Computational and synthetic approaches for the discovery of HIV-1 integrase inhibitors

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Non-perturbative renormalisation and improvement of non-singlet tensor currents in Nf=3N_\mathrm{f}=3 QCD

Hadronic matrix elements involving tensor currents play an important r\^ole in decays that allow to probe the consistency of the Standard Model via precision lattice QCD calculations. The non-singlet tensor current is a scale-dependent (anomalous) quantity. We fully resolve its renormalisation group (RG) running in the continuum by carrying out a recursive finite-size scaling technique. In this way ambiguities due to a perturbative RG running and matching to lattice data at low energies are eliminated. We provide the total renormalisation factor at a hadronic scale of 233 MeV, which converts the bare current into its RG-invariant form. Our calculation features three flavours of O(a) improved Wilson fermions and tree-level Symanzik-improved gauge action. We employ the (massless) Schr\"odinger functional renormalisation scheme throughout and present the first non-perturbative determination of the Symanzik counterterm $c_\mathrm{T}$ derived from an axial Ward identity. We elaborate on various details of our calculations, including two different renormalisation conditions.Comment: 39 pages, 10 figures, 11 tables

One-step isolation and biochemical characterization of a highlyactive plant PSII monomeric core

We describe a one-step detergent solubilization protocol for isolating a highly active form of Photosystem II (PSII) from Pisum sativum L. Detailed characterization of the preparation showed that the complex was a monomer having no light harvesting proteins attached. This core reaction centre complex had, however, a range of low molecular mass intrinsic proteins as well as the chlorophyll binding proteins CP43 and CP47 and the reaction centre proteins D1 and D2. Of particular note was the presence of a stoichiometric level of PsbW, a low molecular weight protein not present in PSII of cyanobacteria. Despite the high oxygen evolution rate, the core complex did not retain the PsbQ extrinsic protein although there was close to a full complement of PsbO and PsbR and partial level of PsbP. However, reconstitution of PsbP and PsbPQ was possible. The presence of PsbP in absence of LHCII and other chlorophyll a/b binding proteins confirms that LHCII proteins are not a strict requirement for the assembly of this extrinsic polypeptide to the PSII core in contrast with the conclusion of Caffarri et al. (2009)

Molecular dynamics studies of the full-length integrase-DNA complex

We have carried out a molecular dynamics (MD) simulation of full-length HIV-1 integrase (IN) dimer complexed with viral DNA with the aim of gaining information about the enzyme motion and investigating the movement of the catalytic flexible loop (residues 140-149) thought to be essential in the catalytic mechanism of IN. During the simulation, we observed quite a different behavior of this region in the presence or absence of the viral DNA. In particular, the MD results underline the crucial role of the residue Tyr143 in the mechanism of integration of viral DNA into the host chromosome. The present findings confirm the experimental data (e.g., site-directed mutagenesis experiments) showing that the loop is involved in the integration reactions and its mobility is correlated with the catalytic, activity of HIV-1 integrase

Molecular Dynamics Studies of the Wild-Type and Double Mutant HIV-1 Integrase Complexed with the 5CITEP Inhibitor: Mechanism for Inhibition and Drug Resistance

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the virus and is an attractive target for the development of new drugs useful in acquired immunodeficiency syndrome multidrug therapy. Starting from the crystal structure of the 5CITEP inhibitor bound to the active site in the catalytic domain of the HIV-1 IN, two different molecular dynamics simulations in water have been carried out. In the first simulation the wild-type IN was used, whereas in the second one the double mutation T66I/M154I, described to lead to drug resistance, was introduced in the protein. Compelling differences have been observed in these two structures during analyses of the molecular dynamics trajectories, particularly in the inhibitor binding modes and in the conformational flexibility of the loop (residues 138–149) located near the three catalytic residues in the active site (Asp(64), Asp(116), Glu(152)). Because the conformational flexibility of this region is important for efficient biological activity and its behavior is quite different in the two models, we suggest a hypothetical mechanism for the inhibition and drug resistance of HIV-1 IN. These results can be useful for the rational design of more potent and selective integrase inhibitors and may allow for the design of inhibitors that will be more robust against known resistance mutations

Analysis of the full-length integrase DNA complex by a modified approach for DNA docking

A model of the full-length HIV-1 integrase dimer was constructed assembling the experimentally determined structures of the single domains. Subsequently, the three-domain protein-viral DNA complex was generated for the first time through an automated docking algorithm, obtained modifying the ESCHER program, a well-known method for protein-protein docking. A detailed study of the contacts established with DNA by the enzyme revealed that the predicted model reproduced the results of mutagenesis and cross-linking experiments, confirming the validity of our docking approach in predicting the base specificity in the DNA-protein interaction