578 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Jiang et al.
3,30,5,50-tetramethylbenzidine (Sigma) were added sequentially.
Absorbance at 450 nm was measured using an ELISA reader
(Ultra 384, Tecan, Research Triangle Park, NC). The percent
inhibition by the compounds and their EC50 values were calcu-
lated using the software Calcusyn as described above.
(10) Tan, K.; Liu, J.; Wang, J.-H.; Shen, S.; Liu, M. Atomic structure of
a thermostable subdomain of HIV-1 gp41. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 12303–12308.
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prominent cavity in the coiled coil of HIV type 1 gp41 is an
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15613–15617.
Circular Dichroism Analysis. The effect of the compounds on
the coiled coil structure of the gp41 NHR-trimer formed by N46
peptide (we used N46 here rather than N36, since N36 cannot
automatically form NHR-trimer in physiological buffer) and
the 6-HB formed between N46 and C34 peptides was deter-
mined by CD spectroscopy as previously described.21 Briefly,
N46 and C34 peptides were diluted in doubly distilled H2O
(pH7.0) and 50 mM sodium phosphate and 150 mM NaCl (PBS,
pH 7.2), respectively, to a final concentration of 10 μM. For
investigation of the effect of a compound on N-trimer, the mix-
ture N46 and the compound (or PBS as control) was incubated
in a 37 ꢀC water bath for 0.5 h before testing. For investigation of
the effect of a compound on 6-HB formation, the mixture N46
and the compound (or PBS as control) was incubated in a 37 ꢀC
water bath for 0.5 h, followed by addition of C34 peptide and an
additional incubation of 30 min. The spectra of each sample
were acquired on Jasco spectropolarimeter (model J-715, Jasco
Inc., Japan) at room temperature, using a 5.0 nm bandwidth,
0.1 nm resolution, 0.1 cm path length, 4.0 s response time, and a
50 nm/min scanning speed and were corrected by subtraction of
the background corresponding to the solvent. The R-helicity was
calculated from the CD signal by dividing the mean residue
ellipticity at 222 nm by the value expected for 100% helix formation
(i.e., 33000 deg cm2 dmol-1) according to previous studies.38,39
Detection of in Vitro Cytotoxicity. The in vitro cytotoxicity of
compounds on MT-2 cells was measured by XTT assay as pre-
viously described.14 Briefly, an amount of 100 μL of the test
compound at graded concentrations was added to equal volumes
of cells (5 ꢀ 105/mL) in wells of 96-well plates. After incubation at
37 ꢀC for 4 days, 50 μL of XTT solution (1 mg/mL) containing
0.02 μM of phenazine methosulphate was added. After incubation
at 37 ꢀC for 4 h, the absorbance at 450 nm was measured with an
ELISA reader. The CC50 (concentration for 50% cytotoxicity)
values were calculated using the CalcuSyn program.36
(12) Jiang, S.; Lu, H.; Liu, S.; Zhao, Q.; He, Y.; Debnath, A. K.
N-substituted pyrrole derivatives as novel human immunodefi-
ciency virus type 1 entry inhibitors that interfere with the gp41 six-
helix bundle formation and block virus fusion. Antimicrob. Agents
Chemother. 2004, 48, 4349–4359.
(13) Katritzky, A. R.; Tala, S. R.; Lu, H.; Vakulenko, A. V.; Chen,
Q.-Y.; Sivapackiam, J.; Pandya, K.; Jiang, S.; Debnath, A. K.
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methyl)furans as HIV-1 entry inhibitors. J. Med. Chem. 2009, 52,
7631–7639.
(14) Debnath, A. K.; Radigan, L.; Jiang, S. Structure-based identifica-
tion of small molecule antiviral compounds targeted to the gp41
core structure of the human immunodecifiency virus type 1. J. Med.
Chem. 1999, 42, 3203–3209.
(15) He, Y.; Liu, S.; Li, J.; Lu, H.; Qi, Z.; Liu, Z.; Debnath, A. K.; Jiang,
S. Conserved salt bridge between the N- and C-terminal heptad
repeat regions of the human immunodeficiency virus type 1 gp41
core structure is critical for virus entry and inhibition. J. Virol.
2008, 82, 11129–11139.
(16) He, Y.; Liu, S.; Jing, W.; Lu, H.; Cai, D.; Chin, D. J.; Debnath,
A. K.; Kirchoff, F.; Jiang, S. Conserved residue Lys574 in the
cavity of HIV-1 Gp41 coiled-coil domain is critical for six-helix
bundle stability and virus entry. J. Biol. Chem. 2007, 282, 25631–
25639.
(17) Jiang, S.; Debnath, A. K. A salt bridge between an N-terminal
coiled coil of gp41 and an antiviral agent targeted to the gp41 core is
important for anti-HIV-1 activity. Biochem. Biophys. Res. Com-
mun. 2000, 270, 153–157.
(18) Wang, H.; Qi, Z.; Guo, A.; Mao, Q.; Lu, H.; An, X.; Xia, C.; Li, X.;
Debnath, A. K.; Wu, S.; Liu, S.; Jiang, S. ADS-J1 inhibits human
immunodeficiency virus type 1 entry by interacting with the gp41
pocket region and blocking the fusion-active gp41 core formation.
Antimicrob. Agents Chemother. 2009, 53, 4987–4998.
(19) Ikemoto, T.; Nishiguchi, A.; Mitsudera, H.; Wakimasu, M.;
Tomimatsu, K. Convenient efficient synthesis of TAK-779, a non-
peptide CCR5 antagonist: development of preparation of various
ammonium salts using trialkylphosphite and N-halogenosuccinimide.
Tetrahedron 2001, 57, 1525–1529.
(20) Jiang, S.; Lin, K.; Lu, M. A conformation-specific monoclonal
antibody reacting with fusion-active gp41 from the human immu-
nodeficiency virus type 1 envelope glycoprotein. J. Virol. 1998, 72,
10213–10217.
Acknowledgment. This study was supported by NIH Grant
AI046221. We thank Dr. C. D. Hall for helpful discussions.
(21) Liu, S.; Lu, H.; Xu, Y.; Wu, S.; Jiang, S. Different from the HIV
fusion inhibitor C34, the anti-HIV drug Fuzeon (T-20) inhibits
HIV-1 entry by targeting multiple sites in gp41 and gp120. J. Biol.
Chem. 2005, 280, 11259–11273.
Supporting Information Available: Synthetic procedures and
characterization data of compounds. This material is available
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addition of trimethylsilyl azide to nitriles. A novel prepara-
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Jiang, Shibo
