MOTIVATION: RNA molecules are implicated in numerous fundamental biological processes and many human pathologies, such as cancer, neurodegenerative disorders, muscular diseases and bacterial infections. Modulating the mode of action of disease-implicated RNA molecules can lead to the discovery of new therapeutical agents and even address pathologies linked to 'undruggable' protein targets. This modulation can be achieved by direct targeting of RNA with small molecules. As of today, only a few RNA-targeting small molecules are used clinically. One of the main obstacles that have hampered the development of a rational drug design protocol to target RNA with small molecules is the lack of a comprehensive understanding of the molecular mechanisms at the basis of RNA-small molecule (RNA-SM) recognition. RESULTS: Here, we present Harnessing RIBOnucleic acid-Small molecule Structures (HARIBOSS), a curated collection of RNA-SM structures determined by X-ray crystallography, nuclear magnetic resonance spectroscopy and cryo-electron microscopy. HARIBOSS facilitates the exploration of drug-like compounds known to bind RNA, the analysis of ligands and pockets properties and ultimately the development of in silico strategies to identify RNA-targeting small molecules. AVAILABILITY AND IMPLEMENTATION: HARIBOSS can be explored via a web interface available at http://hariboss.pasteur.cloud. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Intracellular uptake of nanoparticles (NPs) may induce phase transitions, restructuring, stretching, or even complete disruption of the cell membrane. Therefore, NP cytotoxicity assessment requires a thorough understanding of the mechanisms by which these engineered nanostructures interact with the cell membrane. In this study, extensive Coarse-Grained Molecular Dynamics (MD) simulations are performed to investigate the partitioning of an anionic, ligand-decorated NP in model membranes containing dipalmitoylphosphatidylcholine (DPPC) phospholipids and different concentrations of cholesterol. Spontaneous fusion and translocation of the anionic NP is not observed in any of the 10-µs unbiased MD simulations, indicating that longer timescales may be required for such phenomena to occur. This picture is supported by the free energy analysis, revealing a considerable free energy barrier for NP translocation across the lipid bilayer. 5-µs unbiased MD simulations with the NP inserted in the bilayer core reveal that the hydrophobic and hydrophilic ligands of the NP surface rearrange to form optimal contacts with the lipid bilayer, leading to the so-called snorkeling effect. Inside cholesterol-containing bilayers, the NP induces rearrangement of the structure of the lipid bilayer in its vicinity from the liquid-ordered to the liquid phase spanning a distance almost twice its core radius (8-10 nm). Based on the physical insights obtained in this study, we propose a mechanism of cellular anionic NP partitioning, which requires structural rearrangements of both the NP and the bilayer, and conclude that the translocation of anionic NPs through cholesterol-rich membranes must be accompanied by formation of cholesterol-lean regions in the proximity of NPs.

The PIK3CA gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), which activates signaling cascades leading to cell proliferation, survival, and cell growth. The most frequent mutation in PIK3CA is H1047R, which results in enzymatic overactivation. Understanding how the H1047R mutation causes the enhanced activity of the protein in atomic detail is central to developing mutant-specific therapeutics for cancer. To this end, Surface Plasmon Resonance (SPR) experiments and Molecular Dynamics (MD) simulations were carried out for both wild-type (WT) and H1047R mutant proteins. An expanded positive charge distribution on the membrane binding regions of the mutant with respect to the WT protein is observed through MD simulations, which justifies the increased ability of the mutated protein variant to bind to membranes rich in anionic lipids in our SPR experiments. Our results further support an auto-inhibitory role of the C-terminal tail in the WT protein, which is abolished in the mutant protein due to loss of crucial intermolecular interactions. Moreover, Functional Mode Analysis reveals that the H1047R mutation alters the twisting motion of the N-lobe of the kinase domain with respect to the C-lobe and shifts the position of the conserved P-loop residues in the vicinity of the active site. These findings demonstrate the dynamical and structural differences of the two proteins in atomic detail and propose a mechanism of overactivation for the mutant protein. The results may be further utilized for the design of mutant-specific PI3Kα inhibitors that exploit the altered mutant conformation.

The application of an in-house developed web server called ChemBioServer to the filtering and selection of drug candidates for the inhibition of the PI3Kα protein is presented. 1000 candidate molecules were initially selected from a virtual screening experiment. Those molecules were then filtered for steric clashes, physicochemical and toxicity properties and grouped into clusters using the ChemBioServer web application. During this filtering process, 400 compounds were rejected and the remaining 600 were clustered in 20 different groups, allowing for a more efficient visual inspection of the compounds. Representatives of these clusters were then selected for further experimental study. Four out of the seven selected molecules inhibited PI3Kα activity in vitro, indicating that the workflow described herein can be successfully applied in drug discovery. ChemBioServer proved to assist the post-processing application of top-ranked molecules resulting from a docking exercise by increasing the efficiency and the quality of compound selection that passed to the experimental test phase.