736 
Induction in vitro of HIV-1 Variants Resistant to a Low-molecular CD4 Mimic Compound, N-(4-Chlorophenyl)-N’-(2,2,6,6-tetramethylpiperidin-4-yl)-oxalamide
Kazuhisa Yoshimura*1, J Shibata1, A Honda1, Y Yamada2, H Masuno2, H Tamamura2, and S Matsushita1
1Ctr for AIDS Res, Kumamoto Univ, Japan and 2Inst of Biomaterials and Bioengineering, Tokyo Med and Dental Univ, Japan
Background: N-(4-Chlorophenyl)-N’-(2,2,6,6-tetramethylpiperidin-4-yl)-oxalamide
(NBD-556) is a low-molecular weight compound that reportedly block the
interaction between the HIV-1 gp120 and its receptor, CD4. The thermodynamic
signature of NBD-556 was similar to that observed for binding of rsCD4 to
gp120. In this study, we induced HIV-1 variants escape from NBD-556 and rsCD4 in
vitro.
Methods: To investigate whether the effect of
binding affinity of anti-gp120 monoclonal antibodies to Env with NBD-556 is
similar to rsCD4, we compared fluorescence-activated cell sorting (FACS)
profiles of each monoclonal antibody binding to Env expressing cell surface. To
select HIV-1 variants resistant to NBD-556 and rsCD4 in vitro, we
exposed PM1-CCR5 cells to HIV-1LAI and the virus was serially
passaged in the presence of increasing concentrations of NBD-556 or rsCD4, up to
50 μM and 20 μg/mL, respectively. We determined the amino acid
sequence of the gp120-encoding region of the HIV-1LAI escape mutant
cultured with NBD-556 or rsCD4. The multi-drug-effect analysis of Chou et al.
was used to analyze the effects of combinations of NBD-556 with anti-gp120 monoclonal
antibodies.
Results: In FACS analysis, the profile of binding of
anti-envelope (CD4 induced and V3) monoclonal antibodies to NBD-556-pretreated
Env expressing cell surface was completely similar to those of rsCD4-pretreated.
At passage 21 in the culture where HIV-1LAI was propagating in the
presence of NBD-556 (50μM), 2 amino acid substitutions, Ser to Asn at
position 375 (S375N, 11 of 11 clones) in C3 and Ala to Thr at position 433
(A433T, 4 of 11 clones) in C4 were identified. On the other hand, in the
selection with rsCD4, 6 mutations (P212, V255E, N280K, S375N, and G380R) were
appeared through the passage. At passage 5, 3 substitutions—V255E (5 of 10
clones), G380R (1 of 10 clones), and G431E (2 of 10 clones)—were remained in 20
μg/mL of rsCD4. These 2 profiles of mutations in the selections of NBD-556
and rsCD4 were very similar in a 3-dimensional position. Moreover, combinations
of NBD-556 with anti-gp120 monoclonal antibodies showed highly synergistic
interactions at the 50, 75, and 90% inhibitory concentrations.
Conclusions: In this study, we observed that NBD-556
could bind a CD4 binding site followed by induction of conformational changes
in gp120 similar to those observed upon rsCD4 binding. We also found highly
synergistic interactions between NBD-556 and anti-gp120 monoclonal antibodies. These
data provide a rational basis for testing of combinations of the NBD compound
and therapeutic monoclonal antibodies, such as KD-247 in vivo.
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