The inhibition process of HIV-1 integrase by diketoacids molecules: Understanding the factors governing the better efficiency of dolutegravir.

The Human Immunodeficiency Virus-1 integrase is responsible for the covalent insertion of a newly synthesized double-stranded viral DNA into the host cells, and is an emerging target for antivirus drug design. Raltegravir (RAL) and elvitegravir (EVG) are the first two integrase strand transfer inhibitors used in therapy. However, treated patients eventually develop detrimental resistance mutations.

A targeted DNA substrate mechanism for the inhibition of HIV-1 integrase by inhibitors with antiretroviral activity.

We recently reported that viral DNA could be the primary target of raltegravir (RAL), an efficient anti-HIV-1 drug, which acts by inhibiting integrase. To elucidate this mechanism, we conducted a comparative analysis of RAL and TB11, a diketoacid abandoned as an anti-HIV-1 drug for its weak efficiency and marked toxicity, and tested the effects of the catalytic cofactor Mg(2+) (5 mm) on drug-binding properties. We used circular dichroism and fluorescence to determine drug affinities for viral DNA long terminal repeats (LTRs) and peptides derived from the integrase active site and DNA retardation assays to assess drug intercalation into DNA base pairs.

Unprocessed viral DNA could be the primary target of the HIV-1 integrase inhibitor raltegravir.

Integration of HIV DNA into host chromosome requires a 3'-processing (3'-P) and a strand transfer (ST) reactions catalyzed by virus integrase (IN). Raltegravir (RAL), commonly used in AIDS therapy, belongs to the family of IN ST inhibitors (INSTIs) acting on IN-viral DNA complexes (intasomes). However, studies show that RAL fails to bind IN alone, but nothing has been reported on the behaviour of RAL toward free viral DNA.

Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target.

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F.

The HIV-1 integrase α4-helix involved in LTR-DNA recognition is also a highly antigenic peptide element.

Monoclonal antibodies (MAbas) constitute remarkable tools to analyze the relationship between the structure and the function of a protein. By immunizing a mouse with a 29mer peptide (K159) formed by residues 147 to 175 of the HIV-1 integrase (IN), we obtained a monoclonal antibody (MAba4) recognizing an epitope lying in the N-terminal portion of K159 (residues 147-166 of IN). The boundaries of the epitope were determined in ELISA assays using peptide truncation and amino acid substitutions.

HIV-1 integrase and virus and cell DNAs: complex formation and perturbation by inhibitors of integration.

HIV-1 integrase (IN) catalyzes integration of viral DNA into cell DNA through 3'-processing of viral DNA and strand transfer reactions. To learn on binding of IN to DNAs and IN inhibition we applied spectroscopy (circular dichroism, fluorescence) in a simplified model consisting in a peptide analogue (K156) of alpha4 helix involved in recognition of viral and cell DNA; an oligonucleotide corresponding to the U5' LTR DNA end; and an inhibitor (TB11) of the diketo acid (DKA) family. Results extrapolated to IN show that: the enzyme binds viral DNA with high affinity and specificity, but cell DNA with low affinity and specificity; the affinity of TB11 for IN is high enough to impair the binding of IN to cell DNA, but not to viral DNA.

Chirality in dynamic supramolecular nanotubes induced by a chiral solvent.

Amplification of chirality has been reported in polymeric systems. It has also been shown that related effects can occur in polymer-like dynamic supramolecular aggregates, if a subtle balance between noncovalent interactions allows the coupling between a chiral information and a cooperative aggregation process. In this context, we report a strong majority-rules effect in the formation of chiral dynamic nanotubes from chiral bisurea monomers.

Specificity of LTR DNA recognition by a peptide mimicking the HIV-1 integrase {alpha}4 helix.

HIV-1 integrase integrates retroviral DNA through 3'-processing and strand transfer reactions in the presence of a divalent cation (Mg(2+) or Mn(2+)). The alpha4 helix exposed at the catalytic core surface is essential to the specific recognition of viral DNA. To define group determinants of recognition, we used a model composed of a peptide analogue of the alpha4 helix, oligonucleotides mimicking processed and unprocessed U5 LTR end and 5 mM Mg(2+).

An unusual helix turn helix motif in the catalytic core of HIV-1 integrase binds viral DNA and LEDGF.

Background: Integrase (IN) of the type 1 human immunodeficiency virus (HIV-1) catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149-186) in the crystal structure of the catalytic core (CC) of the IN-Phe185Lys variant that consists of the alpha(4) and alpha(5) helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer.

Identification of intrinsic dynamics in a DNA sequence preferentially cleaved by topoisomerase II enzyme.

Topoisomerase II enzymes are essential enzymes that modulate DNA topology and play a role in chromatin compaction. While these enzymes appear to recognize and cleave the DNA in a nonrandom fashion, factors that underlie enzyme specificity remain an enigma. To gain new insights on these topics, we undertake, using NMR and molecular dynamics methods, studies of the structural and dynamic features of a 21 bp DNA segment preferentially cleaved by topoisomerases II.

C-->G base mutations in the CArG box of c-fos serum response element alter its bending flexibility. Consequences for core-SRF recognition.

By binding to the CArG box sequence, the serum response factor (SRF) activates several muscle-specific genes, as well as genes that respond to mitogens. The core domain of the SRF (core-SRF) binds as a dimer to the CArG box C-5C-4A-3T-2A-1T+1T+2A+3G+4G+5 of the c-fos serum response element (SREfos). However, previous studies using 20-mer DNAs have shown that the binding stoichiometry of core-SRF is significantly altered by mutations C-5-->G (SREGfos) and C-5C-4-->GG (SREGGfos) of the CArG box [A Huet, A Parlakian, M-C Arnaud, J-M Glandières, P Valat, S Fermandjian, D Paulin, B Alpert & C Zentz (2005) FEBS J272, 3105-3119].

Does topoisomerase II specifically recognize and cleave hairpins, cruciforms and crossovers of DNA?

DNA topoisomerase II is an enzyme that specializes in DNA disentanglement. It catalyzes the interconversion of DNA between different topological states. This event requires the passage of one duplex through another one via a transient double-strand break.

Conformations and dynamics of the phosphodiester backbone of a DNA fragment that bears a strong topoisomerase II cleavage site.

The dynamics of the DNA phosphodiester backbone conformations have been studied for a strong topoisomerase II cleavage site (site 22) using molecular dynamics simulations in explicit water and in the presence of sodium ions. We investigated the backbone motions and more particularly the BI/BII transitions involving the epsilon and zeta angles. The consensus cleavage site is adjacent to the phosphate which shows the most important phosphodiester backbone flexibility in the sequence.

Molecular modelling studies on the interactions of human DNA topoisomerase IB with pyridoxal-compounds.

Candida guilliermondii and human DNA topoisomerases I are inhibited by PL (pyridoxal), PLP (pyridoxal 5'-phosphate) and PLP-AMP (pyridoxal 5'-diphospho-5'-adenosine) (PL View Article and Find Full Text PDF

General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures.

(13)C, (15)N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies.

Assessment of adenyl residue reactivity within model nucleic acids by surface enhanced Raman spectroscopy.

We rank the reactivity of the adenyl residues (A) of model DNA and RNA molecules with electropositive subnano size [Ag]n+ sites as a function of nucleic acid primary sequences and secondary structures and in the presence of biological amounts of Cl- and Na+ or Mg2+ ions. In these conditions A is markedly more reactive than any other nucleic acid bases. A reactivity is higher in ribo (r) than in deoxyribo (d) species [pA>pdA and (pA)n>>(pdA)n].

Self-association and DNA binding properties of the human topoisomerase IIA alpha2HTH module.

The eukaryotic topoisomerase II is an ubiquitous nuclear enzyme involved in vital cellular functions. It is also the target for some of the most active anticancer drugs. In the various crystal structures of yeast topoisomerase II, the 701-748 segment homologous to the human topoisomerase II alpha 724-771 segment folds into a compact alpha(2)beta(1)alpha(3)talpha(4) conformation, hereafter termed alpha(2)HTH module (helix turn helix (HTH), alpha(3)talpha(4)).

Inhibitory effect of fucoidan on the adhesion of adenocarcinoma cells to fibronectin.

Fucoidans inhibit tumour cell adhesion to various substrata, but their mechanisms of action are not fully understood. Using 3H-fucoidan, we observed that fucoidan binds to fibronectin, this binding being saturable and sensitive to ionic strength and pH. The interaction occurred on at least four different sites along the polypeptide chain, two of them being the heparin-binding sequences.

A structural study of model peptides derived from HIV-1 integrase central domain.

The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known.

Mechanism of binding of serum response factor to serum response element.

Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle-specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core-SRF (the 124-245 fragment of serum response factor) with specific oligonucleotides from c-fos and desmin promoters, were determined by spectroscopy.

Structural and functional characterizations reveal the importance of a zinc binding domain in Bloom's syndrome helicase.

Bloom's syndrome (BS) is an autosomal recessive human disorder characterized by genomic instability and a predisposition to a wide variety of cancers. The gene mutated in BS, BLM, encodes a protein containing three domains: an N-terminal domain whose function remains elusive, a helicase domain characterized by seven 'signature' motifs conserved in a wide range of helicases and a C-terminal extension that can be further divided into two sub-domains: RecQ-Ct and HRDC. The RecQ-Ct domain appears essential because two point-mutations altering highly conserved cysteine residues within this domain have been found in BS patients.

Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase.

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3'-processing [dinucleotide released from each 3' end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance.

Structure-based virtual screening: an application to human topoisomerase II alpha.

The eukaryotic topoisomerase II is involved in several vital processes, such as replication, transcription, and recombination. Many compounds interfering with the catalytic action of this enzyme are efficient in human cancer chemotherapy. We applied a methodology combining molecular modeling and virtual screening techniques to identify human topoisomerase II alphainhibitors.

Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid.

The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the epsilon-amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (K(i) = 40 microM) that blocks the cleavable complex formation.