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. 1997 Jun 15;324(Pt 3):721–727. doi: 10.1042/bj3240721

Mode of inhibition of HIV reverse transcriptase by 2-hexaprenylhydroquinone, a novel general inhibitor of RNA-and DNA-directed DNA polymerases.

S Loya 1, A Rudi 1, Y Kashman 1, A Hizi 1
PMCID: PMC1218486  PMID: 9210394

Abstract

A natural compound from the Red Sea sponge Ircinia sp., 2-hexaprenylhydroquinone (HPH), has been shown to be a general inhibitor of retroviral reverse transcriptases (from HIV-1, HIV-2 and murine leukaemia virus) as well as of cellular DNA polymerases (Escherichia coli DNA polymerase I, and DNA polymerases alpha and beta). The pattern of inhibition was found to be similar for all DNA polymerases tested. Thus the mode of inhibition was studied in detail for HIV-1 reverse transcriptase. HPH is a non-competitive inhibitor and binds the enzyme irreversibly with high affinity (Ki=0. 62 microM). The polar hydroxy groups have been shown to be of key importance. A methylated derivative, mHPH, which is devoid of these polar moieties, showed a significantly decreased capacity to inhibit all DNA polymerases tested. Like the natural product, mHPH binds the enzyme independently at an allosteric site, but with reduced affinity (Ki=7.4 microM). We show that HPH does not interfere with the first step of the polymerization process, i.e. the physical formation of the reverse-transcriptase-DNA complex. Consequently, we suggest that the natural inhibitor interferes with the subsequent steps of the overall reaction. Since HPH seems not to affect the affinity of dNTP for the enzyme (the Km is unchanged under conditions where the HPH concentration is increased), we speculate that its inhibitory capacity is derived from its effect on the nucleotidyl-transfer catalytic reaction. We suggest that such a mechanism of inhibition is typical of an inhibitor whose mode of inhibition should be common to all RNA- and DNA-directed polymerases.

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Selected References

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  1. Bakhanashvili M., Hizi A. Fidelity of DNA synthesis exhibited in vitro by the reverse transcriptase of the lentivirus equine infectious anemia virus. Biochemistry. 1993 Jul 27;32(29):7559–7567. doi: 10.1021/bi00080a030. [DOI] [PubMed] [Google Scholar]
  2. Bakhanashvili M., Hizi A. Interaction of the reverse transcriptase of human immunodeficiency virus type 1 with DNA. Biochemistry. 1994 Oct 11;33(40):12222–12228. doi: 10.1021/bi00206a027. [DOI] [PubMed] [Google Scholar]
  3. Bakhanashvili M., Hizi A. The fidelity of the reverse transcriptases of human immunodeficiency viruses and murine leukemia virus, exhibited by the mispair extension frequencies, is sequence dependent and enzyme related. FEBS Lett. 1993 Mar 15;319(1-2):201–205. doi: 10.1016/0014-5793(93)80067-5. [DOI] [PubMed] [Google Scholar]
  4. Balzarini J., Pérez-Pérez M. J., San-Félix A., Camarasa M. J., Bathurst I. C., Barr P. J., De Clercq E. Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine (TSAO-T). J Biol Chem. 1992 Jun 15;267(17):11831–11838. [PubMed] [Google Scholar]
  5. Barré-Sinoussi F., Chermann J. C., Rey F., Nugeyre M. T., Chamaret S., Gruest J., Dauguet C., Axler-Blin C., Vézinet-Brun F., Rouzioux C. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science. 1983 May 20;220(4599):868–871. doi: 10.1126/science.6189183. [DOI] [PubMed] [Google Scholar]
  6. Beese L. S., Friedman J. M., Steitz T. A. Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. Biochemistry. 1993 Dec 28;32(51):14095–14101. doi: 10.1021/bi00214a004. [DOI] [PubMed] [Google Scholar]
  7. Clark P. K., Ferris A. L., Miller D. A., Hizi A., Kim K. W., Deringer-Boyer S. M., Mellini M. L., Clark A. D., Jr, Arnold G. F., Lebherz W. B., 3rd HIV-1 reverse transcriptase purified from a recombinant strain of Escherichia coli. AIDS Res Hum Retroviruses. 1990 Jun;6(6):753–764. doi: 10.1089/aid.1990.6.753. [DOI] [PubMed] [Google Scholar]
  8. Davies J. F., 2nd, Almassy R. J., Hostomska Z., Ferre R. A., Hostomsky Z. 2.3 A crystal structure of the catalytic domain of DNA polymerase beta. Cell. 1994 Mar 25;76(6):1123–1133. doi: 10.1016/0092-8674(94)90388-3. [DOI] [PubMed] [Google Scholar]
  9. De Clercq E. Antiviral therapy for human immunodeficiency virus infections. Clin Microbiol Rev. 1995 Apr;8(2):200–239. doi: 10.1128/cmr.8.2.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Clercq E. HIV inhibitors targeted at the reverse transcriptase. AIDS Res Hum Retroviruses. 1992 Feb;8(2):119–134. doi: 10.1089/aid.1992.8.119. [DOI] [PubMed] [Google Scholar]
  11. De Clercq E. HIV-1-specific RT inhibitors: highly selective inhibitors of human immunodeficiency virus type 1 that are specifically targeted at the viral reverse transcriptase. Med Res Rev. 1993 May;13(3):229–258. doi: 10.1002/med.2610130303. [DOI] [PubMed] [Google Scholar]
  12. Debyser Z., Pauwels R., Andries K., De Clercq E. Specific HIV-1 reverse transcriptase inhibitors. J Enzyme Inhib. 1992;6(1):47–53. doi: 10.3109/14756369209041355. [DOI] [PubMed] [Google Scholar]
  13. Debyser Z., Pauwels R., Andries K., Desmyter J., Kukla M., Janssen P. A., De Clercq E. An antiviral target on reverse transcriptase of human immunodeficiency virus type 1 revealed by tetrahydroimidazo-[4,5,1-jk] [1,4]benzodiazepin-2 (1H)-one and -thione derivatives. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1451–1455. doi: 10.1073/pnas.88.4.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  15. Ding J., Das K., Moereels H., Koymans L., Andries K., Janssen P. A., Hughes S. H., Arnold E. Structure of HIV-1 RT/TIBO R 86183 complex reveals similarity in the binding of diverse nonnucleoside inhibitors. Nat Struct Biol. 1995 May;2(5):407–415. doi: 10.1038/nsb0595-407. [DOI] [PubMed] [Google Scholar]
  16. Fischl M. A., Richman D. D., Causey D. M., Grieco M. H., Bryson Y., Mildvan D., Laskin O. L., Groopman J. E., Volberding P. A., Schooley R. T. Prolonged zidovudine therapy in patients with AIDS and advanced AIDS-related complex. AZT Collaborative Working Group. JAMA. 1989 Nov 3;262(17):2405–2410. [PubMed] [Google Scholar]
  17. Gallo R. C., Salahuddin S. Z., Popovic M., Shearer G. M., Kaplan M., Haynes B. F., Palker T. J., Redfield R., Oleske J., Safai B. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science. 1984 May 4;224(4648):500–503. doi: 10.1126/science.6200936. [DOI] [PubMed] [Google Scholar]
  18. Georgiadis M. M., Jessen S. M., Ogata C. M., Telesnitsky A., Goff S. P., Hendrickson W. A. Mechanistic implications from the structure of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase. Structure. 1995 Sep 15;3(9):879–892. doi: 10.1016/S0969-2126(01)00223-4. [DOI] [PubMed] [Google Scholar]
  19. Guyader M., Emerman M., Sonigo P., Clavel F., Montagnier L., Alizon M. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature. 1987 Apr 16;326(6114):662–669. doi: 10.1038/326662a0. [DOI] [PubMed] [Google Scholar]
  20. Hizi A., McGill C., Hughes S. H. Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1218–1222. doi: 10.1073/pnas.85.4.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hizi A., Tal R., Hughes S. H. Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli. Virology. 1991 Jan;180(1):339–346. doi: 10.1016/0042-6822(91)90038-d. [DOI] [PubMed] [Google Scholar]
  22. Hizi A., Tal R., Shaharabany M., Loya S. Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. J Biol Chem. 1991 Apr 5;266(10):6230–6239. [PubMed] [Google Scholar]
  23. Hsieh J. C., Zinnen S., Modrich P. Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase. J Biol Chem. 1993 Nov 25;268(33):24607–24613. [PubMed] [Google Scholar]
  24. Jacobo-Molina A., Ding J., Nanni R. G., Clark A. D., Jr, Lu X., Tantillo C., Williams R. L., Kamer G., Ferris A. L., Clark P. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6320–6324. doi: 10.1073/pnas.90.13.6320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johnson M. S., McClure M. A., Feng D. F., Gray J., Doolittle R. F. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. doi: 10.1073/pnas.83.20.7648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kati W. M., Johnson K. A., Jerva L. F., Anderson K. S. Mechanism and fidelity of HIV reverse transcriptase. J Biol Chem. 1992 Dec 25;267(36):25988–25997. [PubMed] [Google Scholar]
  27. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  28. Loya S., Bakhanashvili M., Kashman Y., Hizi A. Mechanism of inhibition of HIV reverse transcriptase by toxiusol, a novel general inhibitor of retroviral and cellular DNA polymerases. Biochemistry. 1995 Feb 21;34(7):2260–2266. doi: 10.1021/bi00007a021. [DOI] [PubMed] [Google Scholar]
  29. Loya S., Bakhanashvili M., Kashman Y., Hizi A. Peyssonols A and B, two novel inhibitors of the reverse transcriptases of human immunodeficiency virus types 1 and 2. Arch Biochem Biophys. 1995 Feb 1;316(2):789–796. doi: 10.1006/abbi.1995.1105. [DOI] [PubMed] [Google Scholar]
  30. Loya S., Hizi A. The inhibition of human immunodeficiency virus type 1 reverse transcriptase by avarol and avarone derivatives. FEBS Lett. 1990 Aug 20;269(1):131–134. doi: 10.1016/0014-5793(90)81137-d. [DOI] [PubMed] [Google Scholar]
  31. Loya S., Kashman Y., Hizi A. The carotenoid halocynthiaxanthin: a novel inhibitor of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. Arch Biochem Biophys. 1992 Mar;293(2):208–212. doi: 10.1016/0003-9861(92)90386-b. [DOI] [PubMed] [Google Scholar]
  32. Loya S., Rudi A., Tal R., Kashman Y., Loya Y., Hizi A. 3,5,8-Trihydroxy-4-quinolone, a novel natural inhibitor of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. Arch Biochem Biophys. 1994 Mar;309(2):315–322. doi: 10.1006/abbi.1994.1119. [DOI] [PubMed] [Google Scholar]
  33. Loya S., Tal R., Kashman Y., Hizi A. Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother. 1990 Oct;34(10):2009–2012. doi: 10.1128/aac.34.10.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Majumdar C., Abbotts J., Broder S., Wilson S. H. Studies on the mechanism of human immunodeficiency virus reverse transcriptase. Steady-state kinetics, processivity, and polynucleotide inhibition. J Biol Chem. 1988 Oct 25;263(30):15657–15665. [PubMed] [Google Scholar]
  35. Mitsuya H., Broder S. Inhibition of the in vitro infectivity and cytopathic effect of human T-lymphotrophic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 2',3'-dideoxynucleosides. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1911–1915. doi: 10.1073/pnas.83.6.1911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moras D. Polymerases. Two sisters and their cousin. Nature. 1993 Aug 12;364(6438):572–573. doi: 10.1038/364572a0. [DOI] [PubMed] [Google Scholar]
  37. Patel P. H., Jacobo-Molina A., Ding J., Tantillo C., Clark A. D., Jr, Raag R., Nanni R. G., Hughes S. H., Arnold E. Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. Biochemistry. 1995 Apr 25;34(16):5351–5363. doi: 10.1021/bi00016a006. [DOI] [PubMed] [Google Scholar]
  38. Pelletier H., Sawaya M. R., Kumar A., Wilson S. H., Kraut J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science. 1994 Jun 24;264(5167):1891–1903. [PubMed] [Google Scholar]
  39. Perrino F. W., Loeb L. A. Differential extension of 3' mispairs is a major contribution to the high fidelity of calf thymus DNA polymerase-alpha. J Biol Chem. 1989 Feb 15;264(5):2898–2905. [PubMed] [Google Scholar]
  40. Reardon J. E. Human immunodeficiency virus reverse transcriptase: steady-state and pre-steady-state kinetics of nucleotide incorporation. Biochemistry. 1992 May 12;31(18):4473–4479. doi: 10.1021/bi00133a013. [DOI] [PubMed] [Google Scholar]
  41. Reardon J. E., Miller W. H. Human immunodeficiency virus reverse transcriptase. Substrate and inhibitor kinetics with thymidine 5'-triphosphate and 3'-azido-3'-deoxythymidine 5'-triphosphate. J Biol Chem. 1990 Nov 25;265(33):20302–20307. [PubMed] [Google Scholar]
  42. Ren J., Esnouf R., Hopkins A., Ross C., Jones Y., Stammers D., Stuart D. The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design. Structure. 1995 Sep 15;3(9):915–926. doi: 10.1016/S0969-2126(01)00226-X. [DOI] [PubMed] [Google Scholar]
  43. Richman D. D., Fischl M. A., Grieco M. H., Gottlieb M. S., Volberding P. A., Laskin O. L., Leedom J. M., Groopman J. E., Mildvan D., Hirsch M. S. The toxicity of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. A double-blind, placebo-controlled trial. N Engl J Med. 1987 Jul 23;317(4):192–197. doi: 10.1056/NEJM198707233170402. [DOI] [PubMed] [Google Scholar]
  44. Rittinger K., Divita G., Goody R. S. Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):8046–8049. doi: 10.1073/pnas.92.17.8046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sawaya M. R., Pelletier H., Kumar A., Wilson S. H., Kraut J. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. Science. 1994 Jun 24;264(5167):1930–1935. doi: 10.1126/science.7516581. [DOI] [PubMed] [Google Scholar]
  46. Shaharabany M., Hizi A. The DNA-dependent and RNA-dependent DNA polymerase activities of the reverse transcriptases of human immunodeficiency viruses types 1 and 2. AIDS Res Hum Retroviruses. 1991 Nov;7(11):883–888. doi: 10.1089/aid.1991.7.883. [DOI] [PubMed] [Google Scholar]
  47. Smerdon S. J., Jäger J., Wang J., Kohlstaedt L. A., Chirino A. J., Friedman J. M., Rice P. A., Steitz T. A. Structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3911–3915. doi: 10.1073/pnas.91.9.3911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sousa R. Structural and mechanistic relationships between nucleic acid polymerases. Trends Biochem Sci. 1996 May;21(5):186–190. [PubMed] [Google Scholar]

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