of enzymes or toxins. Their carbohydrate-binding sites are specific for glycans located on human epithelia such as the histo/blood group oligosaccharides of ABO and Lewis systems. Molecules that could interfere with such lectin/glycan interaction are therefore of high interest as anti-infectious agents. Similarly, human lectins are involved in chronic diseases related to inflammation and cancer, and consequently the search for lectin inhibitors to interfere with these pathological conditions is of outstanding interest. The project described here aims at the development of the underexplored area of non-carbohydrate drug-like lectin inhibitors. Such glycomimetics could overcome current limitations of carbohydrate-based therapeutics such as poor pharmacokinetic and pharmacodynamic parameters as well as provide solution for synthetic tractability The consortium of two French and two German laboratories proposes to combine a multidisciplinary approach combining virtual and in vitro screening of chemical libraries, using functional assays and NMR approaches, followed by structure (X-ray, NMR), thermodynamics (ITC) and kinetics (SPR, MD) guided lead optimization.In order to explore the neglected area of chemical space for the development of lectin inhibitors, we plan a highly diverse three-pillar screening strategy, using virtual and experimental screening on three representative examples of bacterial lectins. Selected screening hits from these complementary screening campaigns will be analyzed in silico and validated in biochemical secondary assays. Commercial analogs of these hits will then establish a stringent structure-activity relationship supported by chemo-informatics analysis. Next, kinetic and thermo-dynamic characterization and subsequent structure elucidation using highly complementary biophysical techniques will provide monovalent lead struc-tures. This cyclic iterative process then yields monovalent inhibitors that are assembled onto multivalent scaffolds to pave the way for highly potent non-carbohydrate glycomimetics for bacterial lectins All optimized compounds will be assessed for their efficacy in lectin-dependent bacterial adhesion assays. This proposal aims at the establishment of a broadly applicable methodology for the development of non-carbohydrate lectin inhibitors. A set of three diverse and representative bacterial lectins was chosen to explore the potential of the discovery pipeline. While pipeline development is our major concern, generating novel anti-infectives is foreseen (i.e. a problem-solving endeavor). However, this approach will not be limited to bacterial targets and other viral, fungal, or human lectins of high therapeutic significance can be targeted as well. Using our technology, the scientific community and also companies will be able to develop potential drugs for lectin-mediated diseases, e.g. metastasizing cancers, autoimmune disorders or other infectious diseases. This is certainly a significant improvement to currently pharmaceutically neglected lectins and paves the ground for targeting lectins with drugs. Finally, this endeavor will contribute to our fundamental understanding of biological processes involving multivalent receptors such as membrane dynamics and lipid raft-mediated internalization (i.e. a curiosity driven research).

During the last decade, peptides have gained a wide range of applications in medicine. Indeed, more than 60 US Food and Drug Administration (FDA)-approved peptide medicines are on the market and this is expected to grow significantly, with approximately 140 peptide drugs currently in clinical trials and more than 500 therapeutic peptides in preclinical development. In addition, out of the 390 non-olfactory G protein coupled receptors (GPCRs) identified in the genome, 290 have only peptides as ligands (no small organic compounds). However, these peptides are often not directly suitable for use as convenient pharmacological tools or novel therapeutics because they display generally short circulating plasma half-life. Currently, the techniques developed for half-life extension are generally based on the modification of the native peptide backbone (non-natural amino-acids, lactam bridges, stapling, cyclization) which is a long process. The incorporation of Polyethylene glycol (PEG) into peptides has also been used to limit glomerular filtration and thereby increase plasma half-life by limiting the elimination of peptides. However, because of increased safety and tolerability concerns related to the use of PEG as a component of an injectable therapeutic, PEGylation has become a less preferred choice. Another approach is based on the binding of the peptide to the circulating protein albumin used as a vehicle by peptide acylation with hydrocarbon tail as seen in the GLP-1 agonist (Luraglutide). Nevertheless the presence of the hydrocarbon tail makes the peptide much less selective for its target with potential enhanced toxicity. In the FLUOROPEP project, we propose a multidisciplinary research program (organic chemistry, physicochemistry, biophysics, biology) which aims at developing an unprecedented, innovative and convenient approach to improve circulating plasma half-life of endogenous peptides, taking GPCRs peptidic ligands as model system. The strategy adopted in our project relies in the incorporation of a fluorocarbon-chain (F-chain) into N-terminal or C-terminal part of the peptide sequence to force the native peptide to self-organize in aqueous solution. As a consequence, the supramolecular organization should result in the decrease of enzymatic peptides degradation and the enhancement of its in vivo plasma half-life stability. F-chains have unique properties, very different from those of hydrocarbon chains. In particular, they are more stable, stiffer, hydrophobic and significantly more lipophobic which increases their specificity and their bioavailability. In addition, F-chains are biologically inert molecules with no intrinsic immuno-stimulatory activity or immunogenicity. Thereby, fluorocarbons have already been used for various medical applications, including blood substitutes (fluorocarbon emulsions) or more recently as synthetic vaccine (fluoropeptides) demonstrating their absence of toxicity in human. Three GPCR peptides that were previously reported for in vivo unstability will be selected as models: apelin, oxytocin and spexin. The physicochemical and the in vitro and in vivo pharmacology of the resulting fluoropeptides will be fully investigated. The preliminary data demonstrate that the incorporation of a fluorocarbon-chain on apeline peptide enables not only to maintain its pharmacological properties towards APJ receptor but also greatly increases its human plasma half-life and in vivo efficacy. To date, such a strategy has never been evaluated for therapeutic peptides with targets located in the systemic and/or central nervous system. The FLUOROPEP project should open the route to a convenient, safe and general approach to greatly increase the half-life stability of peptides for their in vivo evaluation as pharmacological tools and/or therapeutic agents.