Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS-9137)

Abstract

Integrase (IN), an essential enzyme of human immunodeficiency virus (HIV), is an attractive antiretroviral drug target. The antiviral activity and resistance profile in vitro of a novel IN inhibitor, elvitegravir (EVG) (also known as JTK-303/GS-9137), currently being developed for the treatment of HIV-1 infection are described. EVG blocked the integration of HIV-1 cDNA through the inhibition of DNA strand transfer. EVG inhibited the replication of HIV-1, including various subtypes and multiple-drug-resistant clinical isolates, and HIV-2 strains with a 50% effective concentration in the subnanomolar to nanomolar range. EVG-resistant variants were selected in two independent inductions, and a total of 8 amino acid substitutions in the catalytic core domain of IN were observed. Among the observed IN mutations, T66I and E92Q substitutions mainly contributed to EVG resistance. These two primary resistance mutations are located in the active site, and other secondary mutations identified are proximal to these primary mutations. The EVG-selected IN mutations, some of which represent novel IN inhibitor resistance mutations, conferred reduced susceptibility to other IN inhibitors, suggesting that a common mechanism is involved in resistance and potential cross-resistance. The replication capacity of EVG-resistant variants was significantly reduced relative to both wild-type virus and other IN inhibitor-resistant variants selected by L-870,810. EVG and L-870,810 both inhibited the replication of murine leukemia virus and simian immunodeficiency virus, suggesting that IN inhibitors bind to a conformationally conserved region of various retroviral IN enzymes and are an ideal drug for a range of retroviral infections.

FIG. 1.

Structure of EVG and L-870,810. A dihydroquinoline carboxylic acid derivative, EVG, and a naphthyridine carboxamide derivative, L-870,810 (a representative IN inhibitor), are shown.

FIG. 2.

Mechanism of action of EVG. (A) Quantification of HIV-1 DNA species. MT-2 cells were infected with HIV-1_IIIB in the presence or absence of AZT, L-870,810, and EVG. Unintegrated (2-LTR) (white bars) and integrated (black bars) forms of proviral DNA were quantified by real-time PCR and normalized to the β-globin gene after 24 h of infection. The data are represented as means and standard deviations of value relative to that of the no-inhibitor control from three independent experiments. ND means that the signals were not detected even after 40 cycles of amplification. (B) Inhibitory effect of IN inhibitors on strand transfer activity. Gel electrophoresis shows strand transfer products (STP) generated from preprocessed donor DNA substrate (19-mer) covalently bound to acceptor DNA.

FIG. 3.

Induction of EVG-resistant HIV-1. Data from MT-2 cells are shown. The initial concentrations of EVG were 0.5 nM (A) and 0.1 nM (B). Results are from two identical but independent experiments. At the indicated passage number (black arrowheads), proviral DNA extracted from infected MT-2 cells was sequenced. Amino acid substitutions are shown. The EC₅₀ values of HIV-1 variants selected by EVG at the indicated passage number (white arrowheads) were determined using MAGI assay (A) or the production of p24 in MT-2 cells (B).

FIG. 4.

Effect of EVG-selected mutations on IN strand transfer activity and on the inhibition of strand transfer by IN inhibitors. The strand transfer activities of recombinant IN enzymes carrying EVG-selected mutations were determined using an oligonucleotide-based strand transfer assay. Strand transfer (ST) activity of IN mutants was compared to that of the wild type (WT); results are shown as percentages of wild-type activity. The effect of IN inhibitors on strand transfer was also determined for wild-type and mutant IN enzymes; results are expressed as the increase (n-fold) in IC₅₀ values of inhibitors relative to those of the wild type.

FIG. 5.

Replication kinetics of EVG- and L-870,810-resistant viral variants. The replication kinetics of wild-type and IN inhibitor-resistant viral variants were determined by p24 ELISA. The relationship of replication capacity and change (n-fold) in susceptibility (shown in Table 3) is depicted. Variants are plotted according to the observed order of their emergence during selection experiments in vitro. Replication kinetics of EVG-selected mutants derived from the two independent selection experiments (shown in Fig. 3) are plotted in different colors. WT, wild type.

FIG. 6.

Effect of IN inhibitors on retroviruses. Antiviral activities of EVG (open circles with dashed lines) and L-870,810 (closed circles with solid lines) against HIV-based (A) or MLV-based (B) vectors harboring the luciferase gene were determined by measuring luciferase activity at 48 h posttransduction. Results are expressed as percentages of relative luciferase units (RLU) compared to those of the no-inhibitor control. (C) Anti-SIV activity was determined using the MAGI assay. These results shown are one representative assay from three independent experiments.

FIG. 7.

Location of IN mutations associated with resistance to EVG. EVG in complex with the HIV-1 IN CCD is shown along with the catalytic triad residues (D64, D116, and E152) (green) and a magnesium ion (magenta). Amino acid residues conferring resistance to EVG as primary mutations (T66, E92, F121, Q146, and S147) or as secondary mutations (H51, Q95, E138, and E157) are shown in red and cyan, respectively. The flexible loop (residues 140 to 152) is shown in pink.