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Dinh T, Tber Z, Rey JS, Mengshetti S, Annamalai AS, Haney R, Briganti L, Amblard F, Fuchs JR, Cherepanov P, Kim K, Schinazi RF, Perilla JR, Kim B, Kvaratskhelia M. The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir. mBio 2024; 15:e0046524. [PMID: 39404354 PMCID: PMC11559089 DOI: 10.1128/mbio.00465-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2024] [Accepted: 09/16/2024] [Indexed: 10/23/2024] Open
Abstract
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents that potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into phase 2a clinical trials. Previous cell culture-based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. Although both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect the direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor-mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR-induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared with the WT virus. By rationally modifying PIR, we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was ~14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.IMPORTANCEAntiretroviral therapies save the lives of millions of people living with HIV (PLWH). However, the evolution of multi-drug-resistant viral phenotypes is a major clinical problem, and there are limited or no treatment options for heavily treatment-experienced PLWH. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a novel class of antiretroviral compounds that work by a unique mechanism of binding to the non-catalytic site on the viral protein and inducing aberrant integrase multimerization. Accordingly, ALLINIs potently inhibit both wild-type HIV-1 and all drug-resistant viral phenotypes that have so far emerged against currently used therapies. Pirmitegravir, a highly potent and safe investigational ALLINI, is currently advancing through clinical trials. Here, we have elucidated the structural and mechanistic bases behind the emergence of HIV-1 integrase mutations in infected cells that confer resistance to pirmitegravir. In turn, our findings allowed us to rationally develop an improved ALLINI with substantially enhanced potency against the pirmitegravir-resistant virus.
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Affiliation(s)
- Tung Dinh
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Zahira Tber
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children’s Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Juan S. Rey
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
| | - Seema Mengshetti
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children’s Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Arun S. Annamalai
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Reed Haney
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Lorenzo Briganti
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Franck Amblard
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children’s Healthcare of Atlanta, Atlanta, Georgia, USA
| | - James R. Fuchs
- College of Pharmacy, The Ohio State University, Columbus, Ohio, USA
| | - Peter Cherepanov
- Chromatin Structure & Mobile DNA Laboratory, The Francis Crick Institute, London, United Kingdom
| | | | - Raymond F. Schinazi
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children’s Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Juan R. Perilla
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
| | - Baek Kim
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children’s Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Mamuka Kvaratskhelia
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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Dinh T, Tber Z, Rey JS, Mengshetti S, Annamalai AS, Haney R, Briganti L, Amblard F, Fuchs JR, Cherepanov P, Kim K, Schinazi RF, Perilla JR, Kim B, Kvaratskhelia M. The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.26.577387. [PMID: 38328097 PMCID: PMC10849636 DOI: 10.1101/2024.01.26.577387] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/09/2024]
Abstract
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents which potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into Phase 2a clinical trials. Previous cell culture based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. While both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared to the WT virus. By rationally modifying PIR we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was ~14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.
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Affiliation(s)
- Tung Dinh
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Zahira Tber
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children's Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Juan S Rey
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
| | - Seema Mengshetti
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children's Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Arun S Annamalai
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Reed Haney
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Lorenzo Briganti
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Franck Amblard
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children's Healthcare of Atlanta, Atlanta, Georgia, USA
| | - James R Fuchs
- College of Pharmacy, The Ohio State University, Columbus, Ohio, United States
| | - Peter Cherepanov
- Chromatin Structure & Mobile DNA Laboratory, The Francis Crick Institute, London, United Kingdom
| | | | - Raymond F Schinazi
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children's Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Juan R Perilla
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
| | - Baek Kim
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children's Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Mamuka Kvaratskhelia
- Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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Elliott JL, Kutluay SB. Going beyond Integration: The Emerging Role of HIV-1 Integrase in Virion Morphogenesis. Viruses 2020; 12:E1005. [PMID: 32916894 PMCID: PMC7551943 DOI: 10.3390/v12091005] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 09/03/2020] [Accepted: 09/07/2020] [Indexed: 12/22/2022] Open
Abstract
The HIV-1 integrase enzyme (IN) plays a critical role in the viral life cycle by integrating the reverse-transcribed viral DNA into the host chromosome. This function of IN has been well studied, and the knowledge gained has informed the design of small molecule inhibitors that now form key components of antiretroviral therapy regimens. Recent discoveries unveiled that IN has an under-studied yet equally vital second function in human immunodeficiency virus type 1 (HIV-1) replication. This involves IN binding to the viral RNA genome in virions, which is necessary for proper virion maturation and morphogenesis. Inhibition of IN binding to the viral RNA genome results in mislocalization of the viral genome inside the virus particle, and its premature exposure and degradation in target cells. The roles of IN in integration and virion morphogenesis share a number of common elements, including interaction with viral nucleic acids and assembly of higher-order IN multimers. Herein we describe these two functions of IN within the context of the HIV-1 life cycle, how IN binding to the viral genome is coordinated by the major structural protein, Gag, and discuss the value of targeting the second role of IN in virion morphogenesis.
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Affiliation(s)
| | - Sebla B. Kutluay
- Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110, USA;
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Kleinpeter AB, Freed EO. HIV-1 Maturation: Lessons Learned from Inhibitors. Viruses 2020; 12:E940. [PMID: 32858867 PMCID: PMC7552077 DOI: 10.3390/v12090940] [Citation(s) in RCA: 67] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Revised: 08/19/2020] [Accepted: 08/20/2020] [Indexed: 02/06/2023] Open
Abstract
Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity.
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Affiliation(s)
| | - Eric O. Freed
- HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA;
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Tse C, Wickstrom L, Kvaratskhelia M, Gallicchio E, Levy R, Deng N. Exploring the Free-Energy Landscape and Thermodynamics of Protein-Protein Association. Biophys J 2020; 119:1226-1238. [PMID: 32877664 DOI: 10.1016/j.bpj.2020.08.005] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 08/04/2020] [Accepted: 08/06/2020] [Indexed: 01/30/2023] Open
Abstract
We report the free-energy landscape and thermodynamics of the protein-protein association responsible for the drug-induced multimerization of HIV-1 integrase (IN). Allosteric HIV-1 integrase inhibitors promote aberrant IN multimerization by bridging IN-IN intermolecular interactions. However, the thermodynamic driving forces and kinetics of the multimerization remain largely unknown. Here, we explore the early steps in the IN multimerization by using umbrella sampling and unbiased molecular dynamics simulations in explicit solvent. In direct simulations, the two initially separated dimers spontaneously associate to form near-native complexes that resemble the crystal structure of the aberrant tetramer. Most strikingly, the effective interaction of the protein-protein association is very short-ranged: the two dimers associate rapidly within tens of nanoseconds when their binding surfaces are separated by d ≤ 4.3 Å (less than two water diameters). Beyond this distance, the oligomerization kinetics appears to be diffusion controlled with a much longer association time. The free-energy profile also captured the crucial role of allosteric IN inhibitors in promoting multimerization and explained why several C-terminal domain mutations are remarkably resistant to the drug-induced multimerization. The results also show that at small separation, the protein-protein binding process contains two consecutive phases with distinct thermodynamic signatures. First, interprotein water molecules are expelled to the bulk, resulting in a small increase in entropy, as the solvent entropy gain from the water release is nearly cancelled by the loss of side-chain entropies as the two proteins approach each other. At shorter distances, the two dry binding surfaces adapt to each other to optimize their interaction energy at the expense of further protein configurational entropy loss. Although the binding interfaces feature clusters of hydrophobic residues, overall, the protein-protein association in this system is driven by enthalpy and opposed by entropy.
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Affiliation(s)
- Celine Tse
- Department of Chemistry and Physical Sciences, Pace University, New York, New York
| | - Lauren Wickstrom
- Borough of Manhattan Community College, the City University of New York, Department of Science, New York, New York
| | - Mamuka Kvaratskhelia
- Division of Infectious Diseases, University of Colorado School of Medicine, Aurora, Colorado
| | - Emilio Gallicchio
- Department of Chemistry, Brooklyn College, the City University of New York, Brooklyn, New York; PhD Program in Biochemistry and PhD Program in Chemistry, The Graduate Center of the City University of New York, New York, New York
| | - Ronald Levy
- Center for Biophysics and Computational Biology and Department of Chemistry, Temple University, Philadelphia, Pennsylvania
| | - Nanjie Deng
- Department of Chemistry and Physical Sciences, Pace University, New York, New York.
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Kapusta K, Sizochenko N, Anderson ML, Kolodziejczyk W, Voronkov EO, Saloni J, Leszczynski J, Hill GA. N-arylnaphthylamines as inhibitors of human immunodeficiency virus integrase - lens epithelium-derived growth factor interactions: theoretical studies. J Biomol Struct Dyn 2020; 39:867-880. [PMID: 31965914 DOI: 10.1080/07391102.2020.1719203] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Presented work reports a comprehensive theoretical study on the inhibitory nature of N-arylnaphthylamines in Human Immunodeficiency Virus Integrase (HIV IN) - Lens Epithelium-Derived Growth Factor (LEDGF/p75) complexes. Factors influencing the inhibition efficiency in AlphaScreen% assay are evaluated and explained through the structure- and ligand-based studies; including molecular docking, molecular dynamics calculations, and quantitative structure-activity relationship (QSAR) approach. It has been shown that N-arylnaphthylamines possess a wide variety of binding poses. Three QSAR models have been developed using structural descriptors and descriptors derived from docking calculations. The activity of untested N-arylnaphthylamines have been predicted using the most successful model. Proposed here technique could become a useful tool for ligand selection, accelerating the development of a new generation of anti-HIV medications. [Formula: see text] Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Karina Kapusta
- Interdisciplinary Center for Nanotoxicity, Department of Chemistry, Physics and Atmospheric Sciences, Jackson State University, Jackson, MS, USA
| | | | - Micah L Anderson
- Interdisciplinary Center for Nanotoxicity, Department of Chemistry, Physics and Atmospheric Sciences, Jackson State University, Jackson, MS, USA
| | - Wojciech Kolodziejczyk
- Interdisciplinary Center for Nanotoxicity, Department of Chemistry, Physics and Atmospheric Sciences, Jackson State University, Jackson, MS, USA
| | | | - Julia Saloni
- Interdisciplinary Center for Nanotoxicity, Department of Chemistry, Physics and Atmospheric Sciences, Jackson State University, Jackson, MS, USA
| | - Jerzy Leszczynski
- Interdisciplinary Center for Nanotoxicity, Department of Chemistry, Physics and Atmospheric Sciences, Jackson State University, Jackson, MS, USA
| | - Glake A Hill
- Interdisciplinary Center for Nanotoxicity, Department of Chemistry, Physics and Atmospheric Sciences, Jackson State University, Jackson, MS, USA
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Peese KM, Allard CW, Connolly T, Johnson BL, Li C, Patel M, Sorensen ME, Walker MA, Meanwell NA, McAuliffe B, Minassian B, Krystal M, Parker DD, Lewis HA, Kish K, Zhang P, Nolte RT, Simmermacher J, Jenkins S, Cianci C, Naidu BN. 5,6,7,8-Tetrahydro-1,6-naphthyridine Derivatives as Potent HIV-1-Integrase-Allosteric-Site Inhibitors. J Med Chem 2019; 62:1348-1361. [PMID: 30609350 DOI: 10.1021/acs.jmedchem.8b01473] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
A series of 5,6,7,8-tetrahydro-1,6-naphthyridine derivatives targeting the allosteric lens-epithelium-derived-growth-factor-p75 (LEDGF/p75)-binding site on HIV-1 integrase, an attractive target for antiviral chemotherapy, was prepared and screened for activity against HIV-1 infection in cell culture. Small molecules that bind within the LEDGF/p75-binding site promote aberrant multimerization of the integrase enzyme and are of significant interest as HIV-1-replication inhibitors. Structure-activity-relationship studies and rat pharmacokinetic studies of lead compounds are presented.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | - Robert T Nolte
- Protein Cellular and Structural Sciences , GlaxoSmithKline , 1250 South Collegeville Rd. , Collegeville , Pennsylvania 19426 , United States
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Anderson EM, Maldarelli F. The role of integration and clonal expansion in HIV infection: live long and prosper. Retrovirology 2018; 15:71. [PMID: 30352600 PMCID: PMC6199739 DOI: 10.1186/s12977-018-0448-8] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Accepted: 09/15/2018] [Indexed: 02/07/2023] Open
Abstract
Integration of viral DNA into the host genome is a central event in the replication cycle and the pathogenesis of retroviruses, including HIV. Although most cells infected with HIV are rapidly eliminated in vivo, HIV also infects long-lived cells that persist during combination antiretroviral therapy (cART). Cells with replication competent HIV proviruses form a reservoir that persists despite cART and such reservoirs are at the center of efforts to eradicate or control infection without cART. The mechanisms of persistence of these chronically infected long-lived cells is uncertain, but recent research has demonstrated that the presence of the HIV provirus has enduring effects on infected cells. Cells with integrated proviruses may persist for many years, undergo clonal expansion, and produce replication competent HIV. Even proviruses with defective genomes can produce HIV RNA and may contribute to ongoing HIV pathogenesis. New analyses of HIV infected cells suggest that over time on cART, there is a shift in the composition of the population of HIV infected cells, with the infected cells that persist over prolonged periods having proviruses integrated in genes associated with regulation of cell growth. In several cases, strong evidence indicates the presence of the provirus in specific genes may determine persistence, proliferation, or both. These data have raised the intriguing possibility that after cART is introduced, a selection process enriches for cells with proviruses integrated in genes associated with cell growth regulation. The dynamic nature of populations of cells infected with HIV during cART is not well understood, but is likely to have a profound influence on the composition of the HIV reservoir with critical consequences for HIV eradication and control strategies. As such, integration studies will shed light on understanding viral persistence and inform eradication and control strategies. Here we review the process of HIV integration, the role that integration plays in persistence, clonal expansion of the HIV reservoir, and highlight current challenges and outstanding questions for future research.
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Affiliation(s)
| | - Frank Maldarelli
- HIV Dynamics and Replication Program, NCI, NIH, Frederick, MD, 21702, USA.
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Jentsch NG, Hart AP, Hume JD, Sun J, McNeely KA, Lama C, Pigza JA, Donahue MG, Kessl JJ. Synthesis and Evaluation of Aryl Quinolines as HIV-1 Integrase Multimerization Inhibitors. ACS Med Chem Lett 2018; 9:1007-1012. [PMID: 30344908 DOI: 10.1021/acsmedchemlett.8b00269] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2018] [Accepted: 09/14/2018] [Indexed: 12/25/2022] Open
Abstract
HIV-1 integrase multimerization inhibitors have recently been established as an effective class of antiretroviral agents due to their potent ability to inhibit viral replication. Specifically, quinoline-based inhibitors have been shown to effectively impair HIV-1 replication, highlighting the importance of these heterocyclic scaffolds. Pursuant of our endeavors to further develop a library of quinoline-based candidates, we have implemented a structure-activity relationship study of trisubstituted 4-arylquinoline scaffolds that examined the integrase multimerization properties of substitution patterns at the 4-position of the quinoline. Compounds consisting of substituted phenyl rings, heteroaromatics, or polycyclic moieties were examined utilizing an integrase aberrant multimerization in vitro assay. para-Chloro-4-phenylquinoline 11b and 2,3-benzo[b][1,4]dioxine 15f showed noteworthy EC50 values of 0.10 and 0.08 μM, respectively.
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Affiliation(s)
- Nicholas G. Jentsch
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
| | - Alison P. Hart
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
| | - Jared D. Hume
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
| | - Jian Sun
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
| | - Kaitlin A. McNeely
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
| | - Chiyang Lama
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
| | - Julie A. Pigza
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
| | - Matthew G. Donahue
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
| | - Jacques J. Kessl
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States
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Abstract
Integration of the reverse-transcribed viral cDNA into the host's genome is a critical step in the lifecycle of all retroviruses. Retrovirus integration is carried out by integrase (IN), a virus-encoded enzyme that forms an oligomeric 'intasome' complex with both ends of the linear viral DNA to catalyze their concerted insertions into the backbones of the host's DNA. IN also forms a complex with host proteins, which guides the intasome to the host's chromosome. Recent structural studies have revealed remarkable diversity as well as conserved features among the architectures of the intasome assembly from different genera of retroviruses. This chapter will review how IN oligomerizes to achieve its function, with particular focus on alpharetrovirus including the avian retrovirus Rous sarcoma virus. Another chapter (Craigie) will focus on the structure and function of IN from HIV-1.
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Affiliation(s)
- Duane P Grandgenett
- Saint Louis University Health Sciences Center, Department of Microbiology and Immunology, Institute for Molecular Virology, Doisy Research Center, St. Louis, MO, USA
| | - Hideki Aihara
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
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11
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Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase. J Virol 2016; 91:JVI.02003-16. [PMID: 27795445 DOI: 10.1128/jvi.02003-16] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2016] [Accepted: 10/07/2016] [Indexed: 12/28/2022] Open
Abstract
Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. IMPORTANCE Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.
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12
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Borrenberghs D, Dirix L, De Wit F, Rocha S, Blokken J, De Houwer S, Gijsbers R, Christ F, Hofkens J, Hendrix J, Debyser Z. Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry. Sci Rep 2016; 6:36485. [PMID: 27830755 PMCID: PMC5103197 DOI: 10.1038/srep36485] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2016] [Accepted: 10/04/2016] [Indexed: 11/16/2022] Open
Abstract
Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.
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Affiliation(s)
- Doortje Borrenberghs
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium.,Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Lieve Dirix
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium.,Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Flore De Wit
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Susana Rocha
- Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Jolien Blokken
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Stéphanie De Houwer
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Rik Gijsbers
- Laboratory for Viral Vector Technology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Frauke Christ
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Johan Hofkens
- Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Jelle Hendrix
- Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Zeger Debyser
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
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13
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Deng N, Hoyte A, Mansour YE, Mohamed MS, Fuchs JR, Engelman AN, Kvaratskhelia M, Levy R. Allosteric HIV-1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter-subunit interactions: Structural and thermodynamic modeling studies. Protein Sci 2016; 25:1911-1917. [PMID: 27503276 PMCID: PMC5079246 DOI: 10.1002/pro.2997] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2016] [Accepted: 08/04/2016] [Indexed: 12/19/2022]
Abstract
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) bind at the dimer interface of the IN catalytic core domain (CCD), and potently inhibit HIV-1 by promoting aberrant, higher-order IN multimerization. Little is known about the structural organization of the inhibitor-induced IN multimers and important questions regarding how ALLINIs promote aberrant IN multimerization remain to be answered. On the basis of physical chemistry principles and from our analysis of experimental information, we propose that inhibitor-induced multimerization is mediated by ALLINIs directly promoting inter-subunit interactions between the CCD dimer and a C-terminal domain (CTD) of another IN dimer. Guided by this hypothesis, we have built atomic models of inter-subunit interfaces in IN multimers by incorporating information from hydrogen-deuterium exchange (HDX) measurements to drive protein-protein docking. We have also developed a novel free energy simulation method to estimate the effects of ALLINI binding on the association of the CCD and CTD. Using this structural and thermodynamic modeling approach, we show that multimer inter-subunit interface models can account for several experimental observations about ALLINI-induced multimerization, including large differences in the potencies of various ALLINIs, the mechanisms of resistance mutations, and the crucial role of solvent exposed R-groups in the high potency of certain ALLINIs. Our study predicts that CTD residues Tyr226, Trp235 and Lys266 are involved in the aberrant multimer interfaces. The key finding of the study is that it suggests the possibility of ALLINIs facilitating inter-subunit interactions between an external CTD and the CCD-CCD dimer interface.
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Affiliation(s)
- Nanjie Deng
- Center for Biophysics & Computational Biology/ICMS, Department of Chemistry, Temple University, Philadelphia, Pennsylvania, 19122.
| | - Ashley Hoyte
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, Ohio, 43210
| | - Yara E Mansour
- Division of Medicinal Chemistry & Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, 43210
- Faculty of Pharmacy, Pharmaceutical Organic Chemistry Department, Helwan University, Cairo, Egypt
| | - Mosaad S Mohamed
- Faculty of Pharmacy, Pharmaceutical Organic Chemistry Department, Helwan University, Cairo, Egypt
| | - James R Fuchs
- Division of Medicinal Chemistry & Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, 43210
| | - Alan N Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, 02215
| | - Mamuka Kvaratskhelia
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, Ohio, 43210
| | - Ronald Levy
- Center for Biophysics & Computational Biology/ICMS, Department of Chemistry, Temple University, Philadelphia, Pennsylvania, 19122
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14
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Galilee M, Britan-Rosich E, Griner SL, Uysal S, Baumgärtel V, Lamb DC, Kossiakoff AA, Kotler M, Stroud RM, Marx A, Alian A. The Preserved HTH-Docking Cleft of HIV-1 Integrase Is Functionally Critical. Structure 2016; 24:1936-1946. [PMID: 27692964 DOI: 10.1016/j.str.2016.08.015] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2016] [Revised: 08/19/2016] [Accepted: 08/19/2016] [Indexed: 02/06/2023]
Abstract
HIV-1 integrase (IN) catalyzes viral DNA integration into the host genome and facilitates multifunctional steps including virus particle maturation. Competency of IN to form multimeric assemblies is functionally critical, presenting an approach for anti-HIV strategies. Multimerization of IN depends on interactions between the distinct subunit domains and among the flanking protomers. Here, we elucidate an overlooked docking cleft of IN core domain that anchors the N-terminal helix-turn-helix (HTH) motif in a highly preserved and functionally critical configuration. Crystallographic structure of IN core domain in complex with Fab specifically targeting this cleft reveals a steric overlap that would inhibit HTH-docking, C-terminal domain contacts, DNA binding, and subsequent multimerization. While Fab inhibits in vitro IN integration activity, in vivo it abolishes virus particle production by specifically associating with preprocessed IN within Gag-Pol and interfering with early cytosolic Gag/Gag-Pol assemblies. The HTH-docking cleft may offer a fresh hotspot for future anti-HIV intervention strategies.
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Affiliation(s)
- Meytal Galilee
- Department of Biology, Technion - Israel Institute of Technology, Haifa 320003, Israel
| | - Elena Britan-Rosich
- Department of Immunology and Pathology, The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel
| | - Sarah L Griner
- Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Serdar Uysal
- Department of Biophysics, Bezmialem Vakif University, Istanbul 34093, Turkey
| | - Viola Baumgärtel
- Physical Chemistry, Department of Chemistry, Nanosystem Initiative Munich (NIM), Center for Integrated Protein Science Munich (CiPSM), Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Munich 81377, Germany
| | - Don C Lamb
- Physical Chemistry, Department of Chemistry, Nanosystem Initiative Munich (NIM), Center for Integrated Protein Science Munich (CiPSM), Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Munich 81377, Germany
| | - Anthony A Kossiakoff
- Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
| | - Moshe Kotler
- Department of Immunology and Pathology, The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel
| | - Robert M Stroud
- Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Ailie Marx
- Department of Biology, Technion - Israel Institute of Technology, Haifa 320003, Israel
| | - Akram Alian
- Department of Biology, Technion - Israel Institute of Technology, Haifa 320003, Israel.
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15
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Patel D, Antwi J, Koneru PC, Serrao E, Forli S, Kessl JJ, Feng L, Deng N, Levy RM, Fuchs JR, Olson AJ, Engelman AN, Bauman JD, Kvaratskhelia M, Arnold E. A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crystallographic Fragment Screening of the Catalytic Core Domain. J Biol Chem 2016; 291:23569-23577. [PMID: 27645997 DOI: 10.1074/jbc.m116.753384] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2016] [Indexed: 12/29/2022] Open
Abstract
HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal lens epithelium-derived growth factor (LEDGF)/p75 binding pocket. The most active derivative (5) inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC50 of 72 μm and impaired HIV-1 infection of T cells at an EC50 of 36 μm The identified lead compound, with a relatively small molecular weight (221 Da), provides an optimal building block for developing a new class of inhibitors. Furthermore, although structurally distinct thiophenecarboxylic acid derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead compound, 5, inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs.
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Affiliation(s)
- Disha Patel
- From the Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854
| | - Janet Antwi
- Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy and
| | - Pratibha C Koneru
- Center for Retrovirus Research and College of Pharmacy, Ohio State University, Columbus, Ohio 43210
| | - Erik Serrao
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215
| | - Stefano Forli
- Molecular Graphics Laboratory, Department of Integrative Structural and Computational Biology, MB-112, The Scripps Research Institute, La Jolla, California 92037
| | - Jacques J Kessl
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, and
| | - Lei Feng
- Center for Retrovirus Research and College of Pharmacy, Ohio State University, Columbus, Ohio 43210
| | - Nanjie Deng
- Center for Biophysics and Computational Biology, Temple University, Philadelphia, Pennsylvania 19122
| | - Ronald M Levy
- Center for Biophysics and Computational Biology, Temple University, Philadelphia, Pennsylvania 19122
| | - James R Fuchs
- Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy and
| | - Arthur J Olson
- Molecular Graphics Laboratory, Department of Integrative Structural and Computational Biology, MB-112, The Scripps Research Institute, La Jolla, California 92037
| | - Alan N Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215
| | - Joseph D Bauman
- From the Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854
| | - Mamuka Kvaratskhelia
- Center for Retrovirus Research and College of Pharmacy, Ohio State University, Columbus, Ohio 43210,
| | - Eddy Arnold
- From the Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854,
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16
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Agharbaoui FE, Hoyte AC, Ferro S, Gitto R, Buemi MR, Fuchs JR, Kvaratskhelia M, De Luca L. Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase. Eur J Med Chem 2016; 123:673-683. [PMID: 27517812 DOI: 10.1016/j.ejmech.2016.07.077] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2016] [Revised: 07/25/2016] [Accepted: 07/31/2016] [Indexed: 01/26/2023]
Abstract
Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors.
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Affiliation(s)
- Fatima E Agharbaoui
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy; Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA.
| | - Ashley C Hoyte
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA
| | - Stefania Ferro
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy
| | - Rosaria Gitto
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy
| | - Maria Rosa Buemi
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy
| | - James R Fuchs
- Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA
| | - Mamuka Kvaratskhelia
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA
| | - Laura De Luca
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy.
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17
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Feng L, Dharmarajan V, Serrao E, Hoyte A, Larue RC, Slaughter A, Sharma A, Plumb MR, Kessl JJ, Fuchs JR, Bushman FD, Engelman AN, Griffin PR, Kvaratskhelia M. The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency. ACS Chem Biol 2016; 11:1313-21. [PMID: 26910179 DOI: 10.1021/acschembio.6b00167] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.
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Affiliation(s)
- Lei Feng
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Venkatasubramanian Dharmarajan
- Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, United States
| | - Erik Serrao
- Department
of Cancer Immunology and Virology, Dana-Farber Cancer Institute and
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, United States
| | - Ashley Hoyte
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Ross C. Larue
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Alison Slaughter
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Amit Sharma
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Matthew R. Plumb
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Jacques J. Kessl
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - James R. Fuchs
- Division of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Frederic D. Bushman
- Perelman School of Medicine, Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
| | - Alan N. Engelman
- Department
of Cancer Immunology and Virology, Dana-Farber Cancer Institute and
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, United States
| | - Patrick R. Griffin
- Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, United States
| | - Mamuka Kvaratskhelia
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
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18
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Grandgenett DP, Pandey KK, Bera S, Aihara H. Multifunctional facets of retrovirus integrase. World J Biol Chem 2015; 6:83-94. [PMID: 26322168 PMCID: PMC4549773 DOI: 10.4331/wjbc.v6.i3.83] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Revised: 07/01/2015] [Accepted: 07/27/2015] [Indexed: 02/05/2023] Open
Abstract
The retrovirus integrase (IN) is responsible for integration of the reverse transcribed linear cDNA into the host DNA genome. First, IN cleaves a dinucleotide from the 3’ OH blunt ends of the viral DNA exposing the highly conserved CA sequence in the recessed ends. IN utilizes the 3’ OH ends to catalyze the concerted integration of the two ends into opposite strands of the cellular DNA producing 4 to 6 bp staggered insertions, depending on the retrovirus species. The staggered ends are repaired by host cell machinery that results in a permanent copy of the viral DNA in the cellular genome. Besides integration, IN performs other functions in the replication cycle of several studied retroviruses. The proper organization of IN within the viral internal core is essential for the correct maturation of the virus. IN plays a major role in reverse transcription by interacting directly with the reverse transcriptase and by binding to the viral capsid protein and a cellular protein. Recruitment of several other host proteins into the viral particle are also promoted by IN. IN assists with the nuclear transport of the preintegration complex across the nuclear membrane. With several retroviruses, IN specifically interacts with different host protein factors that guide the preintegration complex to preferentially integrate the viral genome into specific regions of the host chromosomal target. Human gene therapy using retrovirus vectors is directly affected by the interactions of IN with these host factors. Inhibitors directed against the human immunodeficiency virus (HIV) IN bind within the active site of IN containing viral DNA ends thus preventing integration and subsequent HIV/AIDS.
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