Francesca Vasile

Synthesis of Novel DC-SIGN Ligands with an α-Fucosylamide Anchor

The dendritic cell‐specific intercellular adhesion molecule (ICAM) 3‐grabbing nonintegrin (DC‐SIGN) is a C‐type lectin that appears to perform several different functions. Besides mediating adhesion between dendritic cells and T lymphocytes, DC‐SIGN recognizes several pathogens some of which, including HIV, appear to exploit it to invade host organisms. The intriguing diversity of the roles attributed to DC‐SIGN and their therapeutic implications have stimulated the search for new ligands that could be used as biological probes and possibly as lead compounds for drug development. The natural ligands of DC‐SIGN consist of mannose oligosaccharides or fucose‐containing Lewis‐type determinants. Using the known 3D structure of the Lewis‐x trisaccharide, we have identified some monovalent α‐fucosylamides that bind to DC‐SIGN with inhibitory constants 0.4–0.5 mM, as determined by SPR, and have characterized their interaction with the protein by STD NMR spectroscopy. This work establishes for the first time α‐fucosylamides as functional mimics of chemically and enzymatically unstable α‐fucosides and describes interesting candidates for the preparation of multivalent systems able to block the receptor DC‐SIGN with high affinity and with potential biomedical applications.

Cyclic RGD-Peptidomimetics Containing Bifunctional Diketopiperazine Scaffolds as New Potent Integrin Ligands

The synthesis of eight bifunctional diketopiperazine (DKP) scaffolds is described; these were formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP-1-DKP-7) or glutamic acid (DKP-8) and feature an amine and a carboxylic acid functional group. The scaffolds differ in the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogen atoms. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins α(v)β(3) and α(v)β(5), which are involved in tumor angiogenesis. Nanomolar IC(50) values were obtained for the RGD peptidomimetics derived from trans DKP scaffolds (DKP-2-DKP-8). Conformational studies of the cyclic RGD peptidomimetics by (1)H NMR spectroscopy experiments (VT-NMR and NOESY spectroscopy) in aqueous solution and Monte Carlo/Stochastic Dynamics (MC/SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cβ(Arg)-Cβ(Asp) average distance ≥8.8 Å]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin α(v)β(3) complexed with the cyclic pentapeptide, Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex.

Rational design, synthesis and characterization of potent, non-peptidic Smac mimics/XIAP inhibitors as proapoptotic agents for cancer therapy

Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2-BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.

Design, Synthesis, and Biological Evaluation of Novel cRGD–Paclitaxel Conjugates for Integrin-Assisted Drug Delivery

The efficacy of taxane-based antitumor therapy is limited by several drawbacks which result in a poor therapeutic index. Thus, the development of approaches that favor selective delivery of taxane drugs (e.g., paclitaxel, PTX) to the disease area represents a truly challenging goal. On the basis of the strategic role of integrins in tumor cell survival and tumor progression, as well as on integrin expression in tumors, novel molecular conjugates were prepared where PTX is covalently attached to either cyclic AbaRGD (Azabicycloalkane-RGD) or AmproRGD (Aminoproline-RGD) integrin-recognizing matrices via structurally diverse connections. Receptor-binding assays indicated satisfactory-to-excellent α(V)β(3) binding capabilities for most conjugates, while in vitro growth inhibition assays on a panel of human tumor cell lines revealed outstanding cell sensitivity values. Among the nine conjugate ensemble, derivative 21, bearing a robust triazole ring connected to ethylene glycol units by an amide function and showing excellent cell sensitivity properties, was selected for in vivo studies in an ovarian carcinoma model xenografted in immunodeficient mice. Remarkable antitumor activity was attained, superior to that of PTX itself, which was associated with a marked induction of aberrant mitoses, consistent with the mechanism of action of spindle poisons. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted antitumor therapy.

A folding inhibitor of the HIV-1 protease.

Because the human immunodeficiency virus type 1 protease (HIV‐1‐PR) is an essential enzyme in the viral life cycle, its inhibition can control AIDS. The folding of single‐domain proteins, like each of the monomers forming the HIV‐1‐PR homodimer, is controlled by local elementary structures (LES, folding units stabilized by strongly interacting, highly conserved, as a rule hydrophobic, amino acids). These LES have evolved over myriad generations to recognize and strongly attract each other, so as to make the protein fold fast and be stable in its native conformation. Consequently, peptides displaying a sequence identical to those segments of the monomers associated with LES are expected to act as competitive inhibitors and thus destabilize the native structure of the enzyme. These inhibitors are unlikely to lead to escape mutants as they bind to the protease monomers through highly conserved amino acids, which play an essential role in the folding process. The properties of one of the most promising inhibitors of the folding of the HIV‐1‐PR monomers found among these peptides are demonstrated with the help of spectrophotometric assays and circular dichroism spectroscopy. Proteins 2006.

Structure revision of the lantibiotic 97518.

The lantibiotic 97518, produced by a Planomonospora sp., was reported as a 2194 Da polypeptide comprising 24 amino acid residues with five thioether bridges. It was assigned to the mersacidin subgroup of type B lantibiotics by Castiglione et al. (Biochemistry 2007, 46, 5884-5897) and named planosporicin. New analytical, chemical, and genetic data and reinterpretation of the published NMR chemical shifts enable structure revision of 97518. The resulting revision of the 97518 structure involves both a shift of two amino acids and a reorganization of two thioether bridges. With this revision, the lantibiotic 97518 becomes a clear member of the nisin subgroup of compounds.

Both El Tor and classical cholera toxin bind blood group determinants

Cholera is a disease which shows a clear blood group profile, with blood group O individuals experiencing the most severe symptoms. For a long time, the cholera toxin has been suspected to be the main culprit of this blood group dependence. Here, we show that both El Tor and classical cholera toxin B-pentamers do indeed bind blood group determinants (with equal affinities), using Surface Plasmon Resonance and NMR spectroscopy. Together with previous structural data, this confirms our earlier hypothesis as to the molecular basis of cholera blood group dependence, with an interesting twist: the shorter blood group H-determinant characteristic of blood group O individuals binds with similar binding affinity compared to the A-determinant, however, with different kinetics.

STD and trNOESY NMR study of receptor-ligand interactions in living cancer cells.

Integrins are transmembrane receptors that link the cytoskeleton to the extracellular matrix (ECM), mediating cell–cell and cell–matrix adhesion and providing the traction for cell mobility and invasion. They are involved in tumour cell proliferation, migration and survival. Integrin avb3 and avb5 expression is correlated with disease progression in various tumor types (melanoma, prostate, glioblastoma). They bind to the Arg-Gly-Asp (RGD) motif as their primary recognition sequence. In recent years, both preclinical and clinical studies have demonstrated the effectiveness of various integrin antagonists in blocking tumor progression. The cyclic peptide c(ArgGly-Asp-d-Phe-NMe-Val) (cilengitide), developed by Kessler and co-workers, is an avb3 and avb5 antagonist that displayed activity in patients with glioblastoma during phase III clinical trials. The most detailed information so far available for understanding of the structural basis of the interaction is provided by the X-ray structure of cilengitide bound to the avb3 headpiece: the arginine of the RGD motif associates with the av subunit and the aspartate coordinates the bivalent metal ion on the b3 subunit. [5] Because the RGD motif occurs in many extracellular matrix ligands, the recognition specificity would be expected to be modified by other residues and to depend on the conformation of the RGD sequence. Detailed comparison with different ligands for the same integrin binding site might shed light on the essential elements that determine their interactions, specificities and affinities, and might also allow the rational design of new antagonists. The properties of integrins embedded into cell membranes can differ from those of purified receptors, so we have carried out a NMR study (by saturation transfer difference and trNOESY techniques) of the interactions between ECV304 cells (bladder cancer cells in which integrin avb3 is highly expressed ) and the two cyclic RGD mimics 1 and 2 (Scheme 1), with different configurational and structural features. We show that the interactions between these small ligands and membrane-bound proteins can be observed by NMR directly in H2O buffer suspensions of living cells. The data allowed us to identify (by STD) the portions of the ligands in closest contact with the protein and to define (by trNOESY) the preferred conformations of the bound ligands. These NMR techniques focus on the NMR signals of the ligands, exploiting NOE effects between protein and ligands in the protein– ligand complexes. The ligands, the cyclic RGD pentapeptide mimics 1 and 2 (Scheme 1), previously described by workers from our laboratory, are each characterized by a benzylic group connected to carbon 3 of the lactam unit, but differ in the configurations of the C-3 and C-7 lactam stereocentres. As already reported, the conformational preferences of these compounds in the free state are modulated by the configurations of the bicyclic lactam, and so are their affinities for avb3 integrin. These activities were estimated by competition experiments with [I]echistatin for binding to purified avb3 receptors. [7] The resulting IC50 values for 1 and 2 were 6.4 and 154 nm, respectively. Adhesion assay tests were also performed for 1 with ECV304 bladder cancer cells, a cell line derived from solid tumours that highly expresses avb3 integrin. The IC50 values were estimated in competition experiments versus vitronectin (12.5 0.2 mm) and versus fibronectin (7.5 1.9 mm). The micromolar activities obtained in the adhesion assay on ECV cells represent an ideal range for testing interactions between the ligands and the integral membrane protein of intact cells by means of trNOE and STD experiments. NMR experiments : STD and trNOESY experiments were performed with cell suspensions in nondeuterated buffer. The average number of avb3 receptors per ECV304 cell was estimated by fluorescence analysis (FACS) to be 4 10. Approximately 10 cells were used for ligands 1 or 2 (0.7 mmol) in a Scheme 1. Integrin ligands and corresponding avb3 binding affinities.

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization.

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head-head (8), tail-tail (9) or head-tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2-BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail-tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.

Comprehensive analysis of blood group antigen binding to classical and El Tor cholera toxin B-pentamers by NMR

Cholera is a diarrheal disease responsible for the deaths of thousands, possibly even hundreds of thousands of people every year, and its impact is predicted to further increase with climate change. It has been known for decades that blood group O individuals suffer more severe symptoms of cholera compared with individuals with other blood groups (A, B and AB). The observed blood group dependence is likely to be caused by the major virulence factor of Vibrio cholerae, the cholera toxin (CT). Here, we investigate the binding of ABH blood group determinants to both classical and El Tor CTB-pentamers using saturation transfer difference NMR and show that all three blood group determinants bind to both toxin variants. Although the details of the interactions differ, we see no large differences between the two toxin genotypes and observe very similar binding constants. We also show that the blood group determinants bind to a site distinct from that of the primary receptor, GM1. Transferred NOESY data confirm that the conformations of the blood group determinants in complex with both toxin variants are similar to those of reported X-ray and solution structures. Taken together, this detailed analysis provides a framework for the interpretation of the epidemiological data linking the severity of cholera infection and an individuals blood group, and brings us one step closer to understanding the molecular basis of cholera blood group dependence.