Targeting RNAs using small molecules is an emerging field of medicinal chemistry and holds the promise for the discovery of efficient tools for chemical biology. MicroRNAs are particularly interesting targets since they are involved in a number of pathologies such as cancers. Indeed, overexpressed microRNAs in cancer are oncogenic and various series of inhibitors of microRNAs biogenesis have been developed in recent years. Here, we describe the structure-based design of new efficient inhibitors of microRNA-21. Starting from a previously identified hit, we performed biochemical studies and molecular docking to design a new series of optimized conjugates of neomycin aminoglycoside with artificial nucleobases and amino acids. Detailed investigation about the mode of action and the site of the interaction of the newly synthesized compounds allowed for the description of structure-activity relationships and the identification of the most important parameters for miR-21 inhibition.

Exhaustive structure-efficacy relationship studies on nonviral gene delivery systems are often hampered by the ill-defined or polydisperse nature of the formulations. Facial amphiphiles based on rigid cage-type molecular scaffolds offer unique possibilities towards these studies. Taking advantage of regioselective functionalization schemes, we have synthesized a library of cationic cyclodextrin (CD) derivatives combining a range of hydrophilic and lipophilic domains. We have scrutinized how the hydrophilic-lipophilic balance (HLB) around the CD scaffold determines their self-assembly capabilities and the DNA binding and release abilities of the corresponding CD : DNA nanocomplexes (CDplexes). These features have been ultimately correlated with their capabilities to deliver a reporter luciferase-encoding pDNA into COS-7 cells. The ensemble of results demonstrates that fine tuning of the HLB is critical to induce compaction of DNA by the CD-based facial amphiphiles into transfection-productive CDplexes.

The incorporation of carbohydrate functional elements in the architecture of polycationic amphiphilic cyclodextrins (paCDs) provides glycosylated paCDs (pGaCDs) that form transfectious nanocomplexes (glycoCDplexes) with pDNA. In this study, we aimed at elucidating the internalization mechanisms at play and their incidence in transfection efficiency for glycoCDplexes formulated with 6-amino-6-deoxy-b-Dglucopyranosyl-appended pGaCDs in comparison with mannosylated and non-glycosylated congeners.Preliminary data showed a relatively high uptake of the 6-aminoglucosylated nanocomplexes by BNL-CL2 hepatocytes that correlated with a strong affinity towards the galactose-specific peanut agglutinin (PNA) lectin, suggesting that the galactose-binding asialoglycoprotein receptor at the surface of hepatocytes might be involved in glycoCDplex internalization. Transfection kinetics, internalization rates and protein expression data in BNL-CL2 ASGPR-expressing cells and COS-7 ASGPR-devoid epithelial cells in the absence and presence of different inhibitors of clathrin-dependent (chlorpromazine), caveolae-dependent (genistein) and macropinocytosis (amiloride) endocytic routes evidenced significant differences in cell uptake pathways and fate of glycoCDplexes as compared with CDplexes. Most importantly, such differences were dependent on the cell type and on the carbohydrate coating moiety.Clathrin-mediated uptake in BNLCL-2 cells is particularly favored for the 6-amino-6-deoxyglucose CDplexes, supporting the interplay of specific recognition phenomena. Competitive uptake and transfection experiments conducted in the presence of asialofetuin or of a polyclonal ASGPR-antibody, as well as siRNA-mediated ASGPR-specific gene knockdown, supported the involvement of ASGPR, firmly demonstrating the dual role of the 6-amino-6-deoxyglucose motif as DNA and lectin receptor ligand. The results reinforce the use of carbohydrates in glycoCDplexes to modulate cellular uptake and transfection capabilities in a cell-dependent manner.