Abstract
Development of viral resistance to the aminodiol human immunodeficiency virus (HIV) protease inhibitor BMS 186,318 was studied by serial passage of HIV type 1 RF in MT-2 cells in the presence of increasing concentrations of compound. After 11 passages, an HIV variant that showed a 15-fold increase in 50% effective dose emerged. This HIV variant displays low-level cross-resistance to the C2 symmetric inhibitor A-77003 but remains sensitive to the protease inhibitors Ro 31-8959 and SC52151. Genetic analysis of the protease gene from a drug-resistant variant revealed an Ala-to-Thr change at amino acid residue 71 (A71T) and a Val-to-Ala change at residue 82 (V82A). To determine the effects of these mutations on protease and virus drug susceptibility, recombinant protease and proviral HIV type 1 clones containing the single mutations A71T and V82A or double mutation A71T/V82A were constructed. Subsequent drug sensitivity assays on the mutant proteases and viruses indicated that the V82A substitution was responsible for most of the resistance observed. Further genotypic analysis of the protease genes from earlier passages of virus indicated that the A71T mutation emerged prior to the V82A change. Finally, the level of resistance did not increase following continued passage in increasing concentrations of drug, and the resistant virus retained its drug susceptibility phenotype 34 days after drug withdrawal.
Full Text
The Full Text of this article is available as a PDF (192.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barrish J. C., Gordon E., Alam M., Lin P. F., Bisacchi G. S., Chen P., Cheng P. T., Fritz A. W., Greytok J. A., Hermsmeier M. A. Aminodiol HIV protease inhibitors. 1. Design, synthesis, and preliminary SAR. J Med Chem. 1994 Jun 10;37(12):1758–1768. doi: 10.1021/jm00038a005. [DOI] [PubMed] [Google Scholar]
- Bechtold C. M., Patick A. K., Alam M., Greytok J., Tino J. A., Chen P., Gordon E., Ahmad S., Barrish J. C., Zahler R. Antiviral properties of aminodiol inhibitors against human immunodeficiency virus and protease. Antimicrob Agents Chemother. 1995 Feb;39(2):374–379. doi: 10.1128/aac.39.2.374. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Debouck C. The HIV-1 protease as a therapeutic target for AIDS. AIDS Res Hum Retroviruses. 1992 Feb;8(2):153–164. doi: 10.1089/aid.1992.8.153. [DOI] [PubMed] [Google Scholar]
- Fontenot G., Johnston K., Cohen J. C., Gallaher W. R., Robinson J., Luftig R. B. PCR amplification of HIV-1 proteinase sequences directly from lab isolates allows determination of five conserved domains. Virology. 1992 Sep;190(1):1–10. doi: 10.1016/0042-6822(92)91186-x. [DOI] [PubMed] [Google Scholar]
- Getman D. P., DeCrescenzo G. A., Heintz R. M., Reed K. L., Talley J. J., Bryant M. L., Clare M., Houseman K. A., Marr J. J., Mueller R. A. Discovery of a novel class of potent HIV-1 protease inhibitors containing the (R)-(hydroxyethyl)urea isostere. J Med Chem. 1993 Jan 22;36(2):288–291. doi: 10.1021/jm00054a014. [DOI] [PubMed] [Google Scholar]
- Gu Z., Gao Q., Li X., Parniak M. A., Wainberg M. A. Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine. J Virol. 1992 Dec;66(12):7128–7135. doi: 10.1128/jvi.66.12.7128-7135.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heimbach J. C., Garsky V. M., Michelson S. R., Dixon R. A., Sigal I. S., Darke P. L. Affinity purification of the HIV-1 protease. Biochem Biophys Res Commun. 1989 Nov 15;164(3):955–960. doi: 10.1016/0006-291x(89)91762-2. [DOI] [PubMed] [Google Scholar]
- Ho D. D., Toyoshima T., Mo H., Kempf D. J., Norbeck D., Chen C. M., Wideburg N. E., Burt S. K., Erickson J. W., Singh M. K. Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor. J Virol. 1994 Mar;68(3):2016–2020. doi: 10.1128/jvi.68.3.2016-2020.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huff J. R. HIV protease: a novel chemotherapeutic target for AIDS. J Med Chem. 1991 Aug;34(8):2305–2314. doi: 10.1021/jm00112a001. [DOI] [PubMed] [Google Scholar]
- Kageyama S., Weinstein J. N., Shirasaka T., Kempf D. J., Norbeck D. W., Plattner J. J., Erickson J., Mitsuya H. In vitro inhibition of human immunodeficiency virus (HIV) type 1 replication by C2 symmetry-based HIV protease inhibitors as single agents or in combinations. Antimicrob Agents Chemother. 1992 May;36(5):926–933. doi: 10.1128/aac.36.5.926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaplan A. H., Michael S. F., Wehbie R. S., Knigge M. F., Paul D. A., Everitt L., Kempf D. J., Norbeck D. W., Erickson J. W., Swanstrom R. Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5597–5601. doi: 10.1073/pnas.91.12.5597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kempf D. J., Codacovi L., Wang X. C., Kohlbrenner W. E., Wideburg N. E., Saldivar A., Vasavanonda S., Marsh K. C., Bryant P., Sham H. L. Symmetry-based inhibitors of HIV protease. Structure-activity studies of acylated 2,4-diamino-1,5-diphenyl-3-hydroxypentane and 2,5-diamino-1,6-diphenylhexane-3,4-diol. J Med Chem. 1993 Feb 5;36(3):320–330. doi: 10.1021/jm00055a003. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lam P. Y., Jadhav P. K., Eyermann C. J., Hodge C. N., Ru Y., Bacheler L. T., Meek J. L., Otto M. J., Rayner M. M., Wong Y. N. Rational design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors. Science. 1994 Jan 21;263(5145):380–384. doi: 10.1126/science.8278812. [DOI] [PubMed] [Google Scholar]
- Larder B. A., Coates K. E., Kemp S. D. Zidovudine-resistant human immunodeficiency virus selected by passage in cell culture. J Virol. 1991 Oct;65(10):5232–5236. doi: 10.1128/jvi.65.10.5232-5236.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Larder B. A., Kemp S. D. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. doi: 10.1126/science.2479983. [DOI] [PubMed] [Google Scholar]
- Loeb D. D., Swanstrom R., Everitt L., Manchester M., Stamper S. E., Hutchison C. A., 3rd Complete mutagenesis of the HIV-1 protease. Nature. 1989 Aug 3;340(6232):397–400. doi: 10.1038/340397a0. [DOI] [PubMed] [Google Scholar]
- Louis J. M., McDonald R. A., Nashed N. T., Wondrak E. M., Jerina D. M., Oroszlan S., Mora P. T. Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli. Eur J Biochem. 1991 Jul 15;199(2):361–369. doi: 10.1111/j.1432-1033.1991.tb16132.x. [DOI] [PubMed] [Google Scholar]
- Otto M. J., Garber S., Winslow D. L., Reid C. D., Aldrich P., Jadhav P. K., Patterson C. E., Hodge C. N., Cheng Y. S. In vitro isolation and identification of human immunodeficiency virus (HIV) variants with reduced sensitivity to C-2 symmetrical inhibitors of HIV type 1 protease. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7543–7547. doi: 10.1073/pnas.90.16.7543. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Otto M. J., Reid C. D., Garber S., Lam P. Y., Scarnati H., Bacheler L. T., Rayner M. M., Winslow D. L. In vitro anti-human immunodeficiency virus (HIV) activity of XM323, a novel HIV protease inhibitor. Antimicrob Agents Chemother. 1993 Dec;37(12):2606–2611. doi: 10.1128/aac.37.12.2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richman D. D. Resistance of clinical isolates of human immunodeficiency virus to antiretroviral agents. Antimicrob Agents Chemother. 1993 Jun;37(6):1207–1213. doi: 10.1128/aac.37.6.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roberts N. A., Martin J. A., Kinchington D., Broadhurst A. V., Craig J. C., Duncan I. B., Galpin S. A., Handa B. K., Kay J., Kröhn A. Rational design of peptide-based HIV proteinase inhibitors. Science. 1990 Apr 20;248(4953):358–361. doi: 10.1126/science.2183354. [DOI] [PubMed] [Google Scholar]
- Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
- St Clair M. H., Martin J. L., Tudor-Williams G., Bach M. C., Vavro C. L., King D. M., Kellam P., Kemp S. D., Larder B. A. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science. 1991 Sep 27;253(5027):1557–1559. doi: 10.1126/science.1716788. [DOI] [PubMed] [Google Scholar]
- Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vacca J. P., Dorsey B. D., Schleif W. A., Levin R. B., McDaniel S. L., Darke P. L., Zugay J., Quintero J. C., Blahy O. M., Roth E. L-735,524: an orally bioavailable human immunodeficiency virus type 1 protease inhibitor. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):4096–4100. doi: 10.1073/pnas.91.9.4096. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weislow O. S., Kiser R., Fine D. L., Bader J., Shoemaker R. H., Boyd M. R. New soluble-formazan assay for HIV-1 cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS-antiviral activity. J Natl Cancer Inst. 1989 Apr 19;81(8):577–586. doi: 10.1093/jnci/81.8.577. [DOI] [PubMed] [Google Scholar]
- el-Farrash M. A., Kuroda M. J., Kitazaki T., Masuda T., Kato K., Hatanaka M., Harada S. Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor. J Virol. 1994 Jan;68(1):233–239. doi: 10.1128/jvi.68.1.233-239.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]