scholarly journals Unique Thermodynamic Response of Tipranavir to Human Immunodeficiency Virus Type 1 Protease Drug Resistance Mutations

2007 ◽  
Vol 81 (10) ◽  
pp. 5144-5154 ◽  
Author(s):  
S. Muzammil ◽  
A. A. Armstrong ◽  
L. W. Kang ◽  
A. Jakalian ◽  
P. R. Bonneau ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type ◽  
High Potency ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Drug resistance is a major problem affecting the clinical efficacy of antiretroviral agents, including protease inhibitors, in the treatment of infection with human immunodeficiency virus type 1 (HIV-1)/AIDS. Consequently, the elucidation of the mechanisms by which HIV-1 protease inhibitors maintain antiviral activity in the presence of mutations is critical to the development of superior inhibitors. Tipranavir, a nonpeptidic HIV-1 protease inhibitor, has been recently approved for the treatment of HIV infection. Tipranavir inhibits wild-type protease with high potency (Ki = 19 pM) and demonstrates durable efficacy in the treatment of patients infected with HIV-1 strains containing multiple common mutations associated with resistance. The high potency of tipranavir results from a very large favorable entropy change (−TΔS = −14.6 kcal/mol) combined with a favorable, albeit small, enthalpy change (ΔH = −0.7 kcal/mol, 25°C). Characterization of tipranavir binding to wild-type protease, active site mutants I50V and V82F/I84V, the multidrug-resistant mutant L10I/L33I/M46I/I54V/L63I/V82A/I84V/L90M, and the tipranavir in vitro-selected mutant I13V/V32L/L33F/K45I/V82L/I84V was performed by isothermal titration calorimetry and crystallography. Thermodynamically, the good response of tipranavir arises from a unique behavior: it compensates for entropic losses by actual enthalpic gains or by sustaining minimal enthalpic losses when facing the mutants. The net result is a small loss in binding affinity. Structurally, tipranavir establishes a very strong hydrogen bond network with invariant regions of the protease, which is maintained with the mutants, including catalytic Asp25 and the backbone of Asp29, Asp30, Gly48 and Ile50. Moreover, tipranavir forms hydrogen bonds directly to Ile50, while all other inhibitors do so by being mediated by a water molecule.

scholarly journals Susceptibility to PNU-140690 (Tipranavir) of Human Immunodeficiency Virus Type 1 Isolates Derived from Patients with Multidrug Resistance to Other Protease Inhibitors

2000 ◽  
Vol 44 (5) ◽  
pp. 1328-1332 ◽  
Author(s):  
Stefano Rusconi ◽  
Simona La Seta Catamancio ◽  
Paola Citterio ◽  
Semir Kurtagic ◽  
Michela Violin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Amino Acid ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Entire Group ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In our study we examined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activity of a novel HIV-1 protease inhibitor, PNU-140690 (tipranavir), against patient-derived isolates resistant to multiple other protease inhibitors (PIs). The aim of our experiments was to investigate the genotypes and the in vitro phenotypes of drug resistance of PNU-140690. We carried out drug susceptibility tests with peripheral blood mononuclear cells and a fixed amount of infectious virus (1,000 50% tissue culture infective doses) to determine the 50% inhibitory concentration (IC50) and IC90, PCR assays for the detection of drug resistance mutations in RNA in plasma, and direct sequencing of PCR products. Phenotypic resistance to PIs was invariably related to genotypic mutations. The substitutions among the amino acid residues of the protease included L10I, K20R, L24I, M36I, N37D, G48V, I54V, L63P, I64V, A71V, V77I, V82A, I84V, and L90M. Isolates from all of the patients had developed a maximal degree of resistance to indinavir, ritonavir, and nelfinavir (IC50s, >0.1 μM). We also compared these mutations with the amino acid changes previously described in association with in vivo tipranavir administration. The mutations included the following: I15V, E35D, N37D, R41K, D60E, and A71T. Infections with IIIB, 14aPre, and N70 were inhibited by an average drug IC90 of 0.18 ± 0.02 μM in multiple experiments. The average mean ± standard error of mean IC90 for the entire group of multidrug-resistant isolates derived from the mean values for two culture wells with p24 antigen supernatant appeared to be 0.619 ± 0.055 μM (range, 0.31 to 0.86 μM). Tipranavir retained a sustained antiviral activity against PI-MDR clinical isolates and might be useful in combination regimens with other antiretroviral agents for patients who have already failed other PI-containing therapies.

scholarly journals Mutation Patterns and Structural Correlates in Human Immunodeficiency Virus Type 1 Protease following Different Protease Inhibitor Treatments

2003 ◽  
Vol 77 (8) ◽  
pp. 4836-4847 ◽  
Author(s):  
Thomas D. Wu ◽  
Celia A. Schiffer ◽  
Matthew J. Gonzales ◽  
Jonathan Taylor ◽  
Rami Kantor ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease—including 22 not previously associated with drug resistance—were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 Å of each other—many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance.

scholarly journals Pretreatment human immunodeficiency virus type 1 (HIV-1) drug resistance in transmission clusters of the Cologne-Bonn region, Germany

2019 ◽  
Vol 25 (2) ◽  
pp. 253.e1-253.e4 ◽  
Author(s):  
M. Stecher ◽  
A. Chaillon ◽  
A.M. Eis-Hübinger ◽  
C. Lehmann ◽  
G. Fätkenheuer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

scholarly journals The Thiocarboxanilide Nonnucleoside Inhibitor UC781 Restores Antiviral Activity of 3′-Azido-3′-Deoxythymidine (AZT) against AZT-Resistant Human Immunodeficiency Virus Type 1

1999 ◽  
Vol 43 (2) ◽  
pp. 259-263 ◽  
Author(s):  
Gadi Borkow ◽  
Dominique Arion ◽  
Mark A. Wainberg ◽  
Michael A. Parniak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT N-[4-Chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide (UC781) is an exceptionally potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. We found that a 1:1 molar combination of UC781 and 3′-azido-3′-deoxythymidine (AZT) showed high-level synergy in inhibiting the replication of AZT-resistant virus, implying that UC781 can restore antiviral activity to AZT against AZT-resistant HIV-1. Neither the nevirapine plus AZT nor the 2′,5′-bis-O-(t-butyldimethylsilyl)-3′-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide plus AZT combinations had this effect. Studies with purified HIV-1 reverse transcriptase (from a wild type and an AZT-resistant mutant) showed that UC781 was a potent inhibitor of the pyrophosphorolytic cleavage of nucleotides from the 3′ end of the DNA polymerization primer, a process that we have proposed to be critical for the phenotypic expression of AZT resistance. Combinations of UC781 plus AZT did not act in synergy to inhibit the replication of either wild-type virus or UC781-resistant HIV-1. Importantly, the time to the development of viral resistance to combinations of UC781 plus AZT is significantly delayed compared to the time to the development of resistance to either drug alone.

scholarly journals Activity against Human Immunodeficiency Virus Type 1, Intracellular Metabolism, and Effects on Human DNA Polymerases of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

2007 ◽  
Vol 51 (8) ◽  
pp. 2701-2708 ◽  
Author(s):  
Hirotomo Nakata ◽  
Masayuki Amano ◽  
Yasuhiro Koh ◽  
Eiichi Kodama ◽  
Guangwei Yang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Transcriptase Inhibitor ◽  
Multidrug Resistant ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT We examined the intracytoplasmic anabolism and kinetics of antiviral activity against human immunodeficiency virus type 1 (HIV-1) of a nucleoside reverse transcriptase inhibitor, 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), which has potent activity against wild-type and multidrug-resistant HIV-1 strains. When CEM cells were exposed to 0.1 μM [3H]EFdA or [3H]3′-azido-2′,3′-dideoxythymidine (AZT) for 6 h, the intracellular EFdA-triphosphate (TP) level was 91.6 pmol/109 cells, while that of AZT was 396.5 pmol/109 cells. When CEM cells were exposed to 10 μM [3H]EFdA, the amount of EFdA-TP increased by 22-fold (2,090 pmol/109 cells), while the amount of [3H]AZT-TP increased only moderately by 2.4-fold (970 pmol/109 cells). The intracellular half-life values of EFdA-TP and AZT-TP were ∼17 and ∼3 h, respectively. When MT-4 cells were cultured with 0.01 μM EFdA for 24 h, thoroughly washed to remove EFdA, further cultured without EFdA for various periods of time, exposed to HIV-1NL4-3, and cultured for an additional 5 days, the protection values were 75 and 47%, respectively, after 24 and 48 h with no drug incubation, while those with 1 μM AZT were 55 and 9.2%, respectively. The 50% inhibitory concentration values of EFdA-TP against human polymerases α, β, and γ were >100 μM, >100 μM, and 10 μM, respectively, while those of ddA-TP were >100 μM, 0.2 μM, and 0.2 μM, respectively. These data warrant further development of EFdA as a potential therapeutic agent for those patients who harbor wild-type HIV-1 and/or multidrug-resistant variants.

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

2009 ◽  
Vol 90 (11) ◽  
pp. 2777-2787 ◽  
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.

scholarly journals Multiple human immunodeficiency virus type 1 Nef functions contribute to efficient replication in primary human macrophages

2004 ◽  
Vol 85 (6) ◽  
pp. 1463-1469 ◽  
Author(s):  
Amanda Brown ◽  
Shaghayegh Moghaddam ◽  
Thomas Kawano ◽  
Cecilia Cheng-Mayer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Single Point ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Efficient Replication ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) Nef protein has been shown to accelerate viral growth kinetics in primary human T-lymphocytes and macrophages; however, the specific function(s) of Nef responsible for this phenotype in macrophages is unknown. To address this issue, mutants of a molecularly cloned macrophage-tropic isolate, HIV-1SF162, were generated expressing single point mutations that abrogate the ability of Nef to interact with cellular kinases or mediate CD4 down-regulation. Infection of primary monocyte-derived macrophages (MDM) with these mutant viruses revealed that residues in the PXXP motif contribute to efficient replication. Interestingly, viruses expressing alleles of Nef defective in CD4 down-modulation activity retain wild-type levels of infectivity in single-round assays but exhibited delayed replication kinetics and grew to lower titres compared to the wild-type virus in MDM. These data suggest that efficient HIV-1 replication is dependent on the ability of Nef to interact with cellular kinases and remove CD4 from the surface of infected macrophages.

scholarly journals High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

2005 ◽  
Vol 49 (11) ◽  
pp. 4546-4554 ◽  
Author(s):  
Reynel Cancio ◽  
Romano Silvestri ◽  
Rino Ragno ◽  
Marino Artico ◽  
Gabriella De Martino ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mechanism Of Action ◽  
Drug Resistant ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.

scholarly journals Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

2005 ◽  
Vol 49 (5) ◽  
pp. 1761-1769 ◽  
Author(s):  
Anthony J. Smith ◽  
Peter R. Meyer ◽  
Deshratn Asthana ◽  
Margarita R. Ashman ◽  
Walter A. Scott
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type ◽  
Cell Extracts ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3′-azido-3′-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. Cell extracts incubated with HIV-1 RT and [32P]ddAMP-terminated DNA primer/template gave rise to 32P-labeled adenosine 2′,3′-dideoxyadenosine 5′,5′′′−P1,P4-tetraphosphate (Ap4ddA), ddATP, Gp4ddA, and Ap3ddA, corresponding to the transfer of [32P]ddAMP to ATP, PPi, GTP, and ADP, respectively. Incubation with [32P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous 32P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PPi levels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4+ or CD8+ T cells, while PPi was present at 7 to 15 μM. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4+ or CD8+ T cells contained 1.4 to 2.7 mM ATP and 55 to 79 μM PPi. These cellular PPi concentrations are lower than previously reported; nonetheless, the PPi-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PPi-dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.

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