Samia Aci-Sèche

Lesion-induced DNA weak structural changes detected by pulsed EPR spectroscopy combined with site-directed spin labelling

Double electron-electron resonance (DEER) was applied to determine nanometre spin–spin distances on DNA duplexes that contain selected structural alterations. The present approach to evaluate the structural features of DNA damages is thus related to the interspin distance changes, as well as to the flexibility of the overall structure deduced from the distance distribution. A set of site-directed nitroxide-labelled double-stranded DNA fragments containing defined lesions, namely an 8-oxoguanine, an abasic site or abasic site analogues, a nick, a gap and a bulge structure were prepared and then analysed by the DEER spectroscopic technique. New insights into the application of 4-pulse DEER sequence are also provided, in particular with respect to the spin probes’ positions and the rigidity of selected systems. The lesion-induced conformational changes observed, which were supported by molecular dynamics studies, confirm the results obtained by other, more conventional, spectroscopic techniques. Thus, the experimental approaches described herein provide an efficient method for probing lesion-induced structural changes of nucleic acids.

Transmembrane domain targeting peptide antagonizing ErbB2/Neu inhibits breast tumor growth and metastasis.

Breast cancer is still a deadly disease despite major achievements in targeted therapies designed to block ligands or ligand-binding subunits of major tyrosine kinase receptors. Relapse is significant and metastases deleterious, which demands novel strategies for fighting this disease. Here, we report a proof-of-concept experiment demonstrating that small peptides interfering with the transmembrane domain of the tyrosine kinase epidermal growth factor receptor ErbB2 exhibit anticancer properties when used at micromolar dosages in a genetically engineered mouse model of breast cancer. Different assays demonstrate the specificity of the ErbB2-targeting peptide, which induces long-term reduction of ErbB2 phosphorylation and Akt signaling consistent with reduced tumor cell proliferation and increased survival. Microcomputed tomography analysis established the antimetastatic activity of the peptide and its impact on primary tumor growth. This reveals the interior of the cell membrane as an unexplored dimension for drug design.

A Proteometric Analysis of Human Kinome: Insight into Discriminant Conformation-dependent Residues

Because of the success of imatinib, the first type-II kinase inhibitor approved by the FDA in 2001, sustained efforts have been made by the pharmaceutical industry to discover novel compounds stabilizing the inactive conformation of protein kinases. On the seven type-II inhibitors having reached the market, four were released in 2012, suggesting an acceleration of the research of such a class of compounds. Still, they represent less than a third of the protein kinase inhibitors available to patients today. The identification of key residues involved in the binding of this type of ligands in the kinase active site might ease the design of potent and selective type-II inhibitors. In order to identify those discriminant residues, we have developed a proteometric approach combining residue descriptors of protein kinase sequences and biological activities of various type-II kinase inhibitors. We applied Partial Least Squares (PLS) regression to identify 29 key residues that influence the binding of four type-II inhibitors to most proteins of the kinome. The gatekeeper residue was found to be the most relevant, confirming an essential role in ligand binding as well as in protein kinase conformational changes. Using the newly developed proteometric model, we predicted the propensity of each protein kinase to be inhibited by type-II ligands. The model was further validated using an external data set of protein/ligand activity pairs. Other residues present in the kinase domain, and more specifically in the binding site, have been highlighted by this approach, but their role in biological mechanisms is still unknown.

Ligand entry pathways in the ligand binding domain of PPARγ receptor

To address the question of ligand entry process, we report targeted molecular dynamics simulations of the entry of the flexible ionic ligand GW0072 in the ligand binding domain of the nuclear receptor PPARγ. Starting with the ligand outside the receptor the simulations led to a ligand docked inside the binding pocket resulting in a structure very close to the holo‐form of the complex. The results showed that entry process is guided by hydrophobic interactions and that entry pathways are very similar to exit pathways. We suggest that TMD method may help in discriminating between ligands generated by in silico docking.

VSPrep: A General KNIME Workflow for the Preparation of Molecules for Virtual Screening

Over the past decades, virtual screening has proved itself to be a valuable asset to identify new bioactive compounds. The vast majority of commonly used techniques can be described in three steps: pre‐processing the dataset i. e. small (ligands) and eventually larger (receptors) molecules, execute the method and finally analyse the results. Hence, the preparation of ligands is a critical step for success of commonly used virtual screening approaches such as protein‐ligand docking, similarity or pharmacophore search. We present here a new workflow, VSPrep, for the pre‐processing of small molecules; it is based on freely accessible tools for academics and is integrated within the KNIME platform. It can be used to perform several chemoinformatics tasks such as molecular database cleaning, tautomer and stereoisomer enumeration, focused library design and conformer generation. Additionally, graphical reports of the results are provided to the user as a convenient analysis tool.

Advanced molecular dynamics simulation methods for kinase drug discovery.

Interest in the application of molecular dynamics (MD) simulations has increased in the field of protein kinase (PK) drug discovery. PKs belong to an important drug target class because they are directly involved in a number of diseases, including cancer. MD methods simulate dynamic biological and chemical events at an atomic level. This information can be combined with other in silico and experimental methods to efficiently target selected receptors. In this review, we present common and advanced methods of MD simulations and we focus on the recent applications of MD-based methodologies that provided significant insights into the elucidation of biological mechanisms involving PKs and into the discovery of novel kinase inhibitors.

Inhibition of PlexA1-mediated brain tumor growth and tumor-associated angiogenesis using a transmembrane domain targeting peptide

The neuropilin-plexin receptor complex regulates tumor cell migration and proliferation and thus is an interesting therapeutic target. High expression of neuropilin-1 is indeed associated with a bad prognosis in glioma patients. Q-RTPCR and tissue-array analyses showed here that Plexin-A1 is highly expressed in glioblastoma and that the highest level of expression correlates with the worse survival of patients. We next identified a developmental and tumor-associated pro-angiogenic role of Plexin-A1. Hence, by using molecular simulations and a two-hybrid like assay in parallel with biochemical and cellular assays we developed a specific Plexin-A1 peptidic antagonist disrupting transmembrane domain-mediated oligomerization of the receptor and subsequent signaling and functional activity. We found that this peptide exhibits anti-tumor activity in vivo on different human glioblastoma models including glioma cancer stem cells. Thus, screening Plexin-A1 expression and targeting Plexin-A1 in glioblastoma patients exhibit diagnostic and therapeutic value.