Mutagenesis of Lysines 156 and 159 in Human Immunodeficiency Virus Type 1 Integrase (IN) Reveals Differential Interactions between these Residues and Different IN Inhibitors
Free accessResearch articleFirst published online January, 2015
Mutagenesis of Lysines 156 and 159 in Human Immunodeficiency Virus Type 1 Integrase (IN) Reveals Differential Interactions between these Residues and Different IN Inhibitors
Human immunodeficiency virus (HIV) type 1 integrase (IN) active site, and viral DNA-binding residues K156 and K159 are predicted to interact both with strand transfer-selective IN inhibitors (STI), e.g. L-731,988, Elvitegravir (EVG), and the FDA-approved IN inhibitor, Raltegravir (RGV), and strand transfer non-selective inhibitors, e.g. dicaffeoyltartaric acids (DCTAs), e.g. L-chicoric acid (L-CA). To test posited roles for these two lysine residues in inhibitor action we assayed the potency of L-CA and several STI against a panel of K156 and K159 mutants. Mutagenesis of K156 conferred resistance to L-CA and mutagenesis of either K156 or K159 conferred resistance to STI indicating that the cationic charge at these two viral DNA-binding residues is important for inhibitor potency. IN K156N, a reported polymorphism associated with resistance to RGV, conferred resistance to L-CA and STI as well. To investigate the apparent preference L-CA exhibits for interactions with K156, we assayed the potency of several hybrid inhibitors containing combinations of DCTA and STI pharmacophores against recombinant IN K156A or K159A. Although K156A conferred resistance to diketo acid-branched bis-catechol hybrid inhibitors, neither K156A nor K159A conferred resistance to their monocatechol counterparts, suggesting that bis-catechol moieties direct DCTAs toward K156. In contrast, STI were more promiscuous in their interaction with K156 and K159. Taken together, the results of this study indicate that DCTAs interact with IN in a manner different than that of STI and suggest that DCTAs are an attractive candidate chemotype for development into drugs potent against STI-resistant IN.
BastienJW, Abdel-MalekS, QiJ, ReineckeMG, CorbettTH, ValerioteFA, MahlerWF, RobinsonWEJr., Zalles-AsinJ. (1996) Kallawaya-Bolivian herbalists; analysis of their medicinal plants for leads to anti-HIV, anti-tumor, anti-Chagas drugs. In Ethnobiology in Human Welfare, [Proceedings, IV International Congress of Ethnobiology, Lucknow, India, 17-21 November 1994]. JainSK (Ed). Deep Publications, New Dehli, India, 211–222.
2.
Abdel-MalekS, BastienJW, MahlerWF, QiJ, ReineckeMG, RobinsonWEJr., ShuY, Zalles-AsinJ. (1996) Drug leads from the Kallawaya herbalists of Bolivia; 1. Background, rationale, protocol and anti-HIV activity. Journal of Ethnopharmacology, 50, 157–166.
3.
RobinsonWEJr., ReineckeMG, Abdel-MalekS, QiJ, ChowSA. (1996) Inhibitors of HIV-1 replication that inhibit HIV integrase. Proceedings of the National Academy of Sciences USA, 93, 6326–6331
4.
CrosbyDC and RobinsonWEJr., (2011) Dicaffeoyltartaric acid and dicaffeoylquinic acid HIV integrase inhibitors. In HIV-1 Integrase Mechanism and Inhibitor Design, NeamatiN. (Ed). Wiley, Hoboken, New Jersey, USA, 341–362.
5.
GarridoC, VillacianJ, ZahoneroN, PatteryT, GarciaF, GutierrezF, CaballeroE, Van HoutteM, SorianoV, de MendozaC. (2012) Broad phenotypic cross-resistance to Elvitegravir in HIV-infected patients failing on Raltegravir-containing regimens. Antimicrobial Agents and Chemotherapy, 56, 2873–2878.
6.
JenkinsTM, HickmanAB, DydaF, GhirlandoR, DaviesDR, CraigieR. (1995) Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues. Proceedings of the National Academy of Sciences USA, 92, 6057–6061.
7.
CherepanovP, MaertensGN, HareS. (2011) Structural insights into the retroviral DNA integration apparatus. Current Opinion in Structural Biology, 21, 249–256.
8.
HareS, GuptaSS, ValkovE, EngelmanA, CherepanovP. (2010) Retroviral intasome assembly and inhibition of DNA strand transfer. Nature, 464, 232–236.
9.
HareS, SmithSJ, MetifiotM, Jaxa-ChamiecA, PommierY, HughesSH, CherepanovP. (2011) Structural and functional analyses of the second-generation integrase strand transfer inhibitor Dolutegravir (S/GSK1349572). Molecular Pharmacology, 80, 565–572.
10.
LaFeminaRL, SchneiderCL, RobbinsHL, CallahanPL, LeGrowK, RothE, SchleifWA, EminiEA. (1992) Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells. Journal of Virology, 66, 7414–7419.
11.
RobinsonWEJr. (1998) HIV Integrase: the next target?Infections in Medicine, 15, 129–137.
12.
BukrinskyM. (2004) A hard way to the nucleus. Molecular Medicine, 10, 1–5.
13.
YangW, SteitzTA. (1995) Recombining the structures of HIV integrase, RuvC and RNaseH. Structure, 3, 131–134.
14.
EijkelenboomAP, van den EntFM, VosA, DoreleijersJF, HardK, Tullius PlasterkRH, KapteinR, BoelensR. (1997) The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc. Current Biology, 7, 739–746.
15.
EllisonV, GertonJ, VincentKA, BrownPO. (1995) An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer. Journal of Biological Chemistry, 270, 3320–3326.
16.
GronenbornAM, CloreGM, CraigieR, CaffreyM, ZhengR, CaiM. (1997) Solution structure of the N-terminal zinc binding domain of HIV-1 integrase. Nature Structural Biology, 4, 567–577.
17.
ZhengR, JenkinsT, CraigieR. (1996) Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization, and enhances catalytic activity. Proceedings of the National Academy of Sciences USA, 93, 13659–13664.
18.
EngelmanA, EnglundG, OrensteinJM, MartinMA, CraigieR. (1995) Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. Journal of Virology, 69, 2729–2736.
19.
NakamuraT, MasudaT, GotoT, SanoK, NakaiM, HaradaS. (1997) Lack of infectivity of HIV-1 integrase zinc finger-like domain mutant with morphologically normal maturation. Biochemical and Biophysical Research Communications, 239, 715–722.
20.
BushmanF, EngelmanA, PalmerI, WingfieldP, CraigieR. (1993) Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding. Proceedings of the National Academy of Sciences USA, 90, 3428–3432.
21.
YiJ, Asante-AppiahE, SkalkaAM. (1999) Divalent cations stimulate preferential recognition of a viral DNA end by HIV-1 integrase. Biochemistry, 38, 8458–8468.
22.
EijkelenboomAP, SprangersR, HardK, Puras LutzkeRA, PlasterkRH, BoelensR, KapteinR. (1999) Refined solution structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase. Proteins, 36, 556–564.
23.
HårdK, KapteinR, PlasterkRH, BoelensR, LutzkeRP, EijkelenboomA. (1995) The DNA-binding domain of HIV-1 integrase has an SH3-like fold. Nature Structural Biology, 2, 807–810.
24.
EngelmanA, HickmanAB, CraigieR. (1994) The core and carboxyl-terminal domains of the integrase protein of human-immunodeficiency-virus type-1 each contribute to nonspecific DNA-binding. Journal of Virology, 68, 5911–5917.
25.
Marcus-SekuraCJ, LevinJG, KlutchM, WoernerAM. (1992) Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein. AIDS Research and Human Retroviruses, 8, 297–304.
26.
VinkC, GroenegerAA, PlasterkRH. (1993) Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein. Nucleic Acids Research, 21, 1419–1425.
27.
VinkC, LutzkeRP, PlasterkRH. (1994) Formation of a stable complex between the human immunodeficiency virus integrase protein and viral DNA. Nucleic Acids Research, 22, 4103–4110.
28.
CraigieRF, BushmanF, EngelmanA. (1993) Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex. EMBO Journal, 12, 3269–3275.
29.
LeavittAD, ShiueL, VarmusHE. (1993) Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro. Journal of Biological Chemistry, 268, 2113–2119.
30.
PlasterkRH, GroenegerAA, VinkC, GentDC. (1993) Complementation between HIV integrase proteins mutated in different domains. EMBO Journal, 12, 3261–3267.
31.
HealyEF, SandersJ, KingPJ, RobinsonWEJr. (2009) A docking study of L-chicoric acid with HIV-1 integrase. Journal of Molecular Graphics and Modeling, 27, 584–589.
32.
LeeDJ, RobinsonWEJr. (2006) Preliminary mapping of a putative inhibitor-binding pocket for human immunodeficiency virus type 1 integrase inhibitors. Antimicrobial Agents and Chemotherapy, 50, 134–142.
33.
RobinsonWEJr., CordeiroM, Abdel-MalekS, JiaQ, ChowSA, ReineckeMG, MitchellWM. (1996). Dicaffeoylquinic acid inhibitors of human immunodeficiency virus integrase: inhibition of the core catalytic domain of human immunodeficiency virus integrase. Molecular Pharmacology, 50, 846–855.
34.
SotrifferCA, NiH, McCammonJA. (2000) Active site binding modes of HIV-1 integrase inhibitors. Journal of Medicinal Chemistry, 43, 4109–4117.
35.
AlvesCN, MartiS, CastilloR, AndresJ, MolinerV, TunonI, SillaE. (2008) A quantum mechanic/molecular mechanic study of the wild-type and N155S mutant HIV-1 integrase complexed with diketo acid. Biophysical Journal, 94, 2443–2451.
36.
ChenX, TsiangM, YuF, HungM, JonesGS, ZeynalzadeganA, QiX, JinH, KimCU, SwaminathanS, ChenJM. (2008) Modeling, analysis, and validation of a novel HIV integrase structure provide insights into the binding modes of potent integrase inhibitors. Journal of Molecular Biology, 380, 504–519.
37.
DayamR, NeamatiN. (2004) Active site binding modes of the β-diketoacids: a multi-active site approach in HIV-1 integrase inhibitor design. Bioorganic and Medicinal Chemistry, 12, 6371–6381.
38.
De LucaL, De GraziaS, FerroS, GittoR, ChristF, DebyserZ, ChimirriA. (2011) HIV-1 integrase strand-transfer inhibitors: design, synthesis and molecular modeling investigation. European Journal of Medicinal Chemistry, 46, 756–764.
39.
SharmaH, ChengX, BuolamwiniJK. (2012) Homology model-guided 3D-QSAR studies of HIV-1 integrase inhibitors. Journal of Chemical Information and Modeling, 52, 515–544.
40.
PandeyKK, BeraS, ZahmJ, VoraA, StillmockK, HazudaD, GrandgenettDP. (2007) Inhibition of human immunodeficiency virus type 1 concerted integration by strand transfer inhibitors which recognize a transient structural intermediate. Journal of Virology, 81, 12189–12199.
41.
ReinkeRA, LeeDJ, McdougallBR, KingPJ, VictoriaJ, MaoYQ, LeiXY, ReineckeMG, RobinsonWEJr. (2004) L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro. Virology, 326, 203–219.
42.
GoldgurY, CraigieR, CohenGH, FujiwaraT, YoshinagaT, FujishitaT, SugimotoH, EndoT, MuraiH, DaviesDR. (1999) Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: a platform for antiviral drug design. Proceedings of the National Academy of Sciences USA, 96, 13040–13043.
43.
BrownPO, HeuerTS. (1997) Mapping features of HIV-1 integrase near selected sites on viral and target DNA molecules in an active enzyme-DNA complex by photo-cross-linking. Biochemistry36, 10655–10665.
44.
DrakeRR, NeamatiN, HongH, PilonAA, SunthankarP, HumeSD, MilneGW, PommierY. (1998) Identification of a nucleotide binding site in HIV-1 integrase. Proceedings of the National Academy of Sciences USA, 95, 4170–4175.
45.
HeuerTS, BrownPO. (1998) Photo-cross-linking studies suggest a model for the architecture of an active human immunodeficiency virus type 1 integrase-DNA complex. Biochemistry, 37, 6667–6678.
46.
JenkinsTM, EspositoD, EngelmanA, CraigieR. (1997). Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking. EMBO Journal, 16, 6849–6859.
47.
HareS, MaertensGN, CherepanovP. (2012) 3′-Processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO Journal, 31, 3020–3028.
48.
SichtigN, SierraS, KaiserR, DaumerM, ReuterS, SchulterE, AltmannA, FatkenheuerG, DittmerU, PfisterH, EsserS. (2009) Evolution of Raltegravir resistance during therapy. Journal of Antimicrobial Chemotherapy, 64, 25–32.
49.
GarridoC, GerettiAM, ZahoneroN, BoothC, StrangA, SorianoV, De MendozaC. (2010) Integrase variability and susceptibility to HIV integrase inhibitors: impact of subtypes, antiretroviral experience and duration of HIV infection. Journal of Antimicrobial Chemotherapy, 65, 320–326.
50.
MonleauM, AghokengAF, NkanoBA, ChaixML, PeetersM. (2012) Drug resistance mutations of HIV-1 non-B viruses to integrase inhibitors in treatment-naive patients from sub-Saharan countries and discordant interpretations. AIDS Research and Human Retroviruses, 28, 1157–60.
51.
SierraS, LubkeN, WalterH, SchulterE, ReuterS, FatkenheuerG, BickelM, da SilvaH, KaiserR, EsserS, SnoBSG. (2011) The SnoB study: frequency of baseline Raltegravir resistance mutations prevalence in different non-B subtypes. Medical Microbiology and Immunology, 200, 225–232.
52.
WiskerchenM, MuesingMA. (1995) Identification and characterization of a temperature-sensitive mutant of human immunodeficiency virus type 1 by alanine scanning mutagenesis of the integrase gene. Journal of Virology, 69, 597–601.
53.
AlvesCN, MartiS, CastilloR, AndresJ, MolinerV, TunonI, SillaE. (2007) A quantum mechanics/molecular mechanics study of the protein-ligand interaction for inhibitors of HIV-1 integrase. Chemistry – A European Journal, 13, 7715–7724.
CannonPM, WilsonW, BylesE, KingsmanSM, KingsmanAJ. (1994) Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues. Journal of Virology, 68, 4768–4775.
56.
KulkoskyJ, JonesKS, KatzRA, MackJP, SkalkaAM. (1992) Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Molecular Cell Biology, 12, 2331–2338.
57.
SayasithK, SauveG, YelleJ. (2000) Characterization of mutant HIV-1 integrase carrying amino acid changes in the catalytic domain. Molecules and Cells, 10, 525–532.
58.
ShinCG, TaddeoB, HaseltineWA, FarnetCM. (1994) Genetic analysis of the human immunodeficiency virus type 1 integrase protein. Journal of Virology, 68, 1633–1642.
59.
Bar-MagenT, SloanRD, FaltenbacherVH, DonahueDA, KuhlBD, OliveiraM, XuH, WainbergMA. (2009) Comparative biochemical analysis of HIV-1 subtype B and C integrase enzymes. Retrovirology, 6, article 103.
60.
GroblerJA, StillmockK, HuB, WitmerM, FelockP, EspesethAS, WolfeA, EgbertsonM, BourgeoisM, MelamedJ, WaiJS, YoungS, VaccaJ, HazudaDJ. (2002) Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes. Proceedings of the National Academy of Sciences USA, 99, 6661–6666.
61.
PandeyKK, BeraS, VoraAC, GrandgenettDP. (2010) Physical trapping of HIV-1 synaptic complex by different structural classes of integrase strand transfer inhibitors. Biochemistry, 49, 8376–8387.
62.
HareS, VosAM, ClaytonRF, ThuringJW, CummingsMD, CherepanovP. (2010) Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance. Proceedings of the National Academy of Sciences USA, 107, 20057–20062.
63.
CanducciF, SampaoloM, MarinozziMC, BoeriE, SpagnuoloV, GalliA, CastagnaA, LazzarinA, ClementiM, GianottiN. (2009), Dynamic patterns of human immunodeficiency virus type 1 integrase gene evolution in patients failing Raltegravir-based salvage therapies. AIDS, 23, 455–460.
64.
CrosbyDC, LeiXY, GibbsCG, McDougallBR, RobinsonWEJr., ReineckeMG. (2010) Design, synthesis, and biological evaluation of novel hybrid dicaffeoyltartaric/diketo acid and tetrazole-substituted L-chicoric acid analogue inhibitors of human immunodeficiency virus type 1 integrase. Journal of Medicinal Chemistry, 53, 8161–8175.
65.
KingPJ, MaGX, MiaoWF, JiaQ, McDougallBR, ReineckeMG, Cornell KuanJ, KimTR, RobinsonWEJr. (1999) Structure-activity relationships: Analogues of the dicaffeoylquinic and dicaffeoyltartaric acids as potent inhibitors of human immunodeficiency virus type 1 integrase and replication. Journal of Medicinal Chemistry, 42, 497–509.
66.
SelnickHG, HazudaDJ, EgbertsonM, GuareJPJr., WaiJS, YoungSD, ClarkDL, MedinaJC. (1999) Preparation of nitrogen-containing 4-heteroaryl-2,4-dioxobutyric acids useful as HIV integrase inhibitors. PCT Int. Appl.:WO 99/62513.
67.
BelykKM, MorrisonHG, JonesP, SummaV. (2006) Preparation of N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide potassium salts as HIV integrase inhibitors. PCT Int. Appl.:WO2006/060712.
WaiJS, EgbertsonMS, PayneLS, FisherTE, EmbreyMW, TranLO, MelamedJY, LangfordHM, GuareJPJr., ZhuangL, GreyVE, VaccaJP, HollowayMK, Naylor-OlsenAM, HazudaDJ, FelockPJ, WolfeAL, StillmockKA, SchleifWA, GabryelskiLJ, YoungSD. (2000), 4-Aryl-2,4-dioxobutanoic acid inhibitors of HIV-1 integrase and viral replication in cells. Journal of Medicinal Chemistry, 43, 4923–4926.
70.
PaisGC, ZhangX, MarchandC, NeamatiN, CowansageK, SvarovskaiaES, PathakVK, TangY, NicklausM, PommierY, BurkeTRJr. (2002) Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors. Journal of Medicinal Chemistry, 45, 3184–3194.
71.
LeeDJ, RobinsonWEJr. (2004) Human immunodeficiency virus type 1 (HIV-1) integrase: resistance to diketo acid integrase inhibitors impairs HIV-1 replication and integration and confers cross-resistance to L-chicoric acid. Journal of Virology, 78, 5835–5847.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
