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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 . 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 . 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 . 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 . Theoretically,
version 3.0.5 with a Lamarckian genetic algorithm .
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.
protease inhibitors against cytomegalovirus.
Cytomegalovirus colitis in HIV-1-infected patients: a prospec-tive research in 55 patients. AIDS 1994; 8:461–467.
2. Akler ME, Johnson DW, Burman WJ, Johnson SC. Anterior uveitis
This computational study provides evidence for the
and hypotony after intravenous cidofovir for the treatment ofcytomegalovirus retinitis. Ophthalmology 1998; 105:651–657
inhibitory activity of two approved inhibitors, amprenavir
3. Gellrich MM, Baumert E, Rump JA, Vaith P, Hufert FT, Hansen LL.
and indinavir, against the cytomegalovirus protease.
Clinical utility of cytomegalovirus urine cultures for ophthalmic
Including either of these two inhibitors in a HAART
care in patients with HIV. Br J Ophthalmol 1996; 80:818–822.
4. Macdonald JC, Torriani FJ, Morse LS, Karavellas MP, Reed JB,
regimen should help to control the cytomegalovirus viral
Freeman WR. Lack of reactivation of cytomegalovirus (CMV)
load in HIV-1-infected patients. The activity of the
retinitis after stopping CMV maintenance therapy in AIDS
cytomegalovirus protease would be inhibited soon after
patients with sustained elevations in CD4 T cells in responseto highly active antiretroviral therapy. J Infect Dis 1998;
starting HAART, in contrast to inhibition by promoting
immune system restoration, which may take several weeks.
5. Vrabec TR, Baldassano VF, Whitcup SM. Discontinuation of
maintenance therapy in patients with quiescent cytomegalo-virus retinitis and elevated CD4R counts. Ophthalmology 1998;
This study also provides a list of candidate inhibitors that
may be experimentally tested for cytomegalovirus
6. Casado JL, Perez-Elias MJ, Marti-Belda P, Antela A, Suarez M,
Ciancas E, et al. Improved outcome of cytomegalovirus
protease inhibitory activity, and for the further design
retinitis in AIDS patients after introduction of protease inhibi-
of broad-spectrum inhibitors, to control both HIV-1 and
tors. J Acquir Immune Defic Syndr Hum Retrovirol 1998; 19:130–
cytomegalovirus infection. Structural studies of human
7. Jabs DA, Bolton SG, Dunn JP, Palestine AG. Discontinuing
herpes proteases (of which cytomegalovirus is one)
anticytomegalovirus therapy in patients with immune reconsti-
tution after combination antiretroviral therapy. Am J Ophthal-
receptor modifiers for the management of this
8. Deayton J, Mocroft A, Wilson P, Emery VC, Johnson MA, Griffiths
PD. Loss of cytomegalovirus (CMV) viraemia following highlyactive antiretroviral therapy in the absence of specific anti-CMV
Highly active antiretroviral therapy (HAART), a treat-
9. Casado JL, Arrizabalaga J, Montes M, Marti-Belda P, Tural C,
ment consisting of a combination of protease inhibitors
Pinilla J, et al. Incidence and risk factors for developing cyto-
(PI), nucleoside analogue reverse transcriptase inhibitors
megalovirus retinitis in HIV-infected patients receiving proteaseinhibitor therapy. Spanish CMV-AIDS Study Group. AIDS 1999;
and non-nucleoside reverse transcriptase inhibitors, has
dramatically improved the long-term survival of HIV-
10. Macdonald JC, Karavellas MP, Torriani FJ, Morse LS, Smith IL,
infected patients. However, such a therapy is associated
Reed JB, Freeman WR. Highly active antiretroviral therapy-related immune recovery in AIDS patients with cytomegalo-
with a lipodystrophy syndrome, characterized by changes
virus retinitis. Ophthalmology 2000; 107:877–881.
in body fat distribution with several adverse metabolic
11. Reed JB, Briggs JW, McDonald JC, Freeman WR, Morse LS.
Highly active antiretroviral therapy-associated regression of
effects including insulin resistance, glucose intolerance
cytomegalovirus retinitis: long-term results in a small case
and dyslipidemia, which affects patients’ adherence to
therapy and impairs their prognosis by increasing the risk
12. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew
RK, Olson AJ. Automated docking using a Lamarckian genetic
of cardiovascular disease. Epidemiological data have
algorithm and empirical binding free energy function. J Com-
demonstrated an increased risk of HIV-associated
13. Kale L, Skeel R, Bhandarkar M, Brunner R, Gursoy A, Krawetz N,
lipodystrophy in women , and despite abnormalities
et al. NAMD2: greater scalability for parallel molecular dy-
of sex steroid hormone levels being reported in HIV-
namics. J Comput Phys 1999; 151:283–312.
positive patients receiving HAART , their impact on
14. van Aalten DM, Bywater R, Findlay JB, Hendlich M, Hooft RW,
Vriend G. PRODRG, a program for generating molecular
the development of lipodystrophy has never been
topologies and unique molecular descriptors from coordinates
evaluated. Oestrogens and androgens play a critical role
of small molecules. J Comput Aided Mol Des 1996; 10:255–
in adipose cell differentiation and fat distribution by
15. Jenwitheesuk E, Samudrala R. Improved prediction of HIV-1
acting through their specific receptors . The aim of this
protease-inhibitor binding energies by molecular dynamics
study was to investigate whether HAART treatment
simulations. BMC Struct Biol 2003; 3:2.
16. Jenwitheesuk E, Samudrala R. Identifying inhibitors of the SARS
could modulate the expression of sex steroid hormone
coronavirus proteinase. Bioorg Med Chem Lett 2003; 13:3989–
receptors in the adipose tissue of HIV-infected patients.
17. Tong L, Qian C, Massariol M, Deziel R, Yoakim C, Lagace L.
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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