Binding Kinetics of Darunavir to Human Immunodeficiency Virus Type 1 Protease Explain the Potent Antiviral Activity and High Genetic Barrier

We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1 WT ), with 50% effective concentrations (EC 50 s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC 50 ) for GRL-015, -085, and -097 of 80, >100, and >100 M,

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

Aoki

Hayashi

Yedidi

et al. 2016

“…All PI used in antiviral therapy are efficient enzyme inhibitors characterized by low equilibrium dissociation constant (K D ) and K i values, but we reasoned that the potential for washout may be determined primarily by the off-rate of the PI rather than by its K i value. Kinetic binding parameters determined by surface plasmon resonance measurements using purified PR vary significantly between individual compounds, with off-rates differing by up to several orders of magnitude (36)(37)(38). Absolute rate constants and exact differences between off-rates vary between different studies employing different conditions and analysis setups, but the relative order of offrates remains largely constant (summarized in Fig.…”

Section: Induction Of Gag Proteolysis By Inhibitor Washoutmentioning

confidence: 99%

Induced Maturation of Human Immunodeficiency Virus

Matteı

Anders

Konvalinka

et al. 2014

HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion-termed maturation-that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal halflife (t 1/2 ) of ϳ5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity. IMPORTANCECleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.

“…These inhibitors, including darunavir (DRV) and tipranavir (TPV) as well as a series of potent experimental antiretroviral agents such as TMC126 (41), blocked PR dimerization at concentrations of as low as 0.01 M and potently blocked HIV replication in vitro (26). DRV contains a structure-based and designed privileged nonpeptidic P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) (14,15,27), which potently inhibits the enzymatic activity and dimerization of HIV PR (26) and has a high-level genetic barrier against HIV development of resistance to DRV (9,10). Nevertheless, we have witnessed that HIV acquires significant levels of resistance against DRV among HIV-infected individuals who have received long-term combi-nation chemotherapy (33,38).…”

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confidence: 99%

Loss of Protease Dimerization Inhibition Activity of Darunavir Is Associated with the Acquisition of Resistance to Darunavir by HIV-1

Koh

Aoki

Danish

et al. 2011

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (