100
J. M. LaLonde et al. / Bioorg. Med. Chem. 19 (2011) 91–101
4.6. Cell-based infectivity assays
4.6.1. General considerations
Compounds were dissolved in dimethyl sulfoxide (DMSO), and
stored at 10 mM concentrations at À20 °C. The compounds were
diluted in Dulbecco Modified Eagle Medium (DMEM, Invitrogen)
to create 1 mM solutions before use. Soluble CD4 (sCD4) was pur-
chased from ImmunoDiagnostics (Woburn, MA). Human 293T
embryonic kidney and canine Cf2Th thymocytes (ATCC) were
grown at 37 °C and 5% CO2 in DMEM (Invitrogen) containing
Acknowledgments
We thank Irwin Chaiken and Wayne Hendrickson and all the
members of the PO1 Consortium Structure-Based Antagonism of
HIV-1 Envelope Function in Cell Entry. Funding was provided by
NIH GM 56550 to JL, EF, ABS, and JS. A. Sugawara thanks the Japan
Society for the Promotion of Science for research fellowship sup-
port. JL thanks the Pittsburgh Supercomputing Center for an alloca-
tion for computing resources #MCB090108.
10% fetal bovine serum (Sigma) and 100 lg/mL of penicillin–strep-
Supplementary data
tomycin (Meditech, Inc.). Cf2Th cells stably expressing human CD4
and either CCR5 or CXCR442,43 were grown in medium supple-
mented with 0.4 mg/mL of G418 (Invitrogen) and 0.20 mg/mL of
hygromycin B (Roche Diagnostics). Using the Effectene transfec-
tion reagent (Qiagen), 293T human embryonic kidney cells were
Supplementary data (tables of inactive compounds, detailed
synthetic procedures, and spectral data for new compounds) asso-
ciated with this article can be found, in the online version, at
cotransfected with plasmids expressing the pCMVDP1DenvpA
HIV-1 Gag-Pol packaging construct, the wild-type or mutant
HIV-1YU2 envelope glycoproteins or the envelope glycoproteins
of the control amphotropic murine leukemia virus (A-MLV),
and the firefly luciferase-expressing vector at a DNA ratio of
1:1:3 lg. For the production of viruses pseudotyped with the
A-MLV glycoprotein, a rev-expressing plasmid was added. The sin-
gle-round, replication-defective viruses in the supernatants were
harvested 24–30 h after transfection, filtered (0.45 lm), aliquoted,
and frozen at À80 °C until further use. The reverse transcriptase
(RT) activities of all viruses were measured as described
previously.44
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by nonlinear regression of the data.