A Modified P1 Moiety Enhancesin vitroAntiviral Activity against Various Multi-Drug-Resistant HIV-1 Variants andin vitroCNS Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413

We here report that GRL-10413, a novel non-peptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (EC50: 0.00035 - 0.0018 μM) with minimal cytotoxicity (CC50: 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1NL4-3variants selected by up to 5 μM concentrations of atazanavir, lopinavir, or amprenavir (EC50: 0.0021 - 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multi-drug-resistant clinical HIV-1 variants isolated from patients, who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that of APV. In addition, GRL-10413 showed a favorable central nervous system (CNS) penetration property as assessed with anin vitroblood brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active-site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants with favorable CNS-penetration capability and that the newly modified P1-moiety may confer desirable features in designing novel anti-HIV-1 PIs.

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A Novel Tricyclic Ligand-Containing Nonpeptidic HIV-1 Protease Inhibitor, GRL-0739, Effectively Inhibits the Replication of Multidrug-Resistant HIV-1 Variants and Has a Desirable Central Nervous System Penetration PropertyIn Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04757-14 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2625-2635 ◽

Cited By ~ 5

Author(s):

Masayuki Amano ◽

Yasushi Tojo ◽

Pedro Miguel Salcedo-Gómez ◽

Garth L. Parham ◽

Prasanth R. Nyalapatla ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Protease Inhibitor ◽

Serum Proteins ◽

Multidrug Resistant ◽

Highly Active ◽

Cns Penetration ◽

Anti Hiv ◽

Hiv 1

ABSTRACTWe report here that GRL-0739, a novel nonpeptidic HIV-1 protease inhibitor containing a tricycle (cyclohexyl-bis-tetrahydrofuranylurethane [THF]) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0019 to 0.0036 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC50], 21.0 μM). GRL-0739 blocked the infectivity and replication of HIV-1NL4-3variants selected by concentrations of up to 5 μM ritonavir or atazanavir (EC50, 0.035 to 0.058 μM). GRL-0739 was also highly active against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, as well as against the HIV-2RODvariant. The development of resistance against GRL-0739 was substantially delayed compared to that of amprenavir (APV). The effects of the nonspecific binding of human serum proteins on the anti-HIV-1 activity of GRL-0739 were insignificant. In addition, GRL-0739 showed a desirable central nervous system (CNS) penetration property, as assessed using a novelin vitroblood-brain barrier model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and make greater van der Waals contacts with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has desirable features as a compound with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene confers highly desirable anti-HIV-1 potency in the design of novel protease inhibitors with greater CNS penetration profiles.

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Efficient Identification of Human Immunodeficiency Virus Type 1 Mutants Resistant to a Protease Inhibitor by Using a Random Mutant Library

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00687-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5090-5098 ◽

Cited By ~ 1

Author(s):

Sanggu Kim ◽

Yun-Cheol Kim ◽

Hangfei Qi ◽

Kunkai Su ◽

Sherie L. Morrison ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Drug Resistance ◽

Protease Inhibitor ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Drug Resistant ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACTEmergence of drug-resistant mutant viruses during the course of antiretroviral therapy is a major hurdle that limits the success of chemotherapeutic treatment to suppress human immunodeficiency virus type 1 (HIV-1) replication and AIDS progression. Development of new drugs and careful patient management based on resistance genotyping data are important for enhancing therapeutic efficacy. However, identifying changes leading to drug resistance can take years of clinical studies, and conventionalin vitroassays are limited in generating reliable drug resistance data. Here we present an efficientin vitroscreening assay for selecting drug-resistant variants from a library of randomly mutated HIV-1 strains generated by transposon-directed base-exchange mutagenesis. As a test of principle, we screened a library of mutant HIV-1 strains containing random mutations in the protease gene by using a reporter T-cell line in the presence of the protease inhibitor (PI) nelfinavir (NFV). Analysis of replicating viruses from a single round of infection identified 50 amino acid substitutions at 35 HIV-1 protease residue positions. The selected mutant viruses showed specific resistance to NFV and included most of the known NFV resistance mutations. Therefore, the new assay is efficient for identifying changes leading to drug resistance. The data also provide insights into the molecular mechanisms underlying the development of drug resistance.

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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

Journal of Virology ◽

10.1128/jvi.00967-10 ◽

2010 ◽

Vol 84 (22) ◽

pp. 11961-11969 ◽

Cited By ~ 65

Author(s):

Yasuhiro Koh ◽

Masayuki Amano ◽

Tomomi Towata ◽

Matthew Danish ◽

Sofiya Leshchenko-Yashchuk ◽

...

Keyword(s):

Homologous Recombination ◽

In Vitro Selection ◽

Multiple Drug ◽

Drug Resistant ◽

Genetic Barrier ◽

Development Of Resistance ◽

Multiple Drug Resistant ◽

Moderately Resistant ◽

Hiv 1

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.

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GRL-09510, a Unique P2-Crown-Tetrahydrofuranylurethane -Containing HIV-1 Protease Inhibitor, Maintains Its Favorable Antiviral Activity against Highly-Drug-Resistant HIV-1 Variants in vitro

Scientific Reports ◽

10.1038/s41598-017-12052-9 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 5

Author(s):

Masayuki Amano ◽

Pedro Miguel Salcedo-Gómez ◽

Ravikiran S. Yedidi ◽

Nicole S. Delino ◽

Hirotomo Nakata ◽

...

Keyword(s):

Antiviral Activity ◽

Protease Inhibitor ◽

Drug Resistant ◽

Hiv 1

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In Vitro Antiviral Activity and Cross-Resistance Profile of PL-100, a Novel Protease Inhibitor of Human Immunodeficiency Virus Type 1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00149-07 ◽

2007 ◽

Vol 51 (11) ◽

pp. 4036-4043 ◽

Cited By ~ 21

Author(s):

Serge Dandache ◽

Guy Sévigny ◽

Jocelyn Yelle ◽

Brent R. Stranix ◽

Neil Parkin ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antiviral Activity ◽

Highly Active Antiretroviral Therapy ◽

Cross Resistance ◽

Drug Resistant ◽

Resistance Profile ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Despite the success of highly active antiretroviral therapy, the current emergence and spread of drug-resistant variants of human immunodeficiency virus (HIV) stress the need for new inhibitors with distinct properties. We designed, produced, and screened a library of compounds based on an original l-lysine scaffold for their potentials as HIV type 1 (HIV-1) protease inhibitors (PI). One candidate compound, PL-100, emerged as a specific and noncytotoxic PI that exhibited potent inhibition of HIV-1 protease and viral replication in vitro (Ki , ∼36 pM, and 50% effective concentration [EC50], ∼16 nM, respectively). To confirm that PL-100 possessed a favorable resistance profile, we performed a cross-resistance study using a panel of 63 viral strains from PI-experienced patients selected for the presence of primary PI mutations known to confer resistance to multiple PIs now in clinical use. The results showed that PL-100 retained excellent antiviral activity against almost all of these PI-resistant viruses and that its performance in this regard was superior to those of atazanavir, amprenavir, indinavir, lopinavir, nelfinavir, and saquinavir. In almost every case, the increase in the EC50 for PL-100 observed with viruses containing multiple mutations in protease was far less than that obtained with the other drugs tested. These data underscore the potential for PL-100 to be used in the treatment of drug-resistant HIV disease and argue for its further development.

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Mechanism of Action and In Vitro Activity of 1′,3′-Dioxolanylpurine Nucleoside Analogues against Sensitive and Drug-Resistant Human Immunodeficiency Virus Type 1 Variants

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.10.2376 ◽

1999 ◽

Vol 43 (10) ◽

pp. 2376-2382 ◽

Cited By ~ 51

Author(s):

Zhengxian Gu ◽

Mark A. Wainberg ◽

Nghe Nguyen-Ba ◽

Lucille L’Heureux ◽

Jean-Marc de Muys ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Mononuclear Cells ◽

Nucleoside Analogues ◽

Drug Resistant ◽

Immunodeficiency Virus ◽

Blood Mononuclear Cells ◽

Anti Hiv ◽

Hiv 1

ABSTRACT (−)-β-d-1′,3′-Dioxolane guanosine (DXG) and 2,6-diaminopurine (DAPD) dioxolanyl nucleoside analogues have been reported to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1). We have recently conducted experiments to more fully characterize their in vitro anti-HIV-1 profiles. Antiviral assays performed in cell culture systems determined that DXG had 50% effective concentrations of 0.046 and 0.085 μM when evaluated against HIV-1IIIB in cord blood mononuclear cells and MT-2 cells, respectively. These values indicate that DXG is approximately equipotent to 2′,3′-dideoxy-3′-thiacytidine (3TC) but 5- to 10-fold less potent than 3′-azido-2′,3′-dideoxythymidine (AZT) in the two cell systems tested. At the same time, DAPD was approximately 5- to 20-fold less active than DXG in the anti-HIV-1 assays. When recombinant or clinical variants of HIV-1 were used to assess the efficacy of the purine nucleoside analogues against drug-resistant HIV-1, it was observed that AZT-resistant virus remained sensitive to DXG and DAPD. Virus harboring a mutation(s) which conferred decreased sensitivity to 3TC, 2′,3′-dideoxyinosine, and 2′,3′-dideoxycytidine, such as a 65R, 74V, or 184V mutation in the viral reverse transcriptase (RT), exhibited a two- to fivefold-decreased susceptibility to DXG or DAPD. When nonnucleoside RT inhibitor-resistant and protease inhibitor-resistant viruses were tested, no change in virus sensitivity to DXG or DAPD was observed. In vitro drug combination assays indicated that DXG had synergistic antiviral effects when used in combination with AZT, 3TC, or nevirapine. In cellular toxicity analyses, DXG and DAPD had 50% cytotoxic concentrations of greater than 500 μM when tested in peripheral blood mononuclear cells and a variety of human tumor and normal cell lines. The triphosphate form of DXG competed with the natural nucleotide substrates and acted as a chain terminator of the nascent DNA. These data suggest that DXG triphosphate may be the active intracellular metabolite, consistent with the mechanism by which other nucleoside analogues inhibit HIV-1 replication. Our results suggest that the use of DXG and DAPD as therapeutic agents for HIV-1 infection should be explored.

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Novel bis-Tetrahydrofuranylurethane-Containing Nonpeptidic Protease Inhibitor (PI) UIC-94017 (TMC114) with Potent Activity against Multi-PI-Resistant Human Immunodeficiency Virus In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.10.3123-3129.2003 ◽

2003 ◽

Vol 47 (10) ◽

pp. 3123-3129 ◽

Cited By ~ 256

Author(s):

Yasuhiro Koh ◽

Hirotomo Nakata ◽

Kenji Maeda ◽

Hiromi Ogata ◽

Geoffrey Bilcer ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitor ◽

Therapeutic Agent ◽

Close Contact ◽

Wide Spectrum ◽

Antiviral Agents ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We designed, synthesized, and identified UIC-94017 (TMC114), a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing a 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) and a sulfonamide isostere which is extremely potent against laboratory HIV-1 strains and primary clinical isolates (50% inhibitory concentration [IC50], ∼0.003 μM; IC90, ∼0.009 μM) with minimal cytotoxicity (50% cytotoxic concentration for CD4+ MT-2 cells, 74 μM). UIC-94017 blocked the infectivity and replication of each of HIV-1NL4-3 variants exposed to and selected for resistance to saquinavir, indinavir, nelfinavir, or ritonavir at concentrations up to 5 μM (IC50s, 0.003 to 0.029 μM), although it was less active against HIV-1NL4-3 variants selected for resistance to amprenavir (IC50, 0.22 μM). UIC-94017 was also potent against multi-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. Structural analyses revealed that the close contact of UIC-94017 with the main chains of the protease active-site amino acids (Asp-29 and Asp-30) is important for its potency and wide spectrum of activity against multi-PI-resistant HIV-1 variants. Considering the favorable pharmacokinetics of UIC-94017 when administered with ritonavir, the present data warrant that UIC-94017 be further developed as a potential therapeutic agent for the treatment of primary and multi-PI-resistant HIV-1 infections.

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