Biomedical subjects

F Hamy

Publications and source records attributed to F Hamy.

At least 19 recordsLinked to original sources

PubMed2000

The chromosomal protein HMG-D binds to the TAR and RBE RNA of HIV-1.

The high mobility group protein HMG-D is known to bind preferentially to DNA of irregular structures with little or no sequence specificity. Upon binding to DNA, this HMG-box protein widens the minor groove of the double helix and induces a significant bending of the helix. We show here that HMG-D can strongly bind to double-stranded RNA. Electrophoretic mobility shift assays show that HMG-D100 interacts with the transactivation response region (TAR) RNA from HIV-1. Strong interaction with a high affinity Rev protein binding element (RBE) RNA was also characterized. Gel shift experiments performed with several TAR RNA constructs lacking the lateral pyrimidine bulge or with modified apical loop regions indicate that the protein does not recognize the single-strand domains of the RNA but apparently interacts directly with the double-stranded stem regions. No protein-RNA complexes could be detected when using single-stranded oligoribonucleotides. HMG-D protein could bind to the wide minor groove of the A-form TAR RNA. The comparison of the amino acid sequence of HMG-D with that of known RNA binding proteins suggests that the interaction of the protein with a double-stranded RNA implicates the basic region of HMG-D as well as its HMG-box domain. From the in vitro data reported here, we propose a novel functional role for proteins of the HMG-1 family. The results suggest that architectural HMG proteins can be recruited by double-stranded RNA for the development of HIV-1 in the host cell.

Base Sequence↗

PubMed2000

Retinoic acid switches differential expression of FGF8 isoforms in LNCaP cells.

Retinoic acid (RA) is described as an inhibitor of prostate cancer cell growth. We utilized reverse transcription-polymerase chain reaction (RT-PCR) to analyze expression of different isoforms of fibroblast growth factor 8 (FGF8) in response to RA. Results in the prostate cancer cell line LNCaP show that whereas overall expression levels of FGF8 appear to remain constant, RA addition induces an inversion of the ratio between FGF8a and -b mRNAs. Along with this observed "isoform switch," unexpected expression of retinoic acid receptor alpha was detected. Although preliminary, these data allow one to hypothesize on the existence of a possible link between the morphogenic hormone RA and the regulation of the potent mitogen FGF8.

Base Sequence↗

PubMed2000

An ARE-selective DNA minor groove binder from a combinatorial approach.

A synthetic combinatorial library of 10,000 components mostly containing aromatic amino acids was screened for inhibition of DNase I cleavage at two ARE sequences. Ten amino acid building blocks were used to generate the library in which the N and C terminal residues were fixed and the four central positions of the peptide ligands were varied. The DNase I footprinting assay led, after deconvolution through sublibrary synthesis, to the identification of CGL-6382 as an ARE-selective minor groove binder containing a N-terminal nicotinic acid motif adjacent to a N-methylimidazole unit and three N-methylpyrrole units coupled to a C-terminal argininamide residue. The optimized ligand CGL-6382 was found to recognize a 5'-GC(A/T)(A/T) motif within the two cloned androgen receptors responsive elements. The discovery of CGL-6382 as an ARE-selective ligand augurs well for the use of the DNase I footprinting methodology to identify sequence-specific DNA recognition ligands from large mixtures of small molecules.

Base Sequence↗

PubMed2000

Blocking HIV replication by targeting Tat protein.

BACKGROUND: A rapid development of viral drug resistance poses a serious limitation in the current drug development programs against HIV. In turn, this obstacle forms the basis for new efforts, which utilize alternative viral targets. RESULTS: By aiming at the Tat-driven process of HIV gene regulation, we discovered a new class of compounds as well as a novel target. The candidate compound acts on the one hand by classically inhibiting Tat/TAR complexation, however, without binding to nucleic acids. CONCLUSIONS: Structure and molecular modeling/dynamics suggest that the stilbene derivative CGA137053 directly binds to Tat protein but not TAR RNA. As a completely new, second property, the compound also antagonizes a TAR-independent activity of free Tat protein by preventing the recently described upregulation of the HIV coreceptor CXCR4. With the stilbene CGA137053, we have identified a potent, double-hitting and chemically feasible Tat antagonist. The compound possesses high target specificity and low cytotoxicity, is not restricted to the Tat/TAR axis of HIV inhibition and highly active on HIV-infected, primary human cells.

Amides↗

PubMed2000

A second target for the peptoid Tat/transactivation response element inhibitor CGP64222: inhibition of human immunodeficiency virus replication by blocking CXC-chemokine receptor 4-mediated virus entry.

The peptoid CGP64222 has been previously demonstrated to inhibit the human immunodeficiency virus (HIV) Tat/transactivation response element complex formation. It has previously been shown that CGP64222 selectively inhibits HIV-1 long terminal repeat-driven gene expression and HIV-1(LAV) replication in lymphocytes. Here, we show that CGP64222 inhibits the replication of a wide range of laboratory strains of HIV-1 and HIV-2 in MT-4 cells. However, CGP64222 proved inactive in MT-4 cells against HIV-1 strains that are resistant to the bicyclams. The bicyclams are known to specifically interact with CXC-chemokine receptor 4, the main coreceptor used by T-tropic HIV strains to enter the cells. Mechanism of action studies revealed that CGP64222 can inhibit the HIV replicative cycle, also through a selective interaction with the CXC-chemokine receptor 4 coreceptor.

Anti-HIV Agents↗

PubMed1999

The camptothecin-resistant topoisomerase I mutant F361S is cross-resistant to antitumor rebeccamycin derivatives. A model for topoisomerase I inhibition by indolocarbazoles.

DNA topoisomerase I is a major cellular target for antitumor indolocarbazole derivatives (IND) such as the antibiotic rebeccamycin and the synthetic analogue NB-506 which is undergoing phase I clinical trials. We have investigated the mechanism of topoisomerase I inhibition by a rebeccamycin analogue, R-3, using the wild-type human topoisomerase I and a well-characterized recombinant enzyme, F361S. The catalytic activity of this mutant remains fully intact, but the enzyme is resistant to inhibition by camptothecin (CPT). Here we show that the mutated enzyme is cross-resistant to the rebeccamycin analogue. Despite their profound structural differences, CPT and R-3 interfere similarly with the activity of the wild-type and mutant topoisomerase I enzymes, and the drug-induced cleavable complexes are equally sensitive to the NaCl concentration. CPT and IND likely recognize identical structural elements of the topoisomerase I-DNA covalent complex; however, differences do exist in terms of sequence-specificity of topoisomerase I-mediated DNA cleavage. For the first time, a molecular model showing that CPT and IND share common steric and electronic features is proposed. The model helps to identify a specific pharmacophore for topoisomerase I inhibitors.

Amino Acid Substitution↗

PubMed1999

Molecular basis of HIV-1 TAR RNA specific recognition by an acridine tat-antagonist.

We investigated the interaction of a highly potent acridine-based tat-antagonist with the TAR RNA of HIV-1. The wild type TAR RNA and three mutants with U-->C23, G x C-->C x G26-39 or G x C-->A x U26-39 substitutions were used as substrates to study the molecular basis of drug-TAR RNA complex formation. Melting temperature and RNase protection experiments reveal that the G x C26-39 pair is a critical element for specific major groove recognition of TAR at the pyrimidine bulge. The results provide a rational basis for future design of optimized tat/TAR inhibitors.

Acridines↗

PubMed1999

Inhibition of HIV-1 Tat-TAR interaction by diphenylfuran derivatives: effects of the terminal basic side chains.

A series of four biscationic diphenylfuran derivatives was used to investigate drug binding to the transactivation response element (TAR) RNA. The drugs, which are active against the Pneumocystis carinii pathogen (PCP), differ by the nature of the terminal basic side chains. Furimidazoline (DB60) is more potent at inhibiting binding of the Tat protein to TAR than furamidine (DB75) and the amidine-substituted analogues DB244 and DB226. In vivo studies using the fusion-induced gene stimulation (FIGS) assay entirely agree with the in vitro gel mobility shift data. The capacity of the drugs to antagonize Tat binding correlates with their RNA binding properties determined by melting temperature and RNase protection experiments. Footprinting studies indicate that the bulge region of TAR provides the identity element for the diphenylfurans. Access of the drugs to the major groove cavity at the pyrimidine bulge depends on the bulk of the alkylamine substituents. Experiments using TAR mutants show that the bulge of TAR is critical for drug binding but also reveal that the fit of the drugs into the major groove cavity of TAR does not involve specific contacts with the highly conserved residue U23 or the C x G26-39 base pair. The binding essentially involves shape recognition. The results are also discussed with respect to the known activity of the drug against PCP which is the major cause of mortality in AIDS patients. This study provides guidelines for future development of TAR-targeted anti-HIV-1 drugs.

Amidines↗

PubMed1998

TAR-RNA binding by HIV-1 Tat protein is selectively inhibited by its L-enantiomer.

An oligoribonucleotide, corresponding to the Tat-interactive top half of the HIV-1 TAR RNA stem-loop, was synthesized in both the natural D- and the enantiomeric L-configurations. The affinity of Tat for the two RNAs, assessed by competition binding experiments, was found to be identical and is reduced 10-fold for both, upon replacement of the critical bulge residue U23 with cytidine. It is suggested that this interaction of the flexible Tat protein depends strongly upon the tertiary structure of a binding pocket within TAR, but not upon its handedness, and may be described by a 'hand-in-mitten' model.

Binding, Competitive↗

PubMed1998

A new class of HIV-1 Tat antagonist acting through Tat-TAR inhibition.

The main transcriptional regulator of the human immunodeficiency virus, the Tat protein, recognizes and binds to a small structured RNA element at the 5' end of every viral mRNA, termed TAR. On the basis of published structural data of the molecular interactions between TAR and Tat-related peptides, we defined requirements for potential low-molecular weight inhibitors of TAR recognition by the Tat protein. In accordance with the resulting concept, a series of compounds was synthesized. In vitro evaluation of their potential to directly interfere with Tat-TAR interaction was used to define a new chemical class of potent Tat antagonistic substances. The most active compound competed with Tat-TAR complexation with a competition dose CD50 of 22 nM in vitro and blocked HIV expression in a cellular Tat transactivation system with an IC50 of 1.2 microM. The close relation between structural features of the interaction between TAR and a new type of inhibitory agent, "In-PRiNts" (for inhibitor of protein-ribonucleotide sequences), such as CGP 40336A and those of the Tat-TAR complex was confirmed by RNase A footprinting and by two-dimensional NMR. Structural implications for the complex between this class of compounds and TAR RNA will be presented.

Acridines↗

PubMed1997

Binding of Hoechst 33258 to the TAR RNA of HIV-1. Recognition of a pyrimidine bulge-dependent structure.

The transactivation response region (TAR) RNA is an essential component in transcriptional regulation of the human immunodeficiency virus type-1 (HIV-1) genome. We have examined the interaction between TAR RNA and the bisbenzimidazole derivative Hoechst 33258. Previous studies have shown that this drug, which is well known as an AT-selective DNA minor groove binder, can also interact with GC-rich sequences in DNA as well as with RNA, possibly by intercalation. Absorption spectroscopy, circular dichroism and electric linear dichroism, as well as RNase A footprinting, were employed to compare binding of Hoechst 33258 to wild-type RNA and its analogue lacking the pyrimidine bulge. The uridine bulge, which is an important contributor to the structural stability of TAR, plays an essential role in drug binding. Deletion of the bulge destabilizes both free and drug-bound forms of TAR and markedly affects the orientation of the drug chromophore complexed with the RNA. According to the linear dichroism data, the bisbenzimidazole is oriented more or less perpendicular to the RNA helix axis. The data are compatible with a model in which the bisbenzimidazole chromophore is inserted into the existing cavity created by the pyrimidine bulge. The footprinting experiments, showing that the drug binds to a unique site opposite the unpaired uridine residues, also support this model. The binding of Hoechst 33258 to the sequence 5'-GCUCU, which delimits the cavity, reflects the greater accessibility of that region compared with other sites in the RNA molecule. The identification of a binding site for small molecules in TAR offers promising perspectives for developing drugs that would block the access of TAR RNA to proteins and therefore for the design of anti-HIV agents.

Absorption↗

PubMed1997

An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication.

One of the first steps in HIV gene expression is the recruitment of Tat protein to the transcription machinery after its binding to the RNA response element TAR. Starting from a pool of 3.2 x 10(6) individual chemical entities, we were able to select a hybrid peptoid/peptide oligomer of 9 residues (CGP64222) that was able to block the formation of the Tat/TAR RNA complex in vitro at nanomolar concentrations. NMR studies demonstrated that the compound binds similarly to polypeptides derived from the Tat protein and induces a conformational change in TAR RNA at the Tat-binding site. In addition, 10-30 microM CGP64222 specifically inhibited Tat activity in a cellular Tat-dependent transactivation assay [fusion-induced gene stimulation (FIGS) assay] and blocked HIV-1 replication in primary human lymphocytes. By contrast, peptides of a comparable size and side-chain composition inhibited cell fusion in the FIGS assay and only partially inhibited HIV-1 replication in primary human lymphocytes. Thus, we have discovered a compound, CGP64222, that specifically inhibits the Tat/TAR RNA interaction, both in vitro and in vivo.

Anti-HIV Agents↗

PubMed1996

The binding mode of drugs to the TAR RNA of HIV-1 studied by electric linear dichroism.

For the first time, the interaction between a series of small molecules and the TAR RNA of HIV-1 has been investigated by electric linear dichroism (ELD). The compounds tested include the DNA intercalating drugs proflavine and ethidium bromide and an amsacrine-4-carboxamide DNA-threading intercalator as well as the AT-specific DNA minor groove binders netropsin, Hoechst 33258, berenil and DAPI. In all cases except for netropsin, negative reduced dichroism signals were measured in the drug absorption band. In agreement with previous studies, the results indicate that both classical and threading intercalation can occur with the TAR RNA. The ELD data show that the mode of binding of the drugs Hoechst 33258, berenil and DAPI to the TAR RNA is similar to their binding mode in GC-rich regions of DNA and likely involves intercalation into the A-form TAR RNA helix. The wide and shallow minor groove of the TAR RNA is apparently not accessible to DNA minor groove binding drugs such as netropsin. The ELD technique appears uniquely valuable as a means of investigating the interaction of drugs with the TAR RNA.

Antiviral Agents↗

PubMed1996

Cooperativity in the binding of echinomycin to DNA fragments containing closely spaced CpG sites.

Quantitative footprinting has been used to investigate cooperative binding of the antitumor antibiotic echinomycin to DNA fragments containing closely spaced CpG steps. The sequences of the designed DNA fragments contained two pairs of strong echinomycin binding sites: a pair of ACGT sites together with an ACGT site and a TCGA site, either directly adjacent or separated by two or four A.T base pairs. The results demonstrate that the binding of echinomycin to the sequences ACGTACGT and TCGAACGT is highly cooperative. The extent of cooperativity depends on the nature of the sequences clamped by the antibiotic and diminishes as the distance between the binding sites is increased. Various methods of extracting the information necessary to establish cooperativity have been compared. Beyond the specific interest in echinomycin-DNA interaction, the present quantitative footprinting study provides a model that may be generally applicable for designing investigations into cooperativity in drug-DNA recognition.

Antibiotics, Antineoplastic↗

PubMed1994

Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins.

The HIV-1 regulatory proteins tat and rev are both RNA binding proteins which recognize sequences in duplex RNA which are close to structural distortions. Here we identify phosphate contacts which are critical for each binding reaction by use of a new method. Model RNA binding sites are constructed carrying substitutions of individual phosphodiesters by uncharged methylphosphonate derivatives isolated separately as Rp and Sp diastereoisomers and tested for protein binding by competition assays. In the binding of tat to the trans-activation response region (TAR), three phosphates, P21 and P22 which are adjacent to the U-rich bulge and P40 on the opposite strand, are essential and in each case both isomers inhibit binding. Similarly, in the interaction between the HIV-1 rev protein and the rev-responsive element (RRE) both methylphosphonate isomers at P103, P104, P124 and P125 interfere with rev binding. At P106, only the Rp methylphosphonate isomer is impaired in rev binding ability and it is proposed that the Rp oxygen is hydrogen-bonded to an uncharged amino acid or to a main chain hydrogen atom. Synthetic chemistry techniques also provide evidence for the conformations of non-Watson-Crick G106:G129 and G105:A131 base-pairs in the RRE 'bubble' structure upon rev binding. Almost all functional groups on the 5 bulged residues in the bubble have been ruled out as sites of contact with rev but, by contrast, the N7-positions of each G residue in the flanking base-pairs are identified as sites of likely hydrogen-bonding to rev. The results show that both tat and rev recognize the major groove of distorted RNA helixes and that both proteins make specific contacts with phosphates which are displaced from the sites of base-pair contact.

Base Composition↗

PubMed1994

Localized chemical reactivity in DNA associated with the sequence-specific bisintercalation of echinomycin.

Four complementary footprinting and probing techniques utilizing DNAse I, methidiumpropyl EDTA (MPE).FeII, diethyl pyrocarbonate (DEPC) and KMnO4 as DNA-cleaving or DNA-modifying agents have been applied to investigate the sequence-specific binding to DNA of the antitumour antibiotic echinomycin. A 265 bp EcoRI-PvuII DNA restriction fragment excised from plasmid pBS was used as a substrate. Six regions of protection against DNAase I cleavage were located on the 265-mer: three sites encompass the sequences 5'-TCGA or 5'-GCGT and the three others contain 5'-GpG (CpC) dinucleotide sequences where the inhibition of DNAase I cutting by echinomycin is less pronounced. In contrast, MPE.FeII cleavage allows identification of only three echinomycin-binding sites on the 265-mer: two sites contain the sequence 5'-TCGA and one encompasses the sequence 5'-ACCA. Cleavage of DNA by MPE.FeII in the presence of echinomycin remains practically unaffected at the sequence 5'-GCGT, despite its identification by DNAase I as a strong site for binding the antibiotic, as well as at the two other sequences containing GpG steps. With both DNAase I and MPE.FeII, enhanced DNA cleavage is evident at AT-rich sequences in the presence of echinomycin. Enhanced reactivity towards KMnO4 and DEPC provides clear evidence for sequence-dependent conformational changes in DNA induced by the antibiotic. The experiments reveal that KMnO4 reacts most strongly with thymines located around, but not necessarily adjacent to, an echinomycin-binding site, whereas the carbethoxylation reactions caused by DEPC occur primarily at the adenine residues lying immediately 5' or 3' to the dinucleotide that denotes an echinomycin-binding site. The results reported here demonstrate that DEPC and KMnO4 serve as sensitive probes for different states of the DNA helix. It seems that the reaction with KMnO4 involves transient unstacking events, whereas the carbethoxylation reaction of DEPC requires larger-scale helix opening.

Base Sequence↗

PubMed1993

High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region.

The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA (K1) was competed against 3H-labelled mutant TAR RNA (K2). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1.07 +/- 0.05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26.C39 and A27.U38 reduced that affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22.U40 to U.A reduced tat affinity 5-fold, and mutation of G21.C41 to C.G reduced tat affinity 4-fold. The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23-->C mutation in the bulge, or G26.G39-->C.G mutation in the stem of TAR RNA. By contrast, the ADP-I (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23-->C or G26.C39-->C.G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively. Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compare the binding properties of the tat protein and the tat-derived peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.

Base Sequence↗