Molecular Basis for Increased Susceptibility of Isolates with Atazanavir Resistance-Conferring Substitution I50L to Other Protease Inhibitors

ABSTRACT Protease inhibitors (PIs) are highly effective drugs against the human immunodeficiency virus (HIV), yet long-term therapeutic use is limited by emergence of HIV type 1 (HIV-1) protease substitutions that confer cross-resistance to multiple protease inhibitor drugs. Atazanavir is a highly potent HIV protease inhibitor with a distinct resistance profile that includes effectiveness against most HIV-1 isolates resistant to one or two PIs. The signature resistance substitution for atazanavir is I50L, and it is frequently (53%) accompanied by a compensatory A71V substitution that helps restore viability and increases atazanavir resistance levels. We measured the binding affinities of wild-type (WT) and I50L/A71V HIV-1 proteases to atazanavir and other currently approved PIs (ritonavir, lopinavir, saquinavir, nelfinavir, indinavir, and amprenavir) by isothermal titration calorimetry. Remarkably, we find that all of the PIs have 2- to 10-fold increased affinities for I50L/A71V protease, except for atazanavir. The results are also manifested by thermal stability measures of affinity for WT and I50L/A71V proteases. Additional biophysical and enzyme kinetics experiments show I50L/A71V protease is a stable enzyme with catalytic activity that is slightly reduced (34%) relative to the WT. Computational modeling reveals that the unique resistance phenotype of I50L/A71V protease likely originates from bulky tert-butyl groups at P2 and P2′ (specific to atazanavir) that sterically clash with methyl groups on residue L50. The results of this study provide a molecular understanding of the novel hypersusceptibility of atazanavir-resistant I50L/A71V-containing clinical isolates to other currently approved PIs.

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In Vitro Resistance Profile of the Human Immunodeficiency Virus Type 1 Protease Inhibitor BMS-232632

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.9.2319-2326.2000 ◽

2000 ◽

Vol 44 (9) ◽

pp. 2319-2326 ◽

Cited By ~ 94

Author(s):

Yi-Fei Gong ◽

Brett S. Robinson ◽

Ronald E. Rose ◽

Carol Deminie ◽

Timothy P. Spicer ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitor ◽

Protease Inhibitors ◽

Selection Process ◽

Cross Resistance ◽

Phenotypic Analysis ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT BMS-232632 is an azapeptide human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor that displays potent anti-HIV-1 activity (50% effective concentration [EC50], 2.6 to 5.3 nM; EC90, 9 to 15 nM). In vitro passage of HIV-1 RF in the presence of inhibitors showed that BMS-232632 selected for resistant variants more slowly than nelfinavir or ritonavir did. Genotypic and phenotypic analysis of three different HIV strains resistant to BMS-232632 indicated that an N88S substitution in the viral protease appeared first during the selection process in two of the three strains. An I84V change appeared to be an important substitution in the third strain used. Mutations were also observed at the protease cleavage sites following drug selection. The evolution to resistance seemed distinct for each of the three strains used, suggesting multiple pathways to resistance and the importance of the viral genetic background. A cross-resistance study involving five other protease inhibitors indicated that BMS-232632-resistant virus remained sensitive to saquinavir, while it showed various levels (0.1- to 71-fold decrease in sensitivity)-of cross-resistance to nelfinavir, indinavir, ritonavir, and amprenavir. In reciprocal experiments, the BMS-232632 susceptibility of HIV-1 variants selected in the presence of each of the other HIV-1 protease inhibitors showed that the nelfinavir-, saquinavir-, and amprenavir-resistant strains of HIV-1 remained sensitive to BMS-232632, while indinavir- and ritonavir-resistant viruses displayed six- to ninefold changes in BMS-232632 sensitivity. Taken together, our data suggest that BMS-232632 may be a valuable protease inhibitor for use in combination therapy.

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Human immunodeficiency virus type 1 (HIV-1) protease inhibitors block cell-to-cell HIV-1 endocytosis in dendritic cells

Journal of General Virology ◽

10.1099/vir.0.012609-0 ◽

2009 ◽

Vol 90 (11) ◽

pp. 2777-2787 ◽

Cited By ~ 4

Author(s):

Claudia Muratori ◽

Eliana Ruggiero ◽

Antonella Sistigu ◽

Roberta Bona ◽

Maurizio Federico

Keyword(s):

Human Immunodeficiency Virus ◽

Dendritic Cells ◽

Protease Inhibitors ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Hiv Protease ◽

Donor Cells ◽

Immunodeficiency Virus ◽

Hiv 1

Sexual transmission is now the most frequent means of diffusion of human immunodeficiency virus type 1 (HIV-1). Even if the underlying mechanism is still largely unknown, there is a consensus regarding the key role played by mucosal dendritic cells (DCs) in capturing HIV through contact with infected subepithelial lymphocytes, and their capacity to spread HIV by trans-infection. We found that HIV protease inhibitors (PIs) reduced virion endocytosis strongly in monocyte-derived immature (i) DCs contacting HIV-1-infected cells, and that this phenomenon led to dramatically impaired trans-infection activity. This inhibitory effect was not mediated by the block of viral protease activity, as it was also operative when donor cells were infected with a PI-resistant HIV-1 strain. The block of virus maturation imposed by PIs did not correlate with significant variations in the levels of virus expression in donor cells or of Gag/Env virion incorporation. Also, PIs did not affect the endocytosis activity of DCs. In contrast, we noticed that PI treatment inhibited the formation of cell–cell conjugates whilst reducing the expression of ICAM-1 in target iDCs. Our results contribute to a better delineation of the mechanisms underlying HIV-1 trans-infection activity in DCs, whilst having implications for the development of new anti-HIV microbicide strategies.

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Evaluation of reverse transcriptase and protease inhibitors in two-drug combinations against human immunodeficiency virus replication.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.6.1346 ◽

1996 ◽

Vol 40 (6) ◽

pp. 1346-1351 ◽

Cited By ~ 26

Author(s):

C A Deminie ◽

C M Bechtold ◽

D Stock ◽

M Alam ◽

F Djang ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Combination Therapy ◽

Protease Inhibitor ◽

Reverse Transcriptase ◽

Protease Inhibitors ◽

Nucleoside Analogs ◽

Drug Combinations ◽

Hiv Protease ◽

Human Immunodeficiency Virus Replication ◽

Immunodeficiency Virus

Current treatments for human immunodeficiency virus (HIV) include both reverse transcriptase and protease inhibitors. Results from in vitro and clinical studies suggest that combination therapy can be more effective than single drugs in reducing viral burden. To evaluate compounds for combination therapy, stavudine (d4T), didanosine (ddI), or BMS-186,318, an HIV protease inhibitor, were combined with other clinically relevant compounds and tested in a T-cell line (CEM-SS) that was infected with HIV-RF or in peripheral blood mononuclear cells infected with a clinical HIV isolate. The combined drug effects were analyzed by the methods described by Chou and Talalay (Adv. Enzyme Regul. 22:27-55, 1984) as well as by Prichard et al. (Antimicrob. Agents Chemother. 37:540-545, 1993). The results showed that combining two nucleoside analogs (d4T-ddI, d4T-zidovudine [AZT], and d4T-zalcitabine [ddC]), two HIV protease inhibitors (BMS-186,318-saquinavir, BMS-186,318-SC-52151, and BMS-186,318-MK-639) or a reverse transcriptase and a protease inhibitor (BMS-186,318-d4T, BMS-186,318-ddI, BMS-186,318-AZT, d4T-saquinavir, d4T-MK-639, and ddI-MK-639) yielded additive to synergistic antiviral effects. In general, analysis of data by either method gave consistent results. In addition, combined antiviral treatments involving nucleoside analogs gave slightly different outcomes in the two cell types, presumably because of a difference in phosphorylation patterns. Importantly, no strong antagonism was observed with the drug combinations studied. These data should provide useful information for the design of clinical trials of combined chemotherapy.

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An analysis of the delay-dependent HIV-1 protease inhibitor model

International Journal of Biomathematics ◽

10.1142/s1793524518500316 ◽

2018 ◽

Vol 11 (03) ◽

pp. 1850031

Author(s):

M. Divya ◽

M. Pitchaimani

Keyword(s):

Human Immunodeficiency Virus ◽

Sensitivity Analysis ◽

Stability Analysis ◽

Protease Inhibitor ◽

Numerical Simulations ◽

Humoral Immunity ◽

Hiv Protease ◽

Immunodeficiency Virus ◽

Delay Dependent ◽

Hiv 1

In this paper, we have studied about the sensitivity analysis of the human immunodeficiency virus (HIV) protease inhibitor (PI) model and estimated the length of the delay. We have fabricated an HIV PI model accompanied with humoral immunity. Stability analysis of the constructed model about its steady states has been deliberated. We have evaluated some numerical simulations for PI model with humoral immunity by using the existing patient data.

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Accurate Prediction of Inhibitor Binding to HIV-1 Protease Using CANDOCK

Frontiers in Chemistry ◽

10.3389/fchem.2021.775513 ◽

2022 ◽

Vol 9 ◽

Author(s):

Zackary Falls ◽

Jonathan Fine ◽

Gaurav Chopra ◽

Ram Samudrala

Keyword(s):

Molecular Docking ◽

Characteristic Curve ◽

Scoring Function ◽

Hiv Protease ◽

Binding Affinities ◽

Knowledge Based ◽

Pearson Coefficient ◽

Docking Protocol ◽

Immunodeficiency Virus ◽

Hiv 1

The human immunodeficiency virus 1 (HIV-1) protease is an important target for treating HIV infection. Our goal was to benchmark a novel molecular docking protocol and determine its effectiveness as a therapeutic repurposing tool by predicting inhibitor potency to this target. To accomplish this, we predicted the relative binding scores of various inhibitors of the protease using CANDOCK, a hierarchical fragment-based docking protocol with a knowledge-based scoring function. We first used a set of 30 HIV-1 protease complexes as an initial benchmark to optimize the parameters for CANDOCK. We then compared the results from CANDOCK to two other popular molecular docking protocols Autodock Vina and Smina. Our results showed that CANDOCK is superior to both of these protocols in terms of correlating predicted binding scores to experimental binding affinities with a Pearson coefficient of 0.62 compared to 0.48 and 0.49 for Vina and Smina, respectively. We further leveraged the Database of Useful Decoys: Enhanced (DUD-E) HIV protease set to ascertain the effectiveness of each protocol in discriminating active versus decoy ligands for proteases. CANDOCK again displayed better efficacy over the other commonly used molecular docking protocols with area under the receiver operating characteristic curve (AUROC) of 0.94 compared to 0.71 and 0.74 for Vina and Smina. These findings support the utility of CANDOCK to help discover novel therapeutics that effectively inhibit HIV-1 and possibly other retroviral proteases.

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Structural and Thermodynamic Basis for the Binding of TMC114, a Next-Generation Human Immunodeficiency Virus Type 1 Protease Inhibitor

Journal of Virology ◽

10.1128/jvi.78.21.12012-12021.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 12012-12021 ◽

Cited By ~ 170

Author(s):

Nancy M. King ◽

Moses Prabu-Jeyabalan ◽

Ellen A. Nalivaika ◽

Piet Wigerinck ◽

Marie-Pierre de Béthune ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitor ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

First Generation ◽

Titration Calorimetry ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Substrate Envelope

ABSTRACT TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (Kd = 4.5 × 10−12 M) than APV (Kd = 3.9 × 10−10 M). Our X-ray data (resolution ranging from 2.2 to 1.2 Å) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (−12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope.

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In vitro Sequential Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Reduced Sensitivity to Hydroxyethylurea Protease Inhibitors

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029700800508 ◽

1997 ◽

Vol 8 (5) ◽

pp. 447-456 ◽

Cited By ~ 2

Author(s):

KE Potts ◽

ML Smidt ◽

SP Tucker ◽

TR Stiebel ◽

JJ McDonald ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitors ◽

Hiv Protease ◽

Cross Resistance ◽

Protease Gene ◽

Resistant Virus ◽

Selection Scheme ◽

Sequential Selection ◽

Immunodeficiency Virus

In vitro resistance to the human immunodeficiency virus (HIV) protease inhibitors SC-52151 and SC-55389A was evaluated in an in vitro sequential selection scheme. HIVRF variants were selected for reduced sensitivity to SC-52151 and subsequently passaged in both SC-52151 and a structurally different hydroxyethylurea protease inhibitor, SC-55389A, to select for dual-resistant virus. SC-52151 selection alone resulted in a 23-fold reduction in virus sensitivity whereas selection in both inhibitors resulted in 34- and eightfold reductions in virus sensitivity to SC-52151 and SC-55389A, respectively. Sequence analysis of the protease gene revealed that SC-52151 -resistant virus had a Gly to Val substitution at residue 48 (G48V) and, in 58% of subclones, an accompanying Val to Ala substitution at residue 82 (V82A). Dual-resistant virus had both G48V and V82A substitutions present and, in the majority of subclones, an lle to Thr and/or Leu to Pro substitution at residues 54 and 63, respectively. Drug susceptibility assays with limiting dilution-cloned HIVRFR (G48V/V82A) and HIVRFRR (G48V/154T/L63P/V82A) viruses demonstrated moderate to high-level cross-resistance to additional structurally non-related protease inhibitors. Recombinant HIVHXB2 proviral clones with G48V, L63P and V82A substitutions showed that one active site mutation was permissible, but the presence of both G48V and V82A substitutions together significantly reduced infectious virus production. Insight into the contributions of the observed substitutions to drug resistance is presented in molecular modelling studies.

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Phenotypic Hypersusceptibility to Multiple Protease Inhibitors and Low Replicative Capacity in Patients Who Are Chronically Infected with Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.79.10.5907-5913.2005 ◽

2005 ◽

Vol 79 (10) ◽

pp. 5907-5913 ◽

Cited By ~ 12

Author(s):

Javier Martinez-Picado ◽

Terri Wrin ◽

Simon D. W. Frost ◽

Bonaventura Clotet ◽

Lidia Ruiz ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitor ◽

Protease Inhibitors ◽

Human Immunodeficiency Virus Type ◽

Replication Capacity ◽

Treatment Interruptions ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Increased Susceptibility

ABSTRACT Increased susceptibility to the protease inhibitors saquinavir and amprenavir has been observed in human immunodeficiency virus type 1 (HIV-1) with specific mutations in protease (V82T and N88S). Increased susceptibility to ritonavir has also been described in some viruses from antiretroviral agent-naïve patients with primary HIV-1 infection in association with combinations of amino acid changes at polymorphic sites in the protease. Many of the viruses displaying increased susceptibility to protease inhibitors also had low replication capacity. In this retrospective study, we analyze the drug susceptibility phenotype and the replication capacity of virus isolates obtained at the peaks of viremia during five consecutive structured treatment interruptions in 12 chronically HIV-1-infected patients. Ten out of 12 patients had at least one sample with protease inhibitor hypersusceptibility (change ≤0.4-fold) to one or more protease inhibitor. Hypersusceptibility to different protease inhibitors was observed at variable frequency, ranging from 38% to amprenavir to 11% to nelfinavir. Pairwise comparisons between susceptibilities for the protease inhibitors showed a consistent correlation among all pairs. There was also a significant relationship between susceptibility to protease inhibitors and replication capacity in all patients. Replication capacity remained stable over the course of repetitive cycles of structured treatment interruptions. We could find no association between in vitro replication capacity and in vivo plasma viral load doubling time and CD4+ and CD8+ T-cell counts at each treatment interruption. Several mutations were associated with hypersusceptibility to each protease inhibitor in a univariate analysis. This study extends the association between hypersusceptibility to protease inhibitors and low replication capacity to virus isolated from chronically infected patients and highlights the complexity of determining the genetic basis of this phenomenon. The potential clinical relevance of protease inhibitor hypersusceptibility and low replication capacity to virologic response to protease inhibitor-based therapies deserves to be investigated further.

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A Novel Bis-Tetrahydrofuranylurethane-Containing Nonpeptidic Protease Inhibitor (PI), GRL-98065, Is Potent against Multiple-PI-Resistant Human Immunodeficiency Virus In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01413-06 ◽

2007 ◽

Vol 51 (6) ◽

pp. 2143-2155 ◽

Cited By ~ 49

Author(s):

Masayuki Amano ◽

Yasuhiro Koh ◽

Debananda Das ◽

Jianfeng Li ◽

Sofiya Leschenko ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitor ◽

Close Contact ◽

Wide Spectrum ◽

Antiviral Agents ◽

Cyclic Ether ◽

Hiv Protease ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We designed, synthesized, and identified GRL-98065, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0002 to 0.0005 μM) with minimal cytotoxicity (50% cytotoxicity, 35.7 μM in CD4+ MT-2 cells). GRL-98065 blocked the infectivity and replication of each of the HIV-1NL4-3 variants exposed to and selected by up to a 5 μM concentration of saquinavir, indinavir, nelfinavir, or ritonavir and a 1 μM concentration of lopinavir or atazanavir (EC50, 0.0015 to 0.0075 μM), although it was less active against HIV-1NL4-3 selected by amprenavir (EC50, 0.032 μM). GRL-98065 was also potent against multiple-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, and HIV-2 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active-site amino acids (Asp29 and Asp30) is important for its potency and wide-spectrum activity against multiple-PI-resistant HIV-1 variants. The present data demonstrate that the privileged nonpeptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and that this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.

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GRL-0519, a Novel Oxatricyclic Ligand-Containing Nonpeptidic HIV-1 Protease Inhibitor (PI), Potently Suppresses Replication of a Wide Spectrum of Multi-PI-Resistant HIV-1 VariantsIn Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02189-12 ◽

2013 ◽

Vol 57 (5) ◽

pp. 2036-2046 ◽

Cited By ~ 20

Author(s):

Masayuki Amano ◽

Yasushi Tojo ◽

Pedro Miguel Salcedo-Gómez ◽

Joseph Richard Campbell ◽

Debananda Das ◽

...

Keyword(s):

Protease Inhibitor ◽

Serum Proteins ◽

Wide Spectrum ◽

Hiv Protease ◽

Active Site Residues ◽

Development Of Resistance ◽

Immunodeficiency Virus ◽

Protease Substrate ◽

Binding Pockets ◽

Hiv 1

ABSTRACTWe report that GRL-0519, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containingtris-tetrahydrofuranylurethane (tris-THF) and a sulfonamide isostere, is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0005 to 0.0007 μM) with minimal cytotoxicity (50% cytotoxic concentration [CC50], 44.6 μM). GRL-0519 blocked the infectivity and replication of HIV-1NL4-3variants selected by up to a 5 μM concentration of ritonavir, lopinavir, or atazanavir (EC50, 0.0028 to 0.0033 μM). GRL-0519 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, highly darunavir (DRV)-resistant variants, and HIV-2ROD. The development of resistance against GRL-0519 was substantially delayed compared to other PIs, including amprenavir (APV) and DRV. The effects of nonspecific binding of human serum proteins on GRL-0519's antiviral activity were insignificant. Our analysis of the crystal structures of GRL-0519 (3OK9) and DRV (2IEN) with protease suggested that thetris-THF moiety, compared to thebis-THF moiety present in DRV, has greater water-mediated polar interactions with key active-site residues of protease and that thetris-THF moiety and paramethoxy group effectively fill the S2 and S2′ binding pockets, respectively, of the protease. The present data demonstrate that GRL-0519 has highly favorable features as a potential therapeutic agent for treating patients infected with wild-type and/or multi-PI-resistant variants and that thetris-THF moiety is critical for strong binding of GRL-0519 to the HIV protease substrate binding site and appears to be responsible for its favorable antiretroviral characteristics.

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