scholarly journals A unique conformational escape reaction of HIV-1 against an allosteric integrase inhibitor

2019 ◽  
Author(s):  
Tomofumi Nakamura ◽  
Teruya Nakamura ◽  
Masayuki Amano ◽  
Toshikazu Miyakawa ◽  
Yuriko Yamagata ◽  
...  
Keyword(s):  
Rna Binding ◽  
Integrase Inhibitor ◽  
Catalytic Core ◽  
Viral Particles ◽  
Dimerization Interface ◽  
Conformational Alteration ◽  
Escape Reaction ◽  
Viral Maturation ◽  
Anti Hiv ◽  
Hiv 1

AbstractHIV-1 integrase (IN) contributes to HIV-1 RNA binding, which is required for viral maturation. Non-catalytic site integrase inhibitors (NCINIs) have been developed as allosteric IN inhibitors, which perform anti-HIV-1 activity by disrupting IN multimerization. Here, we show that IN undergoes a novel conformational alteration to escape from NCINIs. We observed that NCINI-resistant HIV-1 variants have accumulated amino acid (AA) mutations in the IN-encoding region. We employed HPLC and thermal stability assays to show that the AA mutations affect the folding and dimerization interface of the IN catalytic core domains, resulting in severely decreased multimerization of full-length IN proteins (IN under-multimerization). The under-multimerization of IN was finally restored by HIV-1 RNA in the viral particles. Our study demonstrates that HIV-1 countervails NCINIs by IN under-multimerization as a novel escape mechanism. Our findings provide information on the understanding of IN multimerization and influence the development of unique anti-HIV-1 strategies.

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

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.

scholarly journals A highly potent and safe pyrrolopyridine-based allosteric HIV-1 integrase inhibitor targeting host LEDGF/p75-integrase interaction site

2021 ◽  
Vol 17 (7) ◽  
pp. e1009671
Author(s):  
Tatsuya Maehigashi ◽  
Seohyun Ahn ◽  
Uk-Il Kim ◽  
Jared Lindenberger ◽  
Adrian Oo ◽  
...  
Keyword(s):  
Therapeutic Index ◽  
Integrase Inhibitor ◽  
Binding Pocket ◽  
Viral Particles ◽  
Viral Maturation ◽  
Biochemical Analyses ◽  
Rna Interaction ◽  
Anti Hiv Agents ◽  
Hiv 1

Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC50 in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.

scholarly journals Study on Suitable Analysis Method for HIV-1 Non-catalytic Integrase Inhibitor

2020 ◽  
Author(s):  
Ki Hoon Park ◽  
Minjee Kim ◽  
Seoung Eun Bae ◽  
Hee Jung Lee ◽  
Kyung-Chang Kim ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Integrase Inhibitor ◽  
Treatment Method ◽  
Strand Transfer ◽  
Inhibition Assay ◽  
Analysis Method ◽  
Catalytic Core ◽  
Host Chromosome ◽  
Inconsistent Result ◽  
Hiv 1

Abstract Background: Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy (HAART) is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain (CCD) of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs. Methods: In this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3'-processing inhibition assay were performed. Results: During screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication. Conclusion: Accordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

scholarly journals Structural-Functional Analysis of 2,1,3-Benzoxadiazoles and Their N-oxides As HIV-1 Integrase Inhibitors

Acta Naturae ◽  
2013 ◽  
Vol 5 (1) ◽  
pp. 63-72 ◽  
Author(s):  
S. P. Korolev ◽  
O. V. Kondrashina ◽  
D. S. Druzhilovsky ◽  
A. M. Starosotnikov ◽  
M. D. Dutov ◽  
...  
Keyword(s):  
Integrase Inhibitor ◽  
Integrase Inhibitors ◽  
Mechanism Of Inhibition ◽  
Immunodeficiency Virus ◽  
Pharmacokinetic Properties ◽  
Nitro Derivatives ◽  
Derivatives Of ◽  
Anti Hiv ◽  
Hiv 1

Human immunodeficiency virus type 1 integrase is one of the most attractive targets for the development of anti-HIV-1 inhibitors. The capacity of a series of 2,1,3-benzoxadiazoles (benzofurazans) and their N-oxides (benzofuroxans) selected using the PASS software to inhibit the catalytic activity of HIV-1 integrase was studied in the present work. Only the nitro-derivatives of these compounds were found to display inhibitory activity. The study of the mechanism of inhibition by nitro-benzofurazans/benzofuroxans showed that they impede the substrate DNA binding at the integrase active site. These inhibitors were also active against integrase mutants resistant to raltegravir, which is the first HIV-1 integrase inhibitor approved for clinical use. The comparison of computer-aided estimations of the pharmacodynamic and pharmacokinetic properties of the compounds studied and raltegravir led us to conclude that these compounds show promise and need to be further studied as potential HIV-1 integrase inhibitors.

scholarly journals Preclinical Profile of BI 224436, a Novel HIV-1 Non-Catalytic-Site Integrase Inhibitor

2014 ◽  
Vol 58 (6) ◽  
pp. 3233-3244 ◽  
Author(s):  
Craig Fenwick ◽  
Ma'an Amad ◽  
Murray D. Bailey ◽  
Richard Bethell ◽  
Michael Bös ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Phase 1 ◽  
Integrase Inhibitor ◽  
Parallel Synthesis ◽  
Cell Permeability ◽  
Strand Transfer ◽  
Primary Resistance ◽  
Catalytic Core ◽  
Hiv 1

ABSTRACTBI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3′-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 μM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-likein vitroabsorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%;F, 82%), and dog (CL, 8%;F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.

scholarly journals Study on Suitable Analysis Method for HIV-1 Non-catalytic Integrase Inhibitor

2020 ◽  
Author(s):  
Ki Hoon Park ◽  
Minjee Kim ◽  
Seoung Eun Bae ◽  
Hee Jung Lee ◽  
Kyung-Chang Kim ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Integrase Inhibitor ◽  
Treatment Method ◽  
Strand Transfer ◽  
Inhibition Assay ◽  
Analysis Method ◽  
Catalytic Core ◽  
Host Chromosome ◽  
Inconsistent Result ◽  
Hiv 1

Abstract Background: Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy (HAART) is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain (CCD) of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs. Methods: In this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3'-processing inhibition assay were performed. Results: During screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication. Conclusion: Accordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

scholarly journals Association of Potent Human Antiviral Cytidine Deaminases with 7SL RNA and Viral RNP in HIV-1 Virions

2010 ◽  
Vol 84 (24) ◽  
pp. 12903-12913 ◽  
Author(s):  
Wenyan Zhang ◽  
Juan Du ◽  
Kevin Yu ◽  
Tao Wang ◽  
Xiong Yong ◽  
...  
Keyword(s):  
Rna Binding ◽  
Signal Recognition Particle ◽  
Viral Inhibition ◽  
Cytidine Deaminases ◽  
Amino Terminal ◽  
7Sl Rna ◽  
Rnp Complex ◽  
Rna Interaction ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT 7SL RNA promotes the formation of the signal recognition particle that targets secretory and membrane proteins to the endoplasmic reticulum. 7SL RNA is also selectively packaged by many retroviruses, including HIV-1. Here, we demonstrate that 7SL RNA is an integral component of the viral ribonucleoprotein (RNP) complex containing Gag, viral genomic RNA, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{3}^{Lys}\) \end{document} . Only the potent anti-HIV-1 cytidine deaminases can bind to 7SL RNA and target to HIV-1 RNP. A conserved motif in the amino-terminal region of A3G is important for 7SL RNA interaction. The weak anti-HIV-1 A3C did not interact with 7SL RNA and failed to target to viral RNPs, despite efficient virion packaging. However, a chimeric construct of A3C plus the 7SL-binding amino terminus of A3G did target to viral RNPs and showed enhanced anti-HIV-1 activity. 7SL RNA binding is a conserved feature of human anti-HIV-1 cytidine deaminases. Thus, potent anti-HIV-1 cytidine deaminases have evolved to possess a unique RNA-binding ability for precise HIV-1 targeting and viral inhibition.

scholarly journals A small molecule, ACAi-028, with anti-HIV-1 activity targets a novel hydrophobic pocket on HIV-1 capsid

2021 ◽  
Author(s):  
Tomofumi Nakamura ◽  
Travis chia ◽  
Masayuki Amano ◽  
Nobutoki Takamune ◽  
Masao Matsuoka ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Reverse Transcriptase ◽  
Small Molecule ◽  
Early Stage ◽  
Integrase Inhibitor ◽  
Ionization Mass ◽  
Hydrophobic Pocket ◽  
Binding Model ◽  
Anti Hiv ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) capsid (CA) is an essential viral component of HIV-1 infection, and an attractive therapeutic target for antivirals. We report that a small molecule, ACAi-028, inhibits HIV-1 replication by targeting a hydrophobic pocket in the N-terminal domain of CA (CA-NTD). ACAi-028 is one of more than 40 candidate anti-HIV-1 compounds identified by in silico screening and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Our binding model showed that ACAi-028 interacts with the Q13, S16, and T19 amino acid residues, via hydrogen bonds, in the targeting pocket of CA-NTD. Using recombinant fusion methods, TZM-bl, time-of-addition, and colorimetric reverse transcriptase (RT) assays, the compound was found to exert anti-HIV-1 activity in the early stage between a reverse transcriptase inhibitor, azidothymidine (AZT), and an integrase inhibitor, raltegravir (RAL), without any effect on RT activity, suggesting that this compound may affect HIV-1 core disassembly (uncoating). Moreover, electrospray ionization mass spectrometry (ESI-MS) also showed that the compound binds directly and non-covalently to the CA monomer. CA multimerization and thermal stability assays showed that ACAi-028 decreased CA multimerization and thermal stability via S16 or T19 residues.

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