Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K(i) ) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in K(i) in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2' subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel beta-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft.

Gilead Sciences and IOCB Research Center Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague 6 Czech Republic

Zobrazit více v PubMed

Baily, S. The CCP4 Suite—programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 1994;50:760–763. PubMed

Betts, M.J., Sternberg, M.J. An analysis of conformational changes on protein–protein association: Implications for predictive docking. Protein Eng. 1999;12:271–283. PubMed

Brynda, J., Řezáčová, P., Fábry, M., Hořejší, M., Štouráčová, R., Sedláček, J., Souček, M., Hradílek, M., Lepšík, M., Konvalinka, J. A phenylnorstatine inhibitor binding to HIV-1 protease: Geometry, protonation, and subsite-pocket interactions analyzed at atomic resolution. J. Med. Chem. 2004a;47:2030–2036. PubMed

Brynda, J., Řezáčová, P., Fábry, M., Hořejší, M., Štouráčová, R., Souček, M., Hradílek, M., Konvalinka, J., Sedláček, J. Inhibitor binding at the protein interface in crystals of a HIV-1 protease complex. Acta Crystallogr. D Biol. Crystallogr. 2004b;60:1943–1948. PubMed

Carrillo, A., Stewart, K.D., Sham, H.L., Norbeck, D.W., Kohlbrenner, W.E., Leonard, J.M., Kempf, D.J., Molla, A. In vitro selection and characterization of human immunodeficiency virus type 1 variants with increased resistance to ABT-378, a novel protease inhibitor. J. Virol. 1998;72:7532–7541. PubMed PMC

Case, D.A., Darden, T.A., Cheatham T.E., III, Simmerling, C.L., Wang, J., Duke, R.E., Luo, R., Merz, K.M., Wang, B., Pearlman, D.A., et al. AMBER 8. University of California; San Francisco, CA: 2004.

Clemente, J.C., Mosse, R.E., Hemrajani, R., Whitford, L.R., Govindasamy, L., Reutzel, R., McKenna, R., Agbandje-McKenna, M., Goodenow, M.M., Dunn, B.M. Comparing the accumulation of active- and nonactive-site mutations in the HIV-1 protease. Biochemistry. 2004;43:12141–12151. PubMed

De Clercq, E. Anti-HIV drugs. Verh. K. Acad. Geneeskd. Belg. 2007;69:81–104. PubMed

de Mendoza, C., Valer, L., Bacheler, L., Pattery, T., Corral, A., Soriano, V. Prevalence of the HIV-1 protease mutation I47A in clinical practice and association with lopinavir resistance. AIDS. 2006;20:1071–1074. PubMed

Doyon, L., Croteau, G., Thibeault, D., Poulin, F., Pilote, L., Lamarre, D. Second locus involved in human immunodeficiency virus type 1 resistance to protease inhibitors. J. Virol. 1996;70:3763–3769. PubMed PMC

Emsley, P., Cowtan, K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004;60:2126–2132. PubMed

Evans, P.R. Data reduction. In: Helliwell J.R., et al., editors. Proceedings of CCP4 Study Weekend, on Data Collection and Processing; Warrington, UK: Daresbury Laboratory; 1993. pp. 114–122.

Friend, J., Parkin, N., Lieger, T., Martin, J.N., Deeks, S.G. Isolated lopinavir resistance after virological rebound of a ritonavir/lopinavir-based regimen. AIDS. 2004;18:1965–1970. PubMed

Gulnik, S.V., Suvorov, L.I., Liu, B., Yu, B., Anderson, B., Mitsuya, H., Erickson, J.W. Kinetic characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug pressure. Biochemistry. 1995;34:9282–9287. PubMed

Hammer, S.M., Saag, M.S., Schechter, M., Montaner, J.S., Schooley, R.T., Jacobsen, D.M., Thompson, M.A., Carpenter, C.C., Fischl, M.A., Gazzard, B.G., et al. Treatment for adult HIV infection: 2006 recommendations of the International AIDS Society-USA panel. Top. HIV Med. 2006;14:827–843. PubMed

Jacobsen, H., Hänggi, M., Ott, M., Duncan, I.B., Owen, S., Andreoni, M., Vella, S., Mous, J. In vivo resistance to a human immunodeficiency virus type 1 proteinase inhibitor: Mutations, kinetics, and frequencies. J. Infect. Dis. 1996;173:1379–1387. PubMed

Kagan, R.M., Shenderovich, M.D., Heseltine, P.N.R., Ramnarayan, K. Structural analysis of an HIV-1 protease I47A mutant resistant to the protease inhibitor lopinavir. Protein Sci. 2005;14:1870–1878. PubMed PMC

Kagan, R.M., Cheung, P.K., Huang, T.K., Lewinski, M.A. Increasing prevalence of HIV-1 protease inhibitor-associated mutations correlates with long-term non-suppressive protease inhibitor treatment. Antiviral Res. 2006;71:42–52. PubMed

Kempf, D.J., Isaacson, J.D., King, M.S., Brun, S.C., Xu, Y., Real, K., Berstein, B.M., Japour, A.J., Sun, E., Rode, R.A. Identification of genotypic changes in human immunodeficiency virus protease that correlate with reduced susceptibility to the protease inhibitor lopinavir among viral isolates from protease inhibitor-experienced patients. J. Virol. 2001;75:7462–7469. PubMed PMC

Kohl, N.E., Emini, E.A., Schleif, W.A., Davis, L.J., Heimbach, J.C., Dixon, R.A., Scolnick, E.M., Sigal, I.S. Active human immunodeficiency virus protease is required for viral infectivity. Proc. Natl. Acad. Sci. 1988;85:4686–4690. PubMed PMC

Konvalinka, J., Litterst, M.A., Welker, R., Kottler, H., Rippmann, F., Heuser, A.M., Kräusslich, H.G. An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity. J. Virol. 1995;69:7180–7186. PubMed PMC

Konvalinka, J., Litera, J., Weber, J., Vondrášek, J., Hradílek, M., Souček, M., Pichová, I., Majer, P., Štrop, P., Sedláček, J., et al. Configurations of diastereomeric hydroxyethylene isosteres strongly affect biological activities of a series of specific inhibitors of human-immunodeficiency-virus proteinase. Eur. J. Biochem. 1997;250:559–566. PubMed

Kovalevsky, A.Y., Chumanevich, A.A., Liu, F., Louis, J.M., Weber, I.T. Caught in the act: The 1.5 Å resolution crystal structures of the HIV-1 protease and the I54V mutant reveal a tetrahedral reaction intermediate. Biochemistry. 2007;46:14854–14864. PubMed PMC

Kožíšek, M., Prejdová, J., Souček, M., Machala, L., Staňková, M., Linka, M., Brůčková, M., Konvalinka, J. Characterisation of mutated proteinases derived from HIV-positive patients: Enzyme activity, vitality and inhibition. Collect. Czech. Chem. Commun. 2004;69:703–714.

Leslie, A.G. Integration of macromolecular diffraction data. Acta Crystallogr. D Biol. Crystallogr. 1999;55:1696–1702. PubMed

Liu, F., Boross, P.I., Wang, Y.F., Tozser, J., Louis, J.M., Harrison, R.W., Weber, I.T. Kinetic, stability, and structural changes in high-resolution crystal structures of HIV-1 protease with drug-resistant mutations L24I, I50V, and G73S. J. Mol. Biol. 2005;354:789–800. PubMed PMC

Loutfly, M.R., Walmsley, S.L. Salvage antiretroviral therapy in HIV infection. Expert Opin. Pharmacother. 2002;3:81–90. PubMed

Mammano, F., Petit, C., Clavel, F. Resistance-associated loss of viral fitness in human immunodeficiency virus type 1: Phenotypic analysis of protease and gag coevolution in protease inhibitor-treated patients. J. Virol. 1998;72:7632–7637. PubMed PMC

Masse, S., Lu, X., Dekhtyar, T., Lu, L., Koev, G., Gao, F., Mo, H., Kempf, D.J., Bernstein, B., Hanna, G.J., et al. In vitro selection and characterization of human immunodeficiency virus type 2 with decreased susceptibility to lopinavir. Antimicrob. Agents Chemother. 2007;51:3075–3080. PubMed PMC

Massova, I., Kollman, P.A. Combined molecular mechanical and continuum solvent approach (MM-PBSA/GBSA) to predict ligand binding. Perspect. Drug Discov. Des. 2000;18:113–135.

Mastrolorenzo, A., Rusconi, S., Scozzafava, A., Supuran, C.T. Inhibitors of HIV-1 protease: 10 years after. Expert Opin. Ther. Pat. 2006;16:1067–1091.

Mo, H.M., King, S., King, K., Molla, A., Brun, S., Kempf, D.J. Selection of resistance in protease inhibitor-experienced, human immunodeficiency virus type 1-infected subjects failing lopinavir-and ritonavir-based therapy: Mutation patterns and baseline correlates. J. Virol. 2005;79:3329–3338. PubMed PMC

Murshudov, G.N., Vagin, A.A., Dodson, E.J. Refinment of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 1997;53:240–255. PubMed

Nijhuis, M., van Maarseveen, N.M., Lastere, S., Schipper, P., Coakley, E., Glass, B., Rovenská, M., de Jong, D., Chappey, C., Goedegebuure, I.W., et al. A novel substrate based HIV-1 protease inhibitor drug resistance mechanism. PLoS Med. 2007;4:152–163. PubMed PMC

Parkin, N.T., Chappey, C., Petropoulos, Ch.J. Improving lopinavir genotype algorithm through phenotype correlations: Novel mutation patterns and amprenavir cross-resistance. AIDS. 2003;17:955–961. PubMed

Patick, A.K., Duran, M., Cao, Y., Shugarts, D., Keller, M.R., Mazabel, E., Knowles, M., Chapman, S., Kuritzkes, D.R., Markowitz, M. Genotypic and phenotypic characterization of human immunodeficiency virus type 1 variants isolated from patients treated with the protease inhibitor nelfinavir. Antimicrob. Agents Chemother. 1998;42:2637–2644. PubMed PMC

Peng, C., Ho, B.K., Chang, N.T. Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity. J. Virol. 1989;63:2550–2556. PubMed PMC

Prejdová, J., Souček, M., Konvalinka, J. Determining and overcoming resistance to HIV protease inhibitors. Curr. Drug Targets Infect. Disord. 2004;4:137–152. PubMed

Richards, A.D., Phylip, L.H., Farmerie, W.G., Scarborough, P.E., Alvarez, A., Dunn, B.M., Hirel, P.H., Konvalinka, J., Štrop, P., Pavlíčková, L., et al. Sensitive, soluble chromogenic substrates for HIV-1 proteinase. J. Biol. Chem. 1990;265:7733–7736. PubMed

Rose, J.R., Babe, L.M., Craik, C.S. Defining the level of human-immunodeficiency-virus type-1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity. J. Virol. 1995;69:2751–2758. PubMed PMC

Rose, R.B., Craik, C.S., Stroud, R.M. Domain flexibility in retroviral proteases: Structural implications for drug resistant mutations. Biochemistry. 1998;37:2607–2621. PubMed

Schmit, J.C., Ruiz, L., Clotet, B., Raventos, A., Tor, J., Leonard, J., Desmyter, J., De Clercq, E., Vandamme, A.M. Resistance-related mutations in the HIV-1 protease gene of patients treated for 1 year with the protease inhibitor ritonavir (ABT-538) AIDS. 1996;10:995–999. PubMed

Sham, H.L., Kempf, D.J., Molla, A., Kennan, C.M., Gondi, N.K., Chin-Ming, Ch., Kati, W., Steward, K., Lal, R., Hsu, A., et al. ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease. Antimicrob. Agents Chemother. 1998;42:3218–3224. PubMed PMC

Stoll, V., Qin, W., Stewart, K.D., Jakob, C., Park, C., Walter, K., Simmer, R.L., Helfrich, R., Bussiere, D., Kao, J., et al. X-ray crystallographic structure of ABT-378 (lopinavir) bound to HIV-1 protease. Bioorg. Med. Chem. 2002;10:2803–2806. PubMed

Stříšovský, K., Tessmer, U., Langner, J., Konvalinka, J., Kräusslich, H.G. Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: Re-thinking the “fireman's grip” hypothesis. Protein Sci. 2000;9:1631–1641. PubMed PMC

Surleraux, D.L., de Kock, H.A., Verschueren, W.G., Pille, G.M., Maes, L.J., Peeters, A., Vendeville, S., De, M.S., Azijn, H., Pauwels, R., et al. Design of HIV-1 protease inhibitors active on multidrug-resistant virus. J. Med. Chem. 2005;48:1965–1973. PubMed

Václavíková, J., Machala, L., Staňková, M., Linka, M., Brůčková, M., Vandasová, J., Konvalinka, J. Response of HIV positive patients to the long-term salvage therapy by lopinavir/ritonavir. J. Clin. Virol. 2005;33:319–323. PubMed

Weber, J., Mesters, J.R., Lepšík, M., Prejdová, J., Švec, M., Šponarová, J., Mlčochová, P., Skalická, K., Stříšovský, K., Uhlíková, T., et al. Unusual binding mode of an HIV-1 protease inhibitor explains its potency against multi-drug-resistant virus strains. J. Mol. Biol. 2002;324:739–754. PubMed

Williams, J.W., Morrison, J.F. The kinetics of reversible tight-binding inhibition. Methods Enzymol. 1979;63:437–467. PubMed

Winn, M.D., Murshudov, M.N., Papiz, M.Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 2003;374:300–321. PubMed

Yin, P.D., Das, D., Mitsuya, H. Overcoming HIV drug resistance through rational drug design based on molecular, biochemical, and structural profiles of HIV resistance. Cell. Mol. Life Sci. 2006;63:1706–1724. PubMed PMC

Zhang, D.W., Zhang, J.Z.H. Full quantum mechanical study of binding of HIV-1 protease drugs. Int. J. Quantum Chem. 2005;103:246–257.

Viral proteases as therapeutic targets

Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor

Mutations in HIV-1 gag and pol compensate for the loss of viral fitness caused by a highly mutated protease

Current and Novel Inhibitors of HIV Protease

Molecular characterization of clinical isolates of human immunodeficiency virus resistant to the protease inhibitor darunavir