Performance of Affinity-Improved DARPin Targeting HIV Capsid Domain in Interference of Viral Progeny Production

Previously, a designed ankyrin repeat protein, AnkGAG1D4, was generated for intracellular targeting of the HIV-1 capsid domain. The efficiency was satisfactory in interfering with the HIV assembly process. Consequently, improved AnkGAG1D4 binding affinity was introduced by substituting tyrosine (Y) for serine (S) at position 45. However, the intracellular anti-HIV-1 activity of AnkGAG1D4-S45Y has not yet been validated. In this study, the performance of AnkGAG1D4 and AnkGAG1D4-S45Y in inhibiting wild-type HIV-1 and HIV-1 maturation inhibitor-resistant replication in SupT1 cells was evaluated. HIV-1 p24 and viral load assays were used to verify the biological activity of AnkGAG1D4 and AnkGAG1D4-S45Y as assembly inhibitors. In addition, retardation of syncytium formation in infected SupT1 cells was observed. Of note, the defense mechanism of both ankyrins did not induce the mutation of target amino acids in the capsid domain. The present data show that the potency of AnkGAG1D4-S45Y was superior to AnkGAG1D4 in interrupting either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain.

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In-Vitro Virucidal and Virustatic anti HIV-1 Effects of Extracts from Persea Americana Mill, (Avocado) Leaves

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029600700401 ◽

1996 ◽

Vol 7 (4) ◽

pp. 179-183 ◽

Cited By ~ 12

Author(s):

M.D. Wigg ◽

A.A. Al-Jabri ◽

S.S. Costa ◽

E. Race ◽

B. Bodo ◽

...

Keyword(s):

Syncytium Formation ◽

Persea Americana ◽

Reverse Phase ◽

Methanolic Extracts ◽

P24 Antigen ◽

Virucidal Effect ◽

First Time ◽

Anti Hiv ◽

Hiv 1

Aqueous (PA1) and methanolic extracts (PA2a–d; PA3) from the tropical tree Persea americana Mill. (Lauraceae), were evaluated for their cellular toxicity and anti-HIV-1 activity both in virustatic and virucidal assays. With the exception of PA3 and PA2d, all extracts showed anti-HIV-1 activity at concentrations which were not toxic for the H9 indicator cells. From the methanol insoluble extract (PA2) four different fractions (PA2a–d) were obtained using reverse-phase column chromatography, and two of the fractions (b and c) showed detectable virucidal effect. One fraction (PA2a) showed virustatic effects inhibiting HIV syncytium formation and viral p24 antigen formation at concentrations which were not toxic for the indicator cells. The results demonstrate for the first time that extracts from P. americana leaves have moderate anti-HIV-1 activity in vitro.

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Novel Protease Inhibitors Containing C-5-Modified bis-Tetrahydrofuranylurethane and Aminobenzothiazole as P2 and P2′ Ligands That Exert Potent Antiviral Activity against Highly Multidrug-Resistant HIV-1 with a High Genetic Barrier against the Emergence of Drug Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00372-19 ◽

2019 ◽

Vol 63 (8) ◽

Cited By ~ 3

Author(s):

Yuki Takamatsu ◽

Manabu Aoki ◽

Haydar Bulut ◽

Debananda Das ◽

Masayuki Amano ◽

...

Keyword(s):

Multidrug Resistant ◽

Van Der Waals Interactions ◽

Wild Type ◽

Genetic Barrier ◽

Mortality And Morbidity ◽

Polar Interactions ◽

Further Development ◽

Anti Hiv ◽

Hiv 1

ABSTRACT Combination antiretroviral therapy has achieved dramatic reductions in the mortality and morbidity in people with HIV-1 infection. Darunavir (DRV) represents a most efficacious and well-tolerated protease inhibitor (PI) with a high genetic barrier to the emergence of drug-resistant HIV-1. However, highly DRV-resistant variants have been reported in patients receiving long-term DRV-containing regimens. Here, we report three novel HIV-1 PIs (GRL-057-14, GRL-058-14, and GRL-059-14), all of which contain a P2-amino-substituted-bis-tetrahydrofuranylurethane (bis-THF) and a P2′-cyclopropyl-amino-benzothiazole (Cp-Abt). These PIs not only potently inhibit the replication of wild-type HIV-1 (50% effective concentration [EC50], 0.22 nM to 10.4 nM) but also inhibit multi-PI-resistant HIV-1 variants, including highly DRV-resistant HIVDRVRP51 (EC50, 1.6 nM to 30.7 nM). The emergence of HIV-1 variants resistant to the three compounds was much delayed in selection experiments compared to resistance to DRV, using a mixture of 11 highly multi-PI-resistant HIV-1 isolates as a starting HIV-1 population. GRL-057-14 showed the most potent anti-HIV-1 activity and greatest thermal stability with wild-type protease, and potently inhibited HIV-1 protease’s proteolytic activity (Ki value, 0.10 nM) among the three PIs. Structural models indicate that the C-5-isopropylamino-bis-THF moiety of GRL-057-14 forms additional polar interactions with the active site of HIV-1 protease. Moreover, GRL-057-14’s P1-bis-fluoro-methylbenzene forms strong hydrogen bonding and effective van der Waals interactions. The present data suggest that the combination of C-5-aminoalkyl-bis-THF, P1-bis-fluoro-methylbenzene, and P2ʹ-Cp-Abt confers highly potent activity against wild-type and multi-PI-resistant HIV strains and warrant further development of the three PIs, in particular, that of GRL-057-14, as potential therapeutic for HIV-1 infection and AIDS.

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Anti-Human Immunodeficiency Virus Activity of YK-FH312 (a Betulinic Acid Derivative), a Novel Compound Blocking Viral Maturation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.4.1225-1230.2001 ◽

2001 ◽

Vol 45 (4) ◽

pp. 1225-1230 ◽

Cited By ~ 83

Author(s):

Taisei Kanamoto ◽

Yoshiki Kashiwada ◽

Kenji Kanbara ◽

Kazuyo Gotoh ◽

Manabu Yoshimori ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Betulinic Acid ◽

Electron Microscopic Observation ◽

Syncytium Formation ◽

Inhibition Assay ◽

Hiv Replication ◽

Electron Microscopic ◽

Immunodeficiency Virus ◽

Anti Hiv ◽

Hiv 1

ABSTRACT Betulinic acid, a triterpenoid isolated from the methyl alcohol extract of the leaves of Syzigium claviflorum, was found to have a potent inhibitory activity against human immunodeficiency virus type 1 (HIV-1). Betulinic acid derivatives were synthesized to enhance the anti-HIV activity. Among the derivatives, 3-O-(3′,3′-dimethylsuccinyl) betulinic acid, designated YK-FH312, showed the highest activity against HIV-induced cytopathic effects in HIV-1-infected MT-4 cells. To determine the step(s) of HIV replication affected by YK-FH312, a syncytium formation inhibition assay in MOLT-4/HIV-1IIIB and MOLT-4 coculture, a multinuclear-activation-of-galactosidase-indicator (MAGI) assay in MAGI-CCR5 cells, electron microscopic observation, and a time-of-addition assay were performed. In the syncytium formation inhibition assay or in the MAGI assay for de novo infection, the compound did not show inhibitory effects against HIV replication. Conversely, no virions were detected in HIV-1-infected cell cultures treated with YK-FH312 either by electron microscopic observation or by viral yield in the supernatant. In accordance with a p24 enzyme-linked immunosorbent assay of culture supernatant in the time-of-addition assay, YK-FH312 inhibited virus expression in the supernatant when it was added 18 h postinfection. However, Western blot analysis of the cells in the time-of-addition assay revealed that the production of viral proteins in the cells was not inhibited completely by YK-FH312. These results suggest that YK-FH312 might affect the step(s) of virion assembly and/or budding of virions, and this is a novel mechanism of action of an anti-HIV compound.

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Negatively charged albumins exhibit a potent invitro anti-HIV-1 activity and a unique inhibitory action on syncytium formation

Antiviral Research ◽

10.1016/0166-3542(91)90157-m ◽

1991 ◽

Vol 15 ◽

pp. 81

Author(s):

R.W. Jansen ◽

G. Molema ◽

R. Pauwels ◽

D. Schols ◽

E. De Clercq ◽

...

Keyword(s):

Inhibitory Action ◽

Syncytium Formation ◽

Anti Hiv ◽

Hiv 1

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GRL-079, a Novel HIV-1 Protease Inhibitor, Is Extremely Potent against Multidrug-Resistant HIV-1 Variants and Has a High Genetic Barrier against the Emergence of Resistant Variants

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02060-17 ◽

2018 ◽

Vol 62 (5) ◽

Cited By ~ 3

Author(s):

Nicole S. Delino ◽

Manabu Aoki ◽

Hironori Hayashi ◽

Shin-ichiro Hattori ◽

Simon B. Chang ◽

...

Keyword(s):

Wide Spectrum ◽

Multidrug Resistant ◽

Strong Hydrogen Bond ◽

Wild Type ◽

Genetic Barrier ◽

Hydrogen Bond Interactions ◽

Immunodeficiency Virus ◽

Anti Hiv ◽

Hiv 1

ABSTRACT We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2′ cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (EC 50 s) were 2.5 to 30 nM against wild-type HIV-1 NL4-3 , 0.3 to 6.7 nM against HIV-2 EHO , and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIV MDR ). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIV DRV r p51 ), with EC 50 s of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (EC 50 s of >1,000 nM) against HIV DRV r p51 . Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2′ Cp-Abt group forms strong hydrogen bonds with Asp30′. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIV MDR strains (HIV 11MIX ), HIV 11MIX became highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV 11MIX failed to replicate at >0.08 μM and >0.2 μM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2′ Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1 NL4-3 and a wide spectrum of HIV MDR strains.

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Profile of Resistance of Human Immunodeficiency Virus to Mannose-Specific Plant Lectins

Journal of Virology ◽

10.1128/jvi.78.19.10617-10627.2004 ◽

2004 ◽

Vol 78 (19) ◽

pp. 10617-10627 ◽

Cited By ~ 72

Author(s):

Jan Balzarini ◽

Kristel Van Laethem ◽

Sigrid Hatse ◽

Kurt Vermeire ◽

Erik De Clercq ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Syncytium Formation ◽

Cross Resistance ◽

Blue Green Algae ◽

Plant Lectins ◽

Glycosylation Sites ◽

Immunodeficiency Virus ◽

Anti Hiv ◽

Hiv 1

ABSTRACT The mannose-specific plant lectins from the Amaryllidaceae family (e.g., Hippeastrum sp. hybrid and Galanthus nivalis) inhibit human immunodeficiency virus (HIV) infection of human lymphocytic cells in the higher nanogram per milliliter range and suppress syncytium formation between persistently HIV type 1 (HIV-1)-infected cells and uninfected CD4+ T cells. These lectins inhibit virus entry. When exposed to escalating concentrations of G. nivalis and Hippeastrum sp. hybrid agglutinin, a variety of HIV-1(IIIB) strains were isolated after 20 to 40 subcultivations which showed a decreased sensitivity to the plant lectins. Several amino acid changes in the envelope glycoprotein gp120, but not in gp41, of the mutant virus isolates were observed. The vast majority of the amino acid changes occurred at the N glycosylation sites and at the S or T residues that are part of the N glycosylation motif. The degree of resistance to the plant lectins was invariably correlated with an increasing number of mutated glycosylation sites in gp120. The nature of these mutations was entirely different from that of mutations that are known to appear in HIV-1 gp120 under the pressure of other viral entry inhibitors such as dextran sulfate, bicyclams (i.e., AMD3100), and chicoric acid, which also explains the lack of cross-resistance of plant lectin-resistant viruses to any other HIV inhibitor including T-20 and the blue-green algae (cyanobacteria)-derived mannose-specific cyanovirin. The plant lectins represent a well-defined class of anti-HIV (microbicidal) drugs with a novel HIV drug resistance profile different from those of other existing anti-HIV drugs.

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A Conformational Escape Reaction of HIV-1 against an Allosteric Integrase Inhibitor

Journal of Virology ◽

10.1128/jvi.00486-20 ◽

2020 ◽

Vol 94 (19) ◽

Author(s):

Tomofumi Nakamura ◽

Teruya Nakamura ◽

Masayuki Amano ◽

Toshikazu Miyakawa ◽

Yuriko Yamagata ◽

...

Keyword(s):

Amino Acid ◽

Mode Of Action ◽

Rna Binding ◽

Drug Resistant ◽

Wild Type ◽

Catalytic Core ◽

Escape Reaction ◽

Amino Acid Mutations ◽

Anti Hiv ◽

Hiv 1

ABSTRACT HIV-1 often acquires drug-resistant mutations in spite of the benefits of antiretroviral therapy (ART). HIV-1 integrase (IN) is essential for the concerted integration of HIV-1 DNA into the host genome. IN further contributes to HIV-1 RNA binding, which is required for HIV-1 maturation. Non-catalytic-site integrase inhibitors (NCINIs) have been developed as allosteric IN inhibitors, which perform anti-HIV-1 activity by a multimodal mode of action such as inhibition of the IN-lens epithelium-derived growth factor (LEDGF)/p75 interaction in the early stage and disruption of functional IN multimerization in the late stage of HIV-1 replication. Here, we show that IN undergoes an adaptable conformational change to escape from NCINIs. We observed that NCINI-resistant HIV-1 variants have accumulated 4 amino acid mutations by passage 26 (P26) in the IN-encoding region. We employed high-performance liquid chromatography (HPLC), thermal stability assays, and X-ray crystallographic analysis to show that some amino acid mutations affect the stability and/or dimerization interface of the IN catalytic core domains (CCDs), potentially resulting in the severely decreased multimerization of full-length IN proteins (IN undermultimerization). This undermultimerized IN via NCINI-related mutations was stabilized by HIV-1 RNA and restored to the same level as that of wild-type HIV-1 in viral particles. Recombinant HIV-1 clones with IN undermultimerization propagated similarly to wild-type HIV-1. Our study revealed that HIV-1 can eventually counteract NCINI-induced IN overmultimerization by IN undermultimerization as one of the escape mechanisms. Our findings provide information on the understanding of IN multimerization with or without HIV-1 RNA and may influence the development of anti-HIV-1 strategies. IMPORTANCE Understanding the mechanism of HIV-1 resistance to anti-HIV-1 drugs could lead to the development of novel drugs with increased efficiency, resulting in more effective ART. ART composed of more potent and long-acting anti-HIV-1 drugs can greatly improve drug adherence and also provide HIV-1 prevention such as preexposure prophylaxis. NCINIs with a multimodal mode of action exert potent anti-HIV-1 effects through IN overmultimerization during HIV-1 maturation. However, HIV-1 can acquire some mutations that cause IN undermultimerization to alleviate NCINI-induced IN overmultimerization. This undermultimerized IN was efficiently stabilized by HIV-1 RNA and restored to the same level as that of wild-type HIV-1. Our findings revealed that HIV-1 eventually acquires such a conformational escape reaction to overcome the unique NCINI actions. The investigation into drug-resistant mutations associated with HIV-1 protein multimerization may facilitate the elucidation of its molecular mechanism and functional multimerization, allowing us to develop more potent anti-HIV-1 drugs and unique treatment strategies.

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Design of Biphenyl-Substituted Diarylpyrimidines with a Cyanomethyl Linker as HIV-1 NNRTIs via a Molecular Hybridization Strategy

Molecules ◽

10.3390/molecules25051050 ◽

2020 ◽

Vol 25 (5) ◽

pp. 1050 ◽

Cited By ~ 1

Author(s):

Yuan Lei ◽

Sheng Han ◽

Yang Yang ◽

Christophe Pannecouque ◽

Erik De Clercq ◽

...

Keyword(s):

Resistant Mutant ◽

Molecular Hybridization ◽

Binding Modes ◽

Wild Type ◽

Hiv Therapy ◽

Mutant Strains ◽

Drug Resistant Mutant ◽

New Compounds ◽

Anti Hiv ◽

Hiv 1

The key problems of human immunodeficiency virus (HIV) therapy are the rapid emergence of drug-resistant mutant strains and significant cumulative drug toxicities. Therefore, there is an urgent demand for new anti-HIV agents with low toxicity and broad-spectrum antiviral potency. A series of biphenyl-substituted diarylpyrimidines with a cyanomethyl linker were designed using a molecular hybridization strategy. The cell-based anti-HIV assay showed that most of the compounds exhibited moderate to good activities against wild-type HIV-1 and clinically relevant mutant strains with a more favorable toxicity, and the enzymatic assay showed they had nanomolar activity against reverse transcriptase (RT). Compound 10p exhibited the best activity against wild-type HIV-1 with an EC50 (50% HIV-1 replication inhibitory concentration) value of 0.027 µM, an acceptable CC50 (50% cytotoxic concentration) value of 36.4 µM, and selectivity index of 1361, with moderate activities against the single mutants (EC50: E138K, 0.17 µM; Y181C, 0.87 µM; K103N, 0.9 µM; L100I, 1.21 µM, respectively), and an IC50 value of 0.059 µM against the RT enzyme, which was six-fold higher than nevirapine (NVP). The preliminary structure–activity relationship (SAR) of these new compounds was concluded. The molecular modeling predicted the binding modes of the new compounds with RT, providing molecular insight for further drug design.

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Scaffold Hopping in Discovery of HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: From CH(CN)-DABOs to CH(CN)-DAPYs

Molecules ◽

10.3390/molecules25071581 ◽

2020 ◽

Vol 25 (7) ◽

pp. 1581

Author(s):

Ting-Ting Li ◽

Christophe Pannecouque ◽

Erik De Clercq ◽

Chun-Lin Zhuang ◽

Fen-Er Chen

Keyword(s):

Reverse Transcriptase ◽

Scaffold Hopping ◽

Wild Type ◽

Reverse Transcriptase Inhibitors ◽

Nucleoside Reverse Transcriptase Inhibitors ◽

Single Digit ◽

Hydrophobic Cavity ◽

Structure Activity ◽

Anti Hiv ◽

Hiv 1

Scaffold hopping is a frequently-used strategy in the development of non-nucleoside reverse transcriptase inhibitors. Herein, CH(CN)-DAPYs were designed by hopping the cyano-methylene linker of our previous published CH(CN)-DABOs onto the etravirine (ETR). Eighteen CH(CN)-DAPYs were synthesized and evaluated for their anti-HIV activity. Most compounds exhibited promising activity against wild-type (WT) HIV-1. Compounds B4 (EC50 = 6 nM) and B6 (EC50 = 8 nM) showed single-digit nanomolar potency against WT HIV-1. Moreover, these two compounds had EC50 values of 0.06 and 0.08 μM toward the K103N mutant, respectively, which were comparable to the reference efavirenz (EFV) (EC50 = 0.08 μM). The preliminary structure–activity relationship (SAR) indicated that introducing substitutions on C2 of the 4-cyanophenyl group could improve antiviral activity. Molecular docking predicted that the cyano-methylene linker was positioned into the hydrophobic cavity formed by Y181/Y188 and V179 residues.

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Dipyridodiazepinone derivatives; synthesis and anti HIV-1 activity

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.5.36 ◽

2009 ◽

Vol 5 ◽

Cited By ~ 2

Author(s):

Nisachon Khunnawutmanotham ◽

Nitirat Chimnoi ◽

Arunee Thitithanyanont ◽

Patchreenart Saparpakorn ◽

Kiattawee Choowongkomon ◽

...

Keyword(s):

Reverse Transcriptase ◽

Reverse Transcriptase Activity ◽

Wild Type ◽

Mutant Type ◽

Transcriptase Activity ◽

Anti Hiv ◽

Hiv 1

Ten dipyridodiazepinone derivatives were synthesized and evaluated for their anti HIV-1 reverse transcriptase activity against wild-type and mutant type enzymes, K103N and Y181C. Two of them were found to be promising inhibitors for HIV-1 RT.

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