Isolation and characterization of human immunodeficiency virus type 1 resistant to the small-molecule CCR5 antagonist TAK-652

Abstract

TAK-652, a novel small-molecule chemokine receptor antagonist, is a highly potent and selective inhibitor of CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) replication in vitro. Since TAK-652 is orally bioavailable and has favorable pharmacokinetic profiles in humans, it is considered a promising candidate for an entry inhibitor of HIV-1. To investigate the resistance to TAK-652, peripheral blood mononuclear cells were infected with the R5 HIV-1 primary isolate KK and passaged in the presence of escalating concentrations of the compound for more than 1 year. After 67 weeks of cultivation, the escape virus emerged even in the presence of a high concentration of TAK-652. This virus displayed more than 200,000-fold resistance to TAK-652 compared with the wild type. The escape virus appeared to have cross-resistance to the structurally related compound TAK-779 but retained full susceptibility to TAK-220, which is from a different class of CCR5 antagonists. Furthermore, the escape virus was unable to use CXCR4 as a coreceptor. Analysis for Env amino acid sequences of escape viruses at certain points of passage revealed that amino acid changes accumulated with an increasing number of passages. Several amino acid changes not only in the V3 region but also in other Env regions seemed to be required for R5 HIV-1 to acquire complete resistance to TAK-652.

FIG. 1.

Structures of TAK-779, TAK-220, and TAK-652.

FIG. 2.

Long-term culture of infected PBMCs with escalating concentrations of TAK-652. PHA-stimulated PBMCs were infected with an R5 HIV-1 clinical isolate (KK strain) and were passaged weekly by replenishing with fresh PBMCs in the presence or absence of TAK-652 at the indicated concentrations. The solid and dotted lines indicate the p24 levels and the concentrations of TAK-652 in culture supernatants, respectively. PBMCs obtained from one healthy volunteer were used throughout the experiment. At passages 8, 43, 56, and 67, viruses were isolated from the supernatants and subjected to phenotypic and genotypic analyses.

FIG. 3.

Antiviral activities of TAK-652, TAK-220, and TAK-779 against the escape virus at passage 67 (KK_652-67). PHA-stimulated PBMCs were infected with the virus and incubated for 4 h. The cells were washed to remove unadsorbed viral particles and seeded into a 96-well plate with culture medium containing various concentrations of test compounds. On day 4 after virus infection, the cells were subcultured at a ratio of 1:2 with fresh culture medium containing the same concentration of the test compounds. On day 7 after infection, the culture supernatants were collected and p24 antigen levels were determined by ELISA. The results of two different experiments are shown. In each experiment, different donors were used for the antiviral assays of the compounds.

FIG.4.

Env amino acid sequences of isolated viruses. Blue letters indicate the amino acid changes identified for both drug escape and control viruses. Green letters indicate the amino acid changes identified only for the control viruses but not for the escape viruses. Red letters indicate the amino acid changes identified only for the escape viruses but not for the control viruses. There was heterogeneity for four amino acids, which are indicated numerically (1, P or A; 2, P or T; 3, M or I; 4, S or L).

FIG.4.

Env amino acid sequences of isolated viruses. Blue letters indicate the amino acid changes identified for both drug escape and control viruses. Green letters indicate the amino acid changes identified only for the control viruses but not for the escape viruses. Red letters indicate the amino acid changes identified only for the escape viruses but not for the control viruses. There was heterogeneity for four amino acids, which are indicated numerically (1, P or A; 2, P or T; 3, M or I; 4, S or L).