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.

Small interfering RNAs (siRNAs) are specific and effective molecules for gene silencing, but fail to enter cells unassisted due to their negative charges and their high molecular weight/size. The conjugation of siRNA to ligands targeting cell surface receptors is a promising approach to silence genes associated with various pathologies. However, besides the external cell barrier, to reach the cytosol where the siRNA associates with the RISC (RNA-induced silencing complex) machinery, the siRNA has to escape from intracellular compartments (ICC). However, despite encouraging perspectives, RNA therapeutic applications are hindered by a low rate of ICC escape. To enhance the cytosolic delivery of siRNAs, one strategy is based on multi-ligand conjugates. Its efficacy has been proven with recently marketed drugs composed of three N-acetylgalactosamine ligands conjugated to siRNA. This bioconjugate has led the way for the use of siRNA conjugates in therapy. However, these conjugates are not transposable to pathologies other than liver diseases. Moreover, mechanisms of escape across ICC remain unknown, the underlying mechanisms relating affinity/avidity/specificity to siRNA cytosolic delivery remain to be elucidated, and active targeting strategies for organs/cells other than liver/hepatocytes, remain to be explored. Our project aims at deciphering and optimizing the delivery of siRNAs mediated by aptamers. Aptamers are nucleic acid molecules which bind to their targets with high affinity and selectivity. When the target is a cell-surface biomarker, aptamers have huge potential as specific cell-targeting ligands. If the receptor is internalized, aptamers can drive conjugated siRNA inside cells. Moreover, their chemical synthesis allows for a variety of modulable constructs. Our project aims to provide strategies based on multivalent/multispecific nucleic acid aptamers as innovative active targeting tools to enhance selective siRNA delivery to targeted cells. We will develop multifunctional molecules, in which the aptamer(s) is/are the ‘cell-targeting part(s)’ and the siRNA is the ‘therapeutic part’. These aptamers conjugated to siRNA are named AsiC for aptamer-siRNA conjugates. This project, based on one siRNA and three aptamers targeting three different cell surface receptors, aims to 1) synthesize conjugates composed of 1-3 aptamers conjugated to siRNA, 2) characterize their interaction kinetics, and 3) follow their intracellular trafficking, to in fine provide insights to improve the delivery of siRNA by active targeting.

Pathogenic bacteria are a permanent threat for Humanity even the discovery of antibiotics led to the fade-out of mass epidemies. Nevertheless humanity faces more and more antibiotic-resistant bacterial strains. Thus, the constant development of innovative antibacterial strategies is crucial, especially against Gram-negative bacteria found in many lethal infections. The current antibiotic arsenal is mainly composed of organic compounds when organometallic derivatives appear to be reserved principally to cancer. However, the toxicity profiles of current last resort antibiotics (colistin, polymyxin, oxazolidinones) make now organometallic derivatives competitive in the benefit/risk balance. Moreover some of these organometallic species proved to have the photophysical properties making them potential photosensitizers (PS) useable in antibacterial photodynamic therapy (PDT). In this context iridium(III) complexes are promising candidates. However, these chemical entities are not able to cross the bacterial envelope of Gram-negative bacteria to reach their intracellular biological targets. Bacterial iron uptake systems are gates through the envelope. Siderophores are small iron(III) chelating molecules secreted by bacteria to promote iron acquisition. Siderophores are able to use bacterial iron uptake systems to cross bacterial membrane reaching therefore bacterial inner space. Siderophores could be thus used as vectors to promote accumulation of PS based on iridium(III) into Gram-negative bacteria using a so called Trojan horse strategy. The recognition of the ferric-siderophore by specific outer membrane transporters will lead to the uptake of the conjugate inside the bacterium. This approach should, at one and at the same time, increase the penetration of organometallic species and reduce the peripheral toxicity of vectorized metals for host cells. Vectors to be used in our approach will be analogs of enterobactin, ferrichrome and pyochelin, three siderophores commonly used by Gram-negative pathogens considered to be critical (WHO list). These vectors will be synthesized by Partner 1 (Dr. Gaëtan MISLIN, UMR7242, Illkirch) and conjugated to Ir(III) complexes prepared by partner 2 (Dr. Sylvain GAILLARD, ENSICaen, Caen). Partner 1 will assess the antibacterial activities (MIC) of the siderophore-Ir(III) conjugates in the presence, and in the absence, of light, on Pseudomonas aeruginosa (planktonic and biofilms). This pathogenic bacterium, responsible for serious infections, is known to be poorly permeable to many xenobiotics. A comparison of results obtained in rich and iron-depleted media will give an overview of metal conjugates activities under various physiological conditions and will provide useful informations on involvement of iron uptake systems. Iron acquisition is completely different in humans and in bacteria: iron homeostasis does not require the presence of siderophores. Actually, eukaryotic membranes are poorly permeable to the ferric siderophores selected in our approach. Thus, metal conjugates are not expected to promote intracellular penetration, and toxicity, of iridium(III) in human cells. To evaluate this point more promising compounds will be tested on a human lung fibroblast infection model in order to evaluate both the cytotoxicity of the conjugates and the robustness of our antibacterial strategy in the presence of eukaryotic cells. Finally, the conception of a perennial therapeutic approach must include a study of the mechanisms potentially involved in the emergence of resistance. To this end, partner 3 (Pr Patrick PLESIAT, Besançon) will investigate the strategies developed by bacteria resistant to our approach. Furthermore this partner also depends on the National Reference Center on Resistances and could enlarge the screening to a wide collection of bacteria including antibiotic-resistant strains and clinical isolates.

Intrinsically disordered proteins (IDPs), comprising a third of the eukaryotic proteome, play key roles in regulatory and signalling processes within cells. The sequences of IDPs frequently contain proline residues, either as part of a polyproline domain or as part of a (Ser/Thr)-Pro phosphorylation site. Currently, the functional importance of proline rich motifs in the regulation of biological functions is not well understood. It is known that subtle conformational changes of the proline ring's structure, as well as changes in the isomerization rate between cis and trans isomers play a role in modulating protein-protein interactions. However, these parameters are difficult to characterize at the level of individual proline residues, let alone at atomic resolution. While molecular disorder precludes the use of X-ray and cryo-EM approaches, the use of Nuclear Magnetic Resonance (NMR) faces the difficult issue of poly-proline homopolymer's assignment. The FLUOPROLINE project's objective is to develop a novel way to solve this issue by the incorporation of fluorine atoms (19F) on selected prolines. 19F-based NMR is highly sensitive and bio-orthogonal which enables straightforward readout of proline's conformation and dynamics. Furthermore, by the introduction of various strereoisomeric fluoro-prolines, it is possible to modify these properties in a controllable way. As a test case, this approach will be applied to decipher the molecular mechanisms underlying the specific recognition of different proline rich motifs (PRM) by the SH3 domain of BIN1 (amphiphysin), a protein whose dysfunction is involved in myopathy. These interactions are involved in particular in regulating the multiple functions of dynamin (DNM2) in membrane remodeling, endocytosis and autophagy. This interdisciplinary project, at the interface between biophysics, chemistry and chemical biology, as well as translational medicine, gathers two teams at IGBMC involved in structural biology (NMR: Bruno Kieffer) and Translational medicine (Jocelyn Laporte) and two teams in chemistry: Vladimir Torbeev specialized in protein chemistry at ISIS, Strasbourg and Bruno Linclau at the University of Southampton specializing in organofluorine chemistry. The proposed work consists of four objectives: (i) The description of conformational and dynamical properties of non-commercial fluorinated prolines able to stabilize either the cis or the trans conformation, as well as the pucker preference using by 19F NMR; (ii) The impact of PRM chemical modifications (fluorination, phosphorylation) on intra and intermolecular interactions between BIN1 SH3 and PRMs from BIN1 and DNM2; (iii) the design of potent peptides able to modulate with the BIN1/ DNM2 interaction; (iv) the development of experimental approaches enabling the observation of BIN1-DNM2 interaction and its modulation in a cellular context. Beside a gain in our fundamental understanding of the specific roles of proline residues in IDPs, the project will lead to methodology developments in the fluorine chemistry, the semi-synthesis of large protein constructs and the studies of molecular interactions by 19F NMR. The characterization of novel fluoroprolines with tuneable molecular properties will extend the current repertoire of chemically modified peptides developed for therapeutic purposes. Peptides modulating the BIN1/DNM2 complex could have medical applications. The strong involvement of two partners of the project in start-up creation will favour an efficient technological transfer for the design of new drugs.

The inhibitor of kB kinase (IKK) is a regulator of inflammatory, immune and apoptotic responses, best known for its role in the activation of nuclear factor kB (NF-kB). This Ser/Thr kinase is an enzymatic complex consisting of two catalytic and one regulatory subunits. The catalytic IKKß subunit accounts for nearly all kinase activity implicated in the activation of NF-kB. It has been proposed that IKK acts as a bridge between inflammation and cancer by operating in a number of pathways that promote tumorigenesis. A recent investigation has shown that IKKß regulates the stability of oncogenic deltaNp73-alpha. This latter protein is an isoform of p73 (a member of the tumor suppressor p53 protein family) that lacks the N-terminal transactivation domain. It exerts anti-apoptotic functions and promotes cellular transformation by competing with full-length p53 proteins for binding at p53 regulatory elements within DNA promoter regions. Massimo Tommasino and Rosita Accardi, who participate to this project, have discovered that IKKß phosphorylation at Ser422 of deltaNp73-alpha leads to the stabilization and a strong nuclear accumulation of deltaNp73-alpha and that this results in the inhibition of the expression of p53-regulated genes. Intriguingly, the interaction between IKKß and deltaNp73-alpha appears to be enhanced by Ser422 phosphorylation. The proposed project aims at characterizing the IKKß/deltaNp73-alpha interaction using an integrated Structural Biology approach, which encompasses x-ray crystallography, NMR spectroscopy and Surface Plasmon Resonance coupled with a number of in vitro and in vivo activity assays and functional studies. Structural studies will provide three-dimensional high-resolution structures or models of IKKß/deltaNp73-alpha complexes, which will be complemented with binding kinetics and kinase activity data. Together structural and activity data will enable to elucidate the mechanisms governing this interaction. Subsequently, specific mutants of IKKß and deltaNp73-alpha will be designed based on knowledge of the interaction mechanisms. These mutants will be tested in a number functional assays in order to better define the role of the IKKß/deltaNp73-alpha interaction in nuclear accumulation of deltaNp73-alpha and subsequent inhibition of the expression of p53-regulated genes. Both IKKß and deltaNp73-alpha have been found to be upregulated in many human malignancies, including breast, prostate, liver, lung and thyroid cancers. For this reason IKKß and deltaNp73-alpha represent potentially important therapeutic targets. We believe that the present project has the potential to unveil important mechanisms regulating cancer and therefore to be of high impact for public heath.