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Virtual screening of HIV-1 protease inhibitors identify the potential binding sites of the inhibitors by against human cytomegalovirus protease using generating a grid box that is big enough to cover the entire surface of the protein. The protein-inhibitor complexes derived from the first ranked docking solutionin the preliminary docking procedure were consequentlysolvated in a TIP3-water shell, and all atoms were allowed The clearance of cytomegalovirus viraemia in HIV- to relax using molecular dynamics simulation. The 1-infected patients may partly result from the molecular dynamics simulation was carried out using the inhibition of cytomegalovirus protease by HIV-1 NAMD software version 2.5b18 [13]. The topology and protease inhibitors contained in highly active anti- parameters for each inhibitor were obtained from the retroviral therapy. We used a computational PRODRG server [14]. One hundred steps of energy method to calculate the binding affinity of six minimization of the protein–inhibitor–water complex HIV-1 protease inhibitors to cytomegalovirus pro- were initially performed, followed by 0.1 picoseconds of tease based on its X-ray crystallography structure.
molecular dynamics simulation at 300 K. The simulations The calculations showed that amprenavir and were repeated with three different starting seeds. The indinavir occupy the substrate-binding site of the trajectories at 0.1 picoseconds were recorded and cytomegalovirus protease with high affinity, and processed in a second docking step using similar docking may be implicated in alleviating cytomegalovirus parameters as used in the preliminary docking procedure.
The primary exception was in the creation of a three-dimensional affinity grid box, in which the C-a atom of Cytomegalovirus is an AIDS-related opportunistic Ser132 of the catalytic triad was set as a grid center, and pathogen that usually infects HIV-1 patients with a high the number of grid points in the x, y, z axes was set to level of plasma HIV-1 RNA and low CD4 cell counts (< 200 cells/ml) [1–3]. Highly active antiretroviraltherapy (HAART), consisting of HIV-1 protease and AutoDock generates three energy terms: intermolecular reverse transcriptase inhibitors, has been shown to lower energy, internal energy of the ligand, and torsional free plasma HIV-1-RNA levels and elevate CD4 cell counts, energy. The final docked energy was calculated from the and is associated with a reduction in cytomegalovirus sum of the intermolecular energy and the internal energy replication and the clearance of cytomegalovirus viraemia of the ligand. The free energy of binding was calculated [4–11]. Reports from several groups have shown that from the sum of the intermolecular and the torsional free immune recovery that results from HAART without any energies, and consequently converted into an inhibitory specific anti-cytomegalovirus therapy is able to suppress constant (Ki) according to Hess’s law. The lowest-energy cytomegalovirus infection in HIV-1-infected patients solution was accepted as the calculated binding energy [8–11]. However, it is unresolved as to whether HIV-1 and its Ki value was used to define the binding affinity of protease inhibitors aid the clearance of cytomegalovirus the inhibitors. Further details of the molecular dynamics viraemia by inhibiting cytomegalovirus protease activity.
simulation and docking protocols are given elsewhere[15,16].
In this study, we used an integrated molecular dynamicssimulation and docking method to calculate the ability of Structural studies of the cytomegalovirus protease show six US Food and Drug Administration approved HIV-1 that it belongs to the serine protease family, with a novel protease inhibitors to bind to the cytomegalovirus Ser132–His63–His157 catalytic triad, with His157 protease in terms of binding mode and binding energy.
representing the third member instead of the typical The X-ray crystallography structures of cytomegalovirus Asp [17]. The substrate-binding site is composed of protease and HIV-1 protease inhibitors were retrieved several subsites: the S1 subsite is formed by residues from the Protein Data Bank (codes: 1NKM for Leu32, Ser132, Leu133, Arg165 and Arg166. The S2 and S4 subsites are fused together, forming a large pocket with 1HSG for indinavir, 1MUI for lopinavir, 1OHR for residues His63 and Asp64 on one side, Ser135 on the nelfinavir, 1HXW for ritonavir and 1C6Z for saquinavir).
other, and Lys156 in the middle. The S3 portion of thispocket is formed by salt bridges between residues Glu31, Docking calculations were carried out using AutoDock Ser135, Arg137, Arg165 and Arg166 [17]. Theoretically, version 3.0.5 with a Lamarckian genetic algorithm [12].
enzymatic activity would be significantly diminished if We first performed preliminary docking experiments to the catalytic triad, or part of the substrate-binding sites, ISSN 0269-9370 Q 2005 Lippincott Williams & Wilkins Table 1. Calculated energies and inhibitory constants (Ki) of six Food and Drug Administration approved HIV-1 protease inhibitors against thecytomegalovirus protease ranked in ascending order of calculated Ki.
PDB, Protein Data Bank.
Amprenavir and indinavir have a high affinity for the cytomegalovirus protease, with a final docked energy 14.00 kcal/mol and calculatedKi < 1 Â 10À8.
were occupied by a small drug molecule or peptidomi- indicates homology among several subtypes [18–20].
Further studies to investigate the interaction and activityof these inhibitors, including approved drugs, against The first ranked docking solution derived from the proteases from human herpesviruses may thus be fruitful preliminary docking procedure showed that all inhibitors in combating opportunistic infections originating in bound to the substrate-binding site of the cytomegalo- virus protease. The binding energy and the calculated Kiobtained after molecular dynamics simulation and secondround docking showed that amprenavir and indinavir had high affinity for the cytomegalovirus protease (asindicated by calculated Ki < 10À8 and final docked The authors would like to thank Tianyun Liu, Kai Wang energy 14.00 kcal/mol) with the inhibitor occupying and Michal Guerquin, as well as other members of the subsites S1, S2 and S3 (Table 1). The other four inhibitors, Samudrala group for valuable discussions and comments.
lopinavir, nelfinavir, ritonavir and saquinavir, only partlyfit into one or two subsites. The docked energy, the Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195, USA.
i and the binding modes of nelfinavir and lopinavir indicated that these two inhibitors bound the cytomegalovirus protease more weakly than the other Sponsorship: This work was partly supported by a inhibitors. Identical results were obtained for all the three Searle Scholar Award to Ram Samudrala.
Received: 10 October 2004; revised: 2 November A number of studies have suggested that protease inhibitors included in the HAART regimen have a significantimpact on cytomegalovirus infection by decreasing theincidence, changing the clinical course, and altering the clinical presentation [4–11]. However, none of themhave identified the inhibitory activity of individual HIV-1 1. Mentec H, Leport C, Leport J, Marche C, Harzic M, Vilde JL.
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Conserved mode of peptidomimetic inhibition and substrate We enrolled 14 male HIV-positive patients (median age recognition of human cytomegalovirus protease. Nat Struct 39 years, range 29–56), including six naive patients who 18. Holwerda BC. Herpesvirus proteases: targets for novel antiviral were starting a HAART protocol (group 1) and eight who were switching from a HAART regimen including 19. Qiu X, Janson CA, Culp JS, Richardson SB, Debouck C, Smith WW, Abdel-Meguid SS. Crystal structure of varicella-zoster PI to a PI-free treatment (group 2). Five patients of virus protease. Proc Natl Acad Sci USA 1997; 94:2874–2879 group 2 had lipodystrophy. All patients gave their 20. Buisson M, Hernandez JF, Lascoux D, Schoehn G, Forest E, informed consent according to the guidelines of the Arlaud G, et al. The crystal structure of the Epstein–Barr virusprotease shows rearrangement of the processed C terminus.
local ethics committee. Patients were evaluated before starting HAART therapy (group 1) or before switching from PI (group 2; time 0 months) and 6 months after thefirst evaluation (time 6 months). Endocrine andmetabolic examinations consisted of a thorough evalua- Do oestrogen receptors play a role in the tion of the hypothalamic–pituitary–adrenal, gonadal and pathogenesis of HIV-associated lipodystrophy? thyroid axes, a determination of the serum lipid profile, Luisa Barzona, Mauro Zambonic, Monia Pacentia, glucose, and insulin, anthropometric measurements, an Gabriella Milanb, Ottavio Boselloc, Giovanni evaluation of body composition by dual energy X-ray Federspilb, Giorgio Palu`a and Roberto Vettorb absorptiometry, an assessment of abdominal fat distribu-tion, total and subcutaneous fat at the level of the thigh bycomputed tomography scan. In order to obtain informa- Epidemiological data show an increased risk of tion on the adipose tissue gene expression profile, all HIV-associated lipodystrophy in women, and sex patients were submitted to a needle biopsy of abdominal hormone abnormalities have been reported with subcutaneous fat at times 0 and 6 months. The expression highly active antiretroviral therapy (HAART). This of the following genes, measured by real-time reverse study, which demonstrates that oestrogen receptor transcriptase–polymerase chain reaction, was investigated: b expression is significantly reduced in the subcu- genes encoding for sex steroid hormone receptors (ERa,ERb, AR, PGR); aromatase (CYP19), the enzyme that taneous adipose tissue of HIV-infected lipody- converts testosterone to oestrogen; LRH-1, a nuclear strophic patients, downregulated by HAART orphan receptor that activates CYP19 expression; factors regimens including protease inhibitors (PI), and produced by adipose tissue and involved in adipogenesis restored after switching from PI, opens perspec- tives for the investigation of selective oestrogen gamma (PPAR-g) 2, vascular endothelial growth factor

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