Loss of the Protease Dimerization Inhibition Activity of Tipranavir (TPV) and Its Association with the Acquisition of Resistance to TPV by HIV-1

Abstract

I54V/V82T most readily developed TPV resistance and acquired E34D, which compromised TPV's dimerization inhibition with the HIV NL4-3 genetic background. The present data demonstrate that certain amino acid substitutions compromise TPV's dimerization inhibition and confer TPV resistance, although the loss of TPV's dimerization inhibition is not always associated with significantly increased TPV resistance. The findings that TPV's dimerization inhibition is compromised with one or two amino acid substitutions may explain at least in part why the genetic barrier of TPV against HIV-1's development of TPV resistance is relatively low compared to that of darunavir.

C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir

“…We next examined whether GRL-015, -085, and -097 were active against a variety of HIV-1 variants that had been selected in vitro with each of six FDA-approved PIs: APV, LPV, ATV, SQV, TPV, and DRV (Table 2). Each HIV-1 variant was selected in vitro by increasing concentrations of each PI and propagating a wild-type HIV-1 NL4-3 for APV, LPV, ATV, and SQV (21, 33) and a mixture of eight or 11 highly multiple-PI-resistant HIV-1 variants for TPV and DRV (21,30). Those variants were confirmed to have acquired multiple amino acid substitutions in their protease region, which have reportedly been associated with viral resistance to PIs (Table 2, ) was highly resistant to the corresponding PI, which the variant was selected against, and the differences in the EC 50 s relative to the EC 50 of each drug against HIV-1 NL4-3 ranged from Ͼ41-to 1,022-fold (Table 2).…”

“…The clinical strains used in the present study contained 8 to 16 amino acid substitutions in the protease-encoding region (see Fig. S1 in the supplemental material), which have reportedly been associated with HIV-1 resistance to various PIs and have been genotypically and phenotypically characterized as multiprotease inhibitor (multi-PI)-resistant HIV-1 (30). Seven recombinant clinical HIV-1 isolates ( rCL HIV-1 F16, rCL HIV-1 F39 , rCL HIV-1 V42 , rCL HIV-1 V44 , rCL HIV-1 T45 , rCL HIV-1 T48 , and rCL HIV-1 F71 ) were kindly provided by Robert Shafer of Stanford University and were produced using recombinant HIV NL4-3 -based infectious molecular clones generated by ligating patient-derived amplicons encompassing approximately 200 nucleotides of Gag (beginning at the unique ApaI restriction site), the entire protease, and the first 72 nucleotides of reverse transcriptase using the expression vector pN-LPFB (a generous gift from Tomozumi Imamichi of the National Institute of Allergy and Infectious Diseases).…”

“…MT-4 cells (10 5 /ml) were exposed to 50 TCID 50 s of infectious molecular HIV-1 clones and PI-selected HIV-1 variants in the presence or absence of various concentrations of drugs and were incubated at 37°C. On day 7 of culture, the supernatant was harvested and the amount of p24 Gag protein was determined by using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse F; Fujirebio Inc., Tokyo, Japan) (30). The drug concentrations that suppressed the production of p24 Gag protein by 50% (50% effective concentration [EC 50 ]) were determined by comparison with the level of p24 production in drug-free control cell cultures.…”

“…Variants resistant to GRL-015, -085, and -097 were generated as previously described (21,30,33) and cultured in the presence of each compound at an initial concentration equivalent to the EC 50 . On the last day of each passage (weeks 1 to 3), 1.5 ml of the cell-free supernatant was harvested and transferred to a culture of fresh uninfected MT-4 cells in the presence of increased concentrations of the drug for the following round of culture.…”

A Cluster Analysis on the Structural Diversity of Protein Crystals, Exemplified by Human Immunodeficiency Virus Type 1 Protease

“…17) This abundance of plenty of information on the structure of HIV-1 protease has been utilized for understanding the roles of respective amino acid residues in enzymatic activity and the fitness to the substrate, and then for the design of novel antiviral agents. 18) Drug resistance is one of the most serious problems in chemotherapy for HIV-1 infectious diseases. The virus bears amino acid mutations to diminish the binding affinity to an Regular Article * To whom correspondence should be addressed.…”