Viral Entry as the Primary Target for the Anti-HIV Activity of Chicoric Acid and Its Tetra-Acetyl Esters

  1. Wim Pluymers1,
  2. Nouri Neamati2,
  3. Christophe Pannecouque1,
  4. Valery Fikkert1,
  5. Christophe Marchand2,
  6. Terrence R. Burke, Jr.2,
  7. Yves Pommier2,
  8. Dominique Schols1,
  9. Erik De Clercq1,
  10. Zeger Debyser1 and
  11. Myriam Witvrouw1
  1. 1Rega Institute for Medical Research, K. U. Leuven, Leuven, Belgium (W.P., C.P., V.F., D.S., E.D.C., Z.D., M.W.); 2Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland (N.N., C.M., T.R.B., Y.P.).

    Abstract

    The antiviral activity of l-chicoric acid against HIV-1 has been attributed previously to the inhibition of HIV-1 integration. This conclusion was based on the inhibition of integrase activity in enzymatic assays and the isolation of a resistant HIV strain with a mutation (G140S) in the integrase gene. Here we show that the primary antiviral target of l-CA and its analogs in cell culture is viral entry. l- and d-chicoric acid (l-CA and d-CA) and their respective tetra-acetyl esters inhibit the replication of HIV-1 (IIIBand NL4.3) and HIV-2 (ROD) in MT-4 cells at a 50% effective concentration (EC50) ranging from 1.7 to 70.6 μM. In a time-of-addition experiment, l-CA, d-CA,l-CATA, and d-CATA were found to interfere with an early event in the viral replication cycle. Moreover,l-CA, d-CA, and their analogs did not inhibit the replication of virus strains that were resistant toward polyanionic and polycationic compounds at subtoxic concentrations. Furthermore, HIV-1 strains resistant to l-CA and d-CA were selected in the presence of l-CA and d-CA, respectively. Mutations were found in the V2, V3, and V4 loop region of the envelope glycoprotein gp120 of the l-CA andd-CA-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain. Recombination of the gp120 gene of the l-CA and d-CA resistant strain in a NL4.3 wild-type molecular clone fully rescued the phenotypic resistance toward l-CA and d-CA. No significant mutations were detected in the integrase gene of the drug-resistant virus strains. Although inhibition of HIV integrase activity byl-CA and its derivatives was confirmed in an oligonucleotide-driven assay, integrase carrying the G140S mutation was inhibited to the same extent as the wild-type integrase.

    Footnotes

    • Send reprint requests to: Dr. Myriam Witvrouw, Rega Institute for Medical Research, K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail:myriam.witvrouw{at}uz.kuleuven.ac.be

    • Wim Pluymers is funded by a grant from the Flemish Institute supporting Scientific-Technological Research in Industry (IWT). Investigations were supported in part by the Biomedical Research Program of the European Commission (EC Biomed2 PL96–2161) and by grants of the Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen (no.G.0104.98) and the Geconcerteerde Onderzoeksacties (GOA no.00/12) from the Flemish Community (“Vlaamse Gemeenschap”).

    • Abbreviations:
      IN
      integrase
      CA
      chicoric acid
      DCQA
      dicaffeoylquinic acid
      SIV
      simian immunodeficiency virus
      DS
      dextran sulfate
      AZT
      3′-azido-3′-deoxythymidine
      PHA
      phytohemagglutinin
      CPE
      cytophatic effect
      PCR
      polymerase chain reaction
      CATA
      tetra-acetate form of chicoric acid
      • Received October 13, 1999.
      • Accepted June 1, 2000.
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    1. Molecular Pharmacology September 1, 2000 vol. 58 no. 3 641-648
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