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. Here, we assessed whether free viral DNA could be a primary target for RAL, assuming that the DNA molecule is a receptor for a huge number of pharmacological agents. Optical spectroscopy, molecular dynamics and free energy calculations, showed that RAL is a tight binder of both processed and unprocessed LTR (long terminal repeat) ends. Complex formation involved mainly van der Waals forces and was enthalpy driven. Dissociation constants (Kds) revealed that RAL affinity for unbound LTRs was stronger than for bound LTRs. Moreover, Kd value for binding of RAL to LTRs and IC50 value (half concentration for inhibition) were in same range, suggesting that RAL binding to DNA and ST inhibition are correlated events. Accommodation of RAL into terminal base-pairs of unprocessed LTR is facilitated by an extensive end fraying that lowers the RAL binding energy barrier. The RAL binding entails a weak damping of fraying and correlatively of 3′-P inhibition. Noteworthy, present calculated RAL structures bound to free viral DNA resemble those found in RAL-intasome crystals, especially concerning the contacts between the fluorobenzyl group and the conserved 5′C4pA33′ step. We propose that RAL inhibits IN, in binding first unprocessed DNA. Similarly to anticancer drug poisons acting on topoisomerases, its interaction with DNA does not alter the cut, but blocks the subsequent joining reaction. We also speculate that INSTIs having viral DNA rather IN as main target could induce less resistance

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. The epitope in K159 or as a free peptide (pep-a4) was mostly a random coil in solution, while in the CCD (catalytic core domain) crystal, the homologous segment displayed an amphipathic helix structure (α4-helix) at the protein surface. Despite this conformational difference, a strong antigenic crossreactivity was observed between pep-a4 and the protein segment, as well as K156, a stabilized analogue of pep-a4 constrained into helix by seven helicogenic mutations, most of them involving hydrophobic residues. We concluded that the epitope is freely accessible to the antibody inside the protein and that its recognition by the antibody is not influenced by the conformation of its backbone and the chemistry of amino acids submitted to helicogenic mutations. In contrast, the AA →Glu mutations of the hydrophilic residues Gln148, Lys156 and Lys159, known for their interactions with LTRs (long terminal repeats) and inhibitors (

CONSTANTES DE COUPLAGE 13C-13C. CONFORMATION DES ACIDES AMINÉS, DES DIPEPTIDES ET TRIPEPTIDES

L'enrichissement à 85 % en carbone 13 de l'alanine, de la valine, de la leucine et de l'isoleucine a conduit à leur étude conformationnelle par la Résonance Magnétique Nucléaire du carbone 13. Dans ces conditions, les constantes de couplage 13C-13C ont pu être mesurées à différents pH. Elles rendent compte d'une part, des degrés d'ionisation et de protonation des groupements C-terminaux, et d'autre part mettent en évidence l'influence spatiale des groupements Net C-terminaux sur les chaînes latérales apolaires. L'introduction d'un acide aminé enrichi dans les peptides peut servir à leur marquage. Ainsi l'étude de la série H2N—Gly—Pro—COOH, Gly—Pro, et H2N—Gly—Pro—Gly—COOH, HH—CO par les mêmes techniques et méthodes met en évidence la présence du résidu prolyl sous ses deux formes cis et trans dans les peptides linéaires, alors que dans la dicétopipérazine seule la forme cis existe. Si l'influence du pH s'exerce sur les proportions des conformations cis et trans dans le cas du dipeptide linéaire, elle est faible dans le cas du tripeptide et nulle pour le dipeptide cyclique. Ces résultats ont été confirmés par ceux de la Résonance Magnétique du proton. L'influence du pH a été également examinée sur les peptides Gly-*Leu et Gly-*Leu-Gly. Dans ces deux cas la présence de plusieurs conformères n'a pu être mise en évidence.85 % 13C-enriched alanine, valine, leucine, isoleucine and proline were studied with 13C-NMR technique in terms of conformation. The l3C-13C coupling constants were measured at several pH ; these data were useful for, on one side to estimate the various degrees of ionisation or protonation of the C-terminal groupement, and on the other side to evidenced the spatial influence of the N- and C-terminal groups on the apolar side chains. The insertion of one 13C- enriched aminoacid in peptides such as H2N—Gly—Pro—COOH, Gly—Pro, and H2N—Gly—Pro—Gly—COOH, HH—CO simplified their spectra and allowed us to determine the presence of two conformations (cis and trans) for the linear peptides, whereas in the case of the diketopiperazine only the cis form existed. Whether the pH effect modified the proportions of the prolyl cis and trans conformations in the case of the linear dipeptide, this effect is weak for the tripeptide and no for the cyclic dipeptide. All these results were confirmed with H-NMR technique. When the pH effect is examined on the Gly-*Leu and Gly-*Leu-Gly peptides, only one conformation was evidenced in the medium