Roba Moumné

Tether influence on the binding properties of tRNALys3 ligands designed by a fragment-based approach

A small library of 1,5-triazole derivatives linking a diaminocyclopentadiol and aromatic ketones has been prepared and screened using NMR and fluorescent techniques against tRNA(Lys)(3), the HIV reverse transcription primer. The comparison of their binding properties to those of their 1,4-triazole isomers, previously discovered in a fragment-based approach, outlines the influence of the linker on affinity and binding selectivity in such an approach.

Investigation of RNA–Ligand Interactions by 19F NMR Spectroscopy Using Fluorinated Probes†

Ribonucleic acid (RNA) is now recognized as playing a key role in many biological functions, and is emerging as an important new drug target. However, its therapeutic potential is still underexploited. Indeed, the limited understanding of the interactions between small molecules and RNA still hampers rational drug development of RNA-targeting molecules. Among the different methods available to investigate binding between small molecules and RNA, NMR spectroscopy is particularly attractive as it can deliver information on molecular interactions at the atomic level, including conformational rearrangements that can occur before or upon binding. The dynamic nature of this interaction is particularly important in RNA-regulated pathways. Many NMR spectroscopy techniques have been developed to visualize dynamic RNA–ligand interactions, most of them based on the observation of either the target or ligand H nuclei. However, some difficulties can occur when studying larger strands of RNA, as the number of detectable signals will increase. Introducing a specific label is one way to overcome this problem. An elegant method based on F NMR spectroscopy was proposed some years ago by Micura and co-workers. Introduction of a fluorine atom at a specific position of RNA allows local monitoring of binding events at this site. One technical difficulty with this approach is the need to chemically modify the RNA, which can be difficult for large RNAs or for RNAs with modified nucleotides. Furthermore, this modification can affect RNA– ligand interactions. We have recently shown that F NMR spectroscopy can be used to monitor the binding of racemic fluorinated molecules to various RNAs and that chiral recognition can be used to monitor the local conformation of the binding site. The use of ligand-based binding-competition NMR screening using fluorinated ligands has been described by Dalvit for the investigation of protein–ligand interactions (FAXS technique), but has not been applied to the study small molecules interacting with RNA. We report herein that competitive binding of fluorinated probes can be used to detect and quantify the interaction between unlabeled RNA and non-fluorinated ligands and to monitor dynamic RNA folding events (Figure 1).

Fluorinated Diaminocyclopentanes as Chiral Sensitive NMR Probes of RNA Structure

The supramolecular chiral recognition between rac-2a and several structured RNA leads to a distinct (19)F NMR signal splitting. The (19)F NMR analysis of the diastereomeric pairs formed upon binding of this racemic probe delivers a topological footprint of the RNA. This phenomenon can be exploited to investigate dynamic events involving structural equilibria, as demonstrated in a melting experiment. This work provides a proof of concept that small fluorinated moderate binders can act as external probes of RNA structures.

3-Substituted Prolines: From Synthesis to Structural Applications, from Peptides to Foldamers

Among the twenty natural proteinogenic amino acids, proline is unique as its secondary amine forms a tertiary amide when incorporated into biopolymers, thus preventing hydrogen bond formation. Despite the lack of hydrogen bonds and thanks to conformational restriction of flexibility linked to the pyrrolidine ring, proline is able to stabilize peptide secondary structures such as β-turns or polyproline helices. These unique conformational properties have aroused a great interest in the development of proline analogues. Among them, proline chimeras are tools combining the proline restriction of flexibility together with the information brought by natural amino acids side chains. This review will focus on the chemical syntheses of 3-substituted proline chimeras of potential use for peptide syntheses and as potential use as tools for SAR studies of biologically active peptides and the development of secondary structure mimetics. Their influence on peptide structure will be briefly described.

Fragment-based design of small RNA binders: promising developments and contribution of NMR.

Fragment-based drug design has become increasingly popular over the last decade. We review here the use of this approach to design small RNA binders. In addition, we discuss the use of NMR to detect the binding of small molecules on RNA targets and to guide chemists in the design of compounds targeting RNA.

CD47 Agonist Peptides Induce Programmed Cell Death in Refractory Chronic Lymphocytic Leukemia B Cells via PLCγ1 Activation: Evidence from Mice and Humans

Background Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides. Methods and Findings In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease. Conclusions Our work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL.

Homooligomers of substituted prolines and β-prolines: syntheses and secondary structure investigation

Homooligomers of enantiomerically pure (2S,3R)-3-methyl-proline, (3R,4R)-4-methyl-β-proline and (3R,4S)-3,4-dimethyl-β-proline were synthesized and studied using circular dichroism (CD) in water, methanol and propanol and using NMR in water. Changes in the far-UV CD spectrum were observed from dimers to hexamers, but little change was observed from hexamers to octa- or nonamers, both in water and methanol. CD and NMR data allowed us to conclude that oligomers of 3-substituted prolines with more than six residues adopt a characteristic PPII secondary structure both in water and aliphatic alcohols. Oligomers of (3R,4R)-4-methyl-β-proline bear the same CD signature as non-substituted β-proline oligomers, suggesting that substitution at position 3 is not sufficient to reduce conformational heterogeneity in β-proline oligomers. In the case of 3,4-disubstituted-β-proline oligomers, an atypical signature with an extra negative band at around 225 nm was observed, together with a concentration dependent CD spectrum indicating association properties. Nevertheless, NMR studies of 13C labelled oligomers of 3,4-disubstituted-β-prolines revealed a complex mixture of cis–trans conformers even for longer oligomers.

Stereoselective synthesis of fluorinated 1,3-cis-diaminocyclopentanes.

Several fluorinated 1,3-diaminocyclopentanes, previously reported to be useful RNA structural probes, can be prepared in a diastereoselective manner from a single bicyclic hydrazine precursor, in 3 to 9 steps.

Thrombospondin-1 Mimetic Agonist Peptides Induce Selective Death in Tumor Cells: Design, Synthesis, and Structure–Activity Relationship Studies

Thrombospondin-1 (TSP-1) is a glycoprotein considered as a key actor within the tumor microenvironment. Its binding to CD47, a cell surface receptor, triggers programmed cell death. Previous studies allowed the identification of 4N1K decapeptide derived from the TSP-1/CD47 binding epitope. Here, we demonstrate that this peptide is able to induce selective apoptosis of various cancer cell lines while sparing normal cells. A structure-activity relationship study led to the design of the first serum stable TSP-1 mimetic agonist peptide able to trigger selective programmed cell death (PCD) of at least lung, breast, and colorectal cancer cells. Altogether, these results will be of valuable interest for further investigation in the design of potent CD47 agonist peptides, opening new perspectives for the development of original anticancer therapies.