In Vitro Selection of Highly Darunavir-Resistant and Replication-Competent HIV-1 Variants by Using a Mixture of Clinical HIV-1 Isolates Resistant to Multiple Conventional Protease Inhibitors

Abstract

We attempted to select HIV-1 variants resistant to darunavir (DRV), which potently inhibits the enzymatic activity and dimerization of protease and has a high genetic barrier to HIV-1 development of resistance to DRV. We conducted selection using a mixture of 8 highly multi-protease inhibitor (PI)-resistant, DRV-susceptible clinical HIV-1 variants (HIV-1MIX) containing 9 to 14 PI resistance-associated amino acid substitutions in protease. HIV-1MIX became highly resistant to DRV, with a 50% effective concentration (EC50) ∼333-fold greater than that against HIV-1NL4-3. HIV-1MIX at passage 51 (HIV-1MIXP51 ) replicated well in the presence of 5 μM DRV and contained 14 mutations. HIV-1MIXP51 was highly resistant to amprenavir, indinavir, nelfinavir, ritonavir, lopinavir, and atazanavir and moderately resistant to saquinavir and tipranavir. HIV-1MIXP51 had a resemblance with HIV-1C of the HIV-1MIX population, and selection using HIV-1C was also performed; however, its DRV resistance acquisition was substantially delayed. The H219Q and I223V substitutions in Gag, lacking in HIV-1CP51 , likely contributed to conferring a replication advantage on HIV-1MIXP51 by reducing intravirion cyclophilin A content. HIV-1MIXP51 apparently acquired the substitutions from another HIV-1 strain(s) of HIV-1MIX through possible homologous recombination. The present data suggest that the use of multiple drug-resistant HIV-1 isolates is of utility in selecting drug-resistant variants and that DRV would not easily permit HIV-1 to develop significant resistance; however, HIV-1 can develop high levels of DRV resistance when a variety of PI-resistant HIV-1 strains are generated, as seen in patients experiencing sequential PI failure, and ensuing homologous recombination takes place. HIV-1MIXP51 should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents.

Multi-step inhibition explains HIV-1 protease inhibitor pharmacodynamics and resistance

“…Koh et al (47) isolated HIV-1 variants from a patient failing PI-containing regimens. These viruses contained 9 to 14 protease mutations associated with PI resistance.…”

“…These viruses contained 9 to 14 protease mutations associated with PI resistance. The viruses were then grown in the presence of DRV for 51 passages and additional mutations in the protease gene accumulated (47). We cloned the full-length env gene from passages 1 and 51 (E-1 and E-51).…”

A Conserved Hydrogen-Bonding Network of P2 bis -Tetrahydrofuran-Containing HIV-1 Protease Inhibitors (PIs) with a Protease Active-Site Amino Acid Backbone Aids in Their Activity against PI-Resistant HIV

“…However, we recently selected a highly DRV-resistant strain of HIV-1 in vitro by using a mixture of multi-PI-resistant (but DRV-sensitive) clinical HIV-1 strains as a starting viral population (7). In an attempt to examine the genetic barrier of GRL008, it was further evaluated against one of the DRV-resistant HIV-1 variants, HIV DRV R P20 (7) and was found to be potent, with only a 2.6-fold increase in its EC 50 above that for HIV WT . Although TPV showed a similar 2.7-fold increase in its EC 50 against HIV DRV R P20 , the absolute EC 50 of GRL008 was 3.5-fold lower than that of TPV, suggesting that GRL008 is overall a better PI than TPV against HIV DRV R P20 .…”

“…1), the latest FDA-approved protease inhibitor (PI), which contains bis-tetrahydrofuranyl urethane (bis-THF) as the P2 moiety, has been shown to have a high genetic barrier (5,6), a feature of a drug or regimen that delays or prevents the occurrence of genetic evolution of HIV-1 to acquire drug resistance-associated mutations, allowing the virus to overcome the antiretroviral activity of the very drug or regimen and to become capable of propagating despite treatment with the very drug or regimen. However, HIV-1 also ultimately develops high levels of resistance to DRV both in vitro and in vivo (7,8). In order to suppress the propagation of such PI-resistant HIV-1 protease variants, the development of novel PIs with greater antiviral activities and higher genetic barriers is urgently needed.…”

“…1) was evaluated against an MDR clinical isolate of HIV-1 (HIV A02 ) (10, 11) that contained eight amino acid substitutions, L10I, K45R, I54V, L63P, A71V, V82T, L90M, and I93L, in its protease (PR A02 ). The antiviral activity of GRL008 was also evaluated against HIV-2 ROD and an HIV-1 variant selected with DRV over 20 passages (HIV DRV R P20 ) (7). In addition, GRL008, DRV, ritonavir (RTV), and tipranavir (TPV) were individually cocrystallized with PR A02 , and a structure-function evaluation of each agent was performed.…”