Targeting the multidrug ABCG2 transporter with flavonoidic inhibitors: in vitro optimization and in vivo validation.

This review describes the breast cancer resistance protein ABCG2 through its structure, functional roles and involvement in cell multidrug resistance, especially in cancer cells resistance to chemotherapeutics. The different types of known inhibitors are described, some being non-selective, since they also bind to other targets, and others being quite specific such as flavonoids. The different classes of active flavonoids and other polyphenols are described, some as plant natural compounds, but most of them being prepared and derivatized through medicinal chemistry. Quantitative structure-activity relationships of the ability of flavones, chalcones, xanthones, acridones and various benzopyrane/benzofurane derivatives to inhibit ABCG2-mediated drug efflux have led to pharmacophores and molecular models allowing to optimize the available hit compounds and to design new-generation lead compounds. Interestingly, inhibitory flavonoids are quite specific for ABCG2 versus ABCB1 and ABCC1, and appear either non-competitive or partially competitive towards mitoxantrone efflux. Most compounds do not inhibit ATPase activity, and are assumed not to be transported themselves by the transporter. Some acridones, firstly optimized in vitro as potent inhibitors, are indeed efficient in vivo, against human xenografts in SCID mice, more efficiently than gefitinib taken as a control. Future developments should open the way to more efficient/targeted modulators including (i) the potential interest of bimodulation by combining two different inhibitors, (ii) computer-assisted ligand-based drug design for getting more potent and more specific inhibitors, (iii) structure-based drug design from ABCG2 molecular models allowing in silico screening and docking of new inhibitors.

Corticosteroid cross-reactivity: clinical and molecular modelling tools.

BACKGROUND: Corticosteroids have been classified into following four cross-reacting groups in function of their contact-allergenic properties: A, B, C and D, the last subdivided into D1 and D2. Recent data indicate that C(16)-methylated and nonmethylated molecules need to be distinguished, the latter selectively binding with arginine to form stable cyclic adducts and producing considerably more positive reactions than the former. This study compares molecular modelling and patch-test results to determine cross-reactivity patterns. METHODS: The patch-test results obtained with 66 corticosteroid molecules in 315 previously sensitized subjects were analysed and correlated with modelling and clustering in function of the electrostatic and steric fields of these molecules. RESULTS: The classification obtained after in silico hydrolysis of C(21) and C(17) esters was selected with an optimal cut into three clusters: the patients who reacted positively to cluster 2 (halogenated molecules from group B, with C(16)/C(17) cis ketal or diol structure) and cluster 3 (halogenated molecules from groups C and D1, C(16)-methylated) also reacted to cluster 1 (molecules mostly from groups A and D2, without C(16)-methyl substitution or halogenation and budesonide). The reverse, however, was not the case. CONCLUSION: Two patient profiles with probably different areas of immune recognition are identified as follows: the profile 1 patients were allergic to the frequently positively reacting cluster 1 only, for whom electrostatic fields (molecular charge) seem important; the profile 2 patients reacted to clusters 1 and 2 and/or 3, for whom steric fields (structure) are determinant and who probably presented a global recognition of the corticosteroid skeleton. A modified classification is thus proposed.

The euHCVdb suite of in silico tools for investigating the structural impact of mutations in hepatitis C virus proteins.

Hepatitis C is a viral infection of the liver that results in acute hepatitis and chronic liver disease, including cirrhosis and liver cancer. An estimated 170 million persons are chronically infected worldwide. The Hepatitis C virus is the pathogen agent responsible for hepatitis C. HCV is an enveloped RNA-positive virus of the flaviviridae family. The HCV genome shows remarkable sequence variability. This variability leads to the classification of HCV into 6 genotypes, numerous subtypes and HCV exists in each infected patient as quasi-species. The genotype may be linked to the severity of the disease and to the efficiency of the combination treatment with interferon and ribavirin. To date, no vaccine to prevent or cure HCV exists. Numerous HCV specific inhibitors have been designed and some are currently under clinical trials. However, resistances of HCV against these inhibitors have been identified. We developed the European Hepatitis C Virus Database (euHCVdb, http://euhcvdb.ibcp.fr/), a collection of functionally and structurally (3D-models) annotated HCV sequences integrated with sequence and structure analysis tools. We show below how the euHCVdb database is a useful in silico tool that can help drug design, combating resistance to drug treatment and understand structural biology of the HCV.

Interactions study between the copper II ion and constitutive elements of chitosan structure by DFT calculation.

Molecular modeling is particularly useful to understand interactions between various kinds of molecules and ions. This study is aimed at studying the interactions between one Cu(2+) ion and one or several glucosamine residues. The geometries and the interaction energies of all of the complexes involving all of the dimers obtained from glucosamine and N-acetylglucosamine were computed by means of density functional theory (DFT) methods. In a first step, for the two dimers A-A and A-B (A for glucosamine and B for N-acetyl glucosamine), a starting geometry was built, and the energies were calculated using a rigid rotation of 30 degrees intervals for each of the dihedral angles (Phi and Psi) of the glycosidic bond, spanning the whole angular range. These calculations allowed us to retrieve the minimal energy conformation and investigate all possible conformations. The results were compared to some experimental data. In a second step, we investigated the interactions of Cu(2+) with the different possible coordination sites of A. For all complexes considered, the Cu(2+) site was completed with H(2)O and/or OH(-) ligands to have a global neutral charge. The calculations confirmed that the most stable interactions involved the free amino site in a "pending complex". Another pending form was possible considering the participation of the heterocyclic O site, but the latter was less favored. On the other hand, we also showed that glucosamine could not act as a bidentate ligand and that N-acetyl glucosamine was not coordinating with Cu(2+). Finally, our results evidenced a cooperative fixation of Cu(2+) ions when considering the complexation of two successive metal ions on the two consecutive glucosamine residues of the dimer A-A.

Antileishmanial activity of polycyclic derivatives.

33 polycyclic derivatives have been studied and tested on Leishmania donovani and L. major promastigotes. Their antileishmanial activity was assessed in vitro and an assay of their cytotoxicity was realized on human myelomonocytic cell line. The reference molecules used in the assays were amphotericin B and pentamidine. Among the compounds tested, 29 possess an antileishmanial activity; 25 of those were more active against L. donovani than amphotericin B, and nine were as effective as amphotericin B against L. major. Many synthesized derivatives were more active against L. donovani than against L. major. The cytotoxicity studies have shown that among the thirty-three derivatives tested, 12 molecules have an IC50 towards THP-1 cells about equal than that reference drugs, the 21 other derivatives are much less toxic. A 3D QSAR study was undertaken and has permitted to predict activity against L. donovani and L. major and to highlight critical area to optimize activity against the two species.

Interaction of new PNA-based molecules with TAR RNA of HIV-1: molecular modelling and biological evaluation.

During the HIV-1 replication process, interactions between the RNA sequence, named TAR RNA, and the viral protein, Tat, permit a fast and efficient transcription of viral DNA into RNA. Based on the NMR structure of TAR RNA from the PDB, two Peptidic Nucleic Analog- (PNA) based molecules were designed by molecular modelling, the first one targeting G32 U31 and the second targeting U31 C30 free loop bases. Before designing the molecules, the flexibility of the TAR RNA was evaluated by molecular dynamics (MD). The molecules studied are composed of three domains: an arginine, a linker, and two PNA bases. First, molecules were designed and the linker length was optimized to fit the TAR RNA; second, a MD simulation on the TAR RNA molecule complex was performed to validate the molecular structure. Optimal molecules were synthesized and tested on infected cells. The experimental results support the choices made in the design of the molecules.

Amphiphilic anionic analogues of galactosylceramide: synthesis, anti-HIV-1 activity, and gp120 binding.

We describe the synthesis together with the results of anti-HIV-1 activity and gp120-monolayer binding experiments of new galactosyl amphiphiles, analogues of galactosylceramide, an alternative receptor used by HIV to infect CD4 negative cells. These compounds consist of single- and double-chain amphiphiles containing one or two galactose residues. To favor their clustering into galactosyl-rich microdomains, their molecular structure contains also an amino group or several hydroxyls or anionic groups, such as carboxylate, sulfate, sulfonate, and phosphate. Among the 12 new galactosylated compounds reported, a specific anti-HIV activity, although moderate (IC(50) from 10 to 50 microM), was detected only for three of them, i.e., I-GalSer[CO2Na][C14], II-GalSer[C14][C7SO3Na], and II-GalSer[C2SO4Na][C14], which contain an anionic group. The marked increase of surface pressure which was observed upon addition of gp120 into the aqueous subphase underneath the monolayers containing these galactolipids indicated gp120 insertion into the monolayers, suggesting that binding of these three derivatives to HIV-1 gp120 may be responsible for their anti-HIV activity.

Synthesis and antiviral activity of ethidium-arginine conjugates directed against the TAR RNA of HIV-1.

The regulatory protein Tat is essential for viral gene expression and replication of the human immunodeficiency virus type 1 (HIV-1). Tat transactivates the HIV-1 long terminal repeat (LTR) via its binding to the transactivation responsive element (TAR) and increases the viral transcription. Studies have shown that the binding of arginine and arginine derivatives induces a conformational change of the TAR RNA at the Tat-binding site. The unpaired A17 residue delimits a small cavity which constitutes a receptor site for small molecules, especially for ethidium bromide. These binding characteristics have prompted us to design a series of ethidium-arginine conjugates capable of interacting with the TAR RNA. Here we report the synthesis of six ethidium derivatives equipped with arginine side chains. These molecules were biologically evaluated, and two compounds (17 and 20) exhibited in vitro anti-HIV-1 activity at micromolar concentration, without toxicity (up to 100 microM concentration). Melting temperature studies indicated that the most active molecule (20) bound strongly to TAR in vitro. RNase protection experiments agreed with the molecular modeling studies which suggested that the ethidium moiety of 20 was inserted next to the A17 residue while the arginine side chain occupied the pyrimidine bulge.

Modeling of the interaction between new ethidium derivatives and TAR RNA of HIV-1.

During the HIV-1 replication process, interactions between the first sequence of RNA synthesized named TAR RNA and a viral protein named Tat permit a fast and efficient transcription of viral DNA in RNA. Based on the NMR structure of TAR RNA found on the PDB, new derivatives of ethidium were designed by molecular modeling to inhibit this interaction. The studied molecules are composed of three domains: an arginine, a linker, and an ethidium. Three linkers of different lengths were considered in the first step, with the TAR RNA-arginine interaction and the intercalation of the ethidium simulated by docking methods. In a second step, the structure of the TAR RNA was completed to obtain a whole ethidium interaction site and docking of the whole studied molecules was investigated. Molecules were synthesized and tested on infected cells. The predicted models and activity are in good agreement with the reported experimental results.