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Determination of Resistance Profile of GRL-02031, a Novel Nonpeptidic Protease Inhibitor Containing a Cyclopentanyltetrahydrofuran Moiety
Yasuhiro Koh*1, H Nakata1, H Ogata-Aoki1, M Nakayama1, S Leschenko2, A Ghosh2, and H Mitsuya1,3
1Kumamoto Univ Sch of Med, Japan; 2Purdue Univ, West Lafayette, IN, US; and 3NCI, NIH, DHHS, Bethesda, MD, US
Background: We have recently generated a
novel nonpeptidic protease inhibitor (PI), GRL-02031, by incorporating a stereochemically
defined fused cyclopentanyltetrahydrofuran (Cp-THF),
which exerts potent activity against a wide spectrum of HIV-1 isolates including
multi-drug-resistant HIV-1 variants (HIVMDR). We attempted to
select GRL-02031-resistant variants by
propagating a laboratory X4 HIV-1 strain (HIV-1NL4-3) in MT-4 cells
in the presence of increasing concentrations of GRL-02031
and determined the activity profile of GRL-02031
against a variety of HIV-1NL4-3-based molecular infectious HIV-1
clones.
Methods: PI-resistant variants were selected in vitro by propagating HIV-1NL4-3
in the presence of increasing concentrations of
various PI using MT-4 cells. Anti-HIV-1 activity of various PI against a
variety of molecular HIV-1 clones was determined by exploiting the p24 assay
using MT-4 cells.
Results: Upon the selection
of GRL-02031-resistant HIV-1 in the presence of
as many as 5 μM of GRL-02031, mutations including L10F, L33F, M46I, I47V, Q58E, V82I, I84V, and I85V
in the protease-encoding region and G62R (p17), L363M (p24/p2 cleavage site), R409K
(p7), and I437T (p7/p1 cleavage site) in the gag-encoding region emerged. No significant changes in IC50
values of GRL-02031 were observed in infectious HIV-1 clones containing one of
such amino acid substitutions. A moderate but significant reduction in drug
susceptibility (~3-fold difference in IC50 values) was seen when the
virus acquired 2 mutations (M46I/I47V or I84V/I85V). Further increase in IC50
values was seen when 4 (L10F/I47V/V82I/I85V) or 5 (L10F/M46I/I47V/V82I/I85V)
substitutions were introduced. We also generated molecular clones containing a
primary mutation such as D30N, G48V, I50V, and L90M, and determined IC50
values of GRL-02031. GRL-02031
was potent against all such molecular clones with IC50 value
differences by 0.7- to 1.7-fold in comparison with the IC50 value
against HIV-1NL4-3, although HIV-1D30N and HIV-1G48V
showed substantial resistance to nelfinavir
(5.6-fold) and saquinavir (5.1-fold), respectively.
Conclusions: The present data suggest that HIV-1
develops substantial resistance to GRL-02031 only
when it acquires >4 mutations in its protease, a unique property considering
the propensity of PI to permit HIV-1 to develop high degrees of drug resistance
with 1 or a few amino acid substitutions.
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