Development of A Fission Yeast Cell-Based Platform for High Throughput Screening of HIV-1 Protease Inhibitors

Author(s): Zsigmond Benko, Jiantao Zhang, Richard Y. Zhao*

Journal Name: Current HIV Research

Volume 17 , Issue 6 , 2019


Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Abstract:

Background: HIV-1 protease inhibitor (PI) is one of the most potent classes of drugs in combinational antiretroviral therapies (cART). When a PI is used in combination with other anti- HIV drugs, cART can often suppress HIV-1 below detection thus prolonging the patient’s lives. However, the challenge often faced by patients is the emergence of HIV-1 drug resistance. Thus, PIs with high genetic-barrier to drug-resistance are needed.

Objective: The objective of this study was to develop a novel and simple fission yeast (Schizosaccharomyces pombe) cell-based system that is suitable for high throughput screening (HTS) of small molecules against HIV-1 protease (PR).

Methods: A fission yeast RE294-GFP strain that stably expresses HIV-1 PR and green fluorescence protein (GFP) under the control of an inducible nmt1 promoter was used. Production of HIV-1 PR induces cellular growth arrest, which was used as the primary endpoint for the search of PIs and was quantified by an absorbance-based method. Levels of GFP production were used as a counter-screen control to eliminate potential transcriptional nmt1 inhibitors.

Results: Both the absorbance-based HIV-1 PR assay and the GFP-based fluorescence assay were miniaturized and optimized for HTS. A pilot study was performed using a small drug library mixed with known PI drugs and nmt1 inhibitors. With empirically adjusted and clearly defined double-selection criteria, we were able to correctly identify the PIs and to exclude all hidden nmt1 inhibitors.

Conclusion: We have successfully developed and validated a fission yeast cell-based HTS platform for the future screening and testing of HIV-1 PR inhibitors.

Keywords: HIV-1 protease (PR), HIV-1 protease inhibitor (PI), fission yeast (Schizosaccharomyces pombe), high throughput screening (HTS), green fluorescence protein (GFP), atazanavir (ATV), transcriptional nmt1 inhibitor (TNI).

[1]
UNAIDS. Global HIV & AIDS statistics fact sheet UNAIDS Report 2019.
[2]
Palmer S, Shafer RW, Merigan TC. Highly drug-resistant HIV-1 clinical isolates are cross-resistant to many antiretroviral compounds in current clinical development. AIDS 1999; 13(6): 661-7.
[http://dx.doi.org/10.1097/00002030-199904160-00006] [PMID: 10397560]
[3]
Logsdon BC, Vickrey JF, Martin P, et al. Crystal structures of a multidrug-resistant human immunodeficiency virus type 1 protease reveal an expanded active-site cavity. J Virol 2004; 78(6): 3123-32.
[http://dx.doi.org/10.1128/JVI.78.6.3123-3132.2004] [PMID: 14990731]
[4]
Rhee SY, Taylor J, Fessel WJ, et al. HIV-1 protease mutations and protease inhibitor cross-resistance. Antimicrob Agents Chemother 2010; 54(10): 4253-61.
[http://dx.doi.org/10.1128/AAC.00574-10] [PMID: 20660676]
[5]
Yang H, Nkeze J, Zhao RY. Effects of HIV-1 protease on cellular functions and their potential applications in antiretroviral therapy. Cell Biosci 2012; 2(1): 32.
[http://dx.doi.org/10.1186/2045-3701-2-32] [PMID: 22971934]
[6]
Calugi C, Guarna A, Trabocchi A. Heterocyclic HIV-protease inhibitors. Curr Med Chem 2013; 20(30): 3693-710.
[http://dx.doi.org/10.2174/09298673113209990135] [PMID: 23746271]
[7]
Pang X, Liu Z, Zhai G. Advances in non-peptidomimetic HIV protease inhibitors. Curr Med Chem 2014; 21(17): 1997-2011.
[http://dx.doi.org/10.2174/0929867321666140217115951] [PMID: 24533811]
[8]
Mallolas J. Darunavir Stands Up as Preferred HIV Protease Inhibitor. AIDS Rev 2017; 19(2): 105-12.
[PMID: 28664942]
[9]
Coffin JM. Response: Plasma Viral Load, CD4+ Cell Counts, and HIV-1 Production by Cells. Science 1996; 271(5249): 671.
[http://dx.doi.org/10.1126/science.271.5249.671] [PMID: 17814908]
[10]
Persaud D, Siberry GK, Ahonkhai A, et al. Continued production of drug-sensitive human immunodeficiency virus type 1 in children on combination antiretroviral therapy who have undetectable viral loads. J Virol 2004; 78(2): 968-79.
[http://dx.doi.org/10.1128/JVI.78.2.968-979.2004] [PMID: 14694128]
[11]
Combescure C, Vallier N, Ledergerber B, et al. Swiss HIV Cohort Study. How reliable is an undetectable viral load? HIV Med 2009; 10(8): 470-6.
[http://dx.doi.org/10.1111/j.1468-1293.2009.00714.x] [PMID: 19459990]
[12]
Wensing AM, van Maarseveen NM, Nijhuis M. Fifteen years of HIV Protease Inhibitors: raising the barrier to resistance. Antiviral Res 2010; 85(1): 59-74.
[http://dx.doi.org/10.1016/j.antiviral.2009.10.003] [PMID: 19853627]
[13]
Côté HC, Brumme ZL, Harrigan PR. Human immunodeficiency virus type 1 protease cleavage site mutations associated with protease inhibitor cross-resistance selected by indinavir, ritonavir, and/or saquinavir. J Virol 2001; 75(2): 589-94.
[http://dx.doi.org/10.1128/JVI.75.2.589-594.2001] [PMID: 11134271]
[14]
Wu TD, Schiffer CA, Gonzales MJ, et al. Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different protease inhibitor treatments. J Virol 2003; 77(8): 4836-47.
[http://dx.doi.org/10.1128/JVI.77.8.4836-4847.2003] [PMID: 12663790]
[15]
Kantor R, Fessel WJ, Zolopa AR, et al. Evolution of primary protease inhibitor resistance mutations during protease inhibitor salvage therapy. Antimicrob Agents Chemother 2002; 46(4): 1086-92.
[http://dx.doi.org/10.1128/AAC.46.4.1086-1092.2002] [PMID: 11897594]
[16]
Voshavar C. Protease Inhibitors for the Treatment of HIV/AIDS: Recent Advances and Future Challenges. Curr Top Med Chem 2019; 19(18): 1571-98.
[http://dx.doi.org/10.2174/1568026619666190619115243] [PMID: 31237209]
[17]
Westby M, Nakayama GR, Butler SL, Blair WS. Cell-based and biochemical screening approaches for the discovery of novel HIV-1 inhibitors. Antiviral Res 2005; 67(3): 121-40.
[http://dx.doi.org/10.1016/j.antiviral.2005.06.006] [PMID: 16112209]
[18]
Molla A, Vasavanonda S, Kumar G, et al. Human serum attenuates the activity of protease inhibitors toward wild-type and mutant human immunodeficiency virus. Virology 1998; 250(2): 255-62.
[http://dx.doi.org/10.1006/viro.1998.9383] [PMID: 9792836]
[19]
Lindsten K, Uhlíková T, Konvalinka J, Masucci MG, Dantuma NP. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity. Antimicrob Agents Chemother 2001; 45(9): 2616-22.
[http://dx.doi.org/10.1128/AAC.45.9.2616-2622.2001] [PMID: 11502538]
[20]
Cheng TJ, Brik A, Wong CH, Kan CC. Model system for high-throughput screening of novel human immunodeficiency virus protease inhibitors in Escherichia coli. Antimicrob Agents Chemother 2004; 48(7): 2437-47.
[http://dx.doi.org/10.1128/AAC.48.7.2437-2447.2004] [PMID: 15215092]
[21]
Hu K, Clément JF, Abrahamyan L, et al. A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer. J Virol Methods 2005; 128(1-2): 93-103.
[http://dx.doi.org/10.1016/j.jviromet.2005.04.012] [PMID: 15951029]
[22]
Fuse T, Watanabe K, Kitazato K, Kobayashi N. Establishment of a new cell line inducibly expressing HIV-1 protease for performing safe and highly sensitive screening of HIV protease inhibitors. Microbes Infect 2006; 8(7): 1783-9.
[http://dx.doi.org/10.1016/j.micinf.2006.02.016] [PMID: 16815068]
[23]
Kräusslich HG, Ingraham RH, Skoog MT, Wimmer E, Pallai PV, Carter CA. Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides. Proc Natl Acad Sci USA 1989; 86(3): 807-11.
[http://dx.doi.org/10.1073/pnas.86.3.807] [PMID: 2644644]
[24]
Kitidee K, Khamaikawin W, Thongkum W, et al. Expedient screening for HIV-1 protease inhibitors using a simplified immunochromatographic assay. J Chromatogr B Analyt Technol Biomed Life Sci 2016; 1021: 153-8.
[http://dx.doi.org/10.1016/j.jchromb.2015.10.003] [PMID: 26490422]
[25]
Ivey FD, Wang L, Demirbas D, Allain C, Hoffman CS. Development of a fission yeast-based high-throughput screen to identify chemical regulators of cAMP phosphodiesterases. J Biomol Screen 2008; 13(1): 62-71.
[http://dx.doi.org/10.1177/1087057107312127] [PMID: 18227226]
[26]
de Medeiros AS, Kwak G, Vanderhooft J, Rivera S, Gottlieb R, Hoffman CS. Fission yeast-based high-throughput screens for PKA pathway inhibitors and activators. Methods Mol Biol 2015; 1263: 77-91.
[http://dx.doi.org/10.1007/978-1-4939-2269-7_6] [PMID: 25618337]
[27]
Nkeze J, Li L, Benko Z, Li G, Zhao RY. Molecular characterization of HIV-1 genome in fission yeast Schizosaccharomyces pombe. Cell Biosci 2015; 5: 47.
[http://dx.doi.org/10.1186/s13578-015-0037-7] [PMID: 26309721]
[28]
Getz RA, Kwak G, Cornell S, et al. A fission yeast platform for heterologous expression of mammalian adenylyl cyclases and high throughput screening. Cell Signal 2019; 60: 114-21.
[http://dx.doi.org/10.1016/j.cellsig.2019.04.010] [PMID: 31026495]
[29]
Zhao Y, Lieberman HB. Schizosaccharomyces pombe: a model for molecular studies of eukaryotic genes. DNA Cell Biol 1995; 14(5): 359-71.
[http://dx.doi.org/10.1089/dna.1995.14.359] [PMID: 7748486]
[30]
Zhao RY. Yeast for virus research. Microb Cell 2017; 4(10): 311-30.
[http://dx.doi.org/10.15698/mic2017.10.592] [PMID: 29082230]
[31]
Li G, Zhao RY. Molecular Cloning and Characterization of Small Viral Genome in Fission Yeast. Methods Mol Biol 2018; 1721: 47-61.
[http://dx.doi.org/10.1007/978-1-4939-7546-4_5] [PMID: 29423846]
[32]
Li G, Poulsen M, Fenyvuesvolgyi C, et al. Characterization of cytopathic factors through genome-wide analysis of the Zika viral proteins in fission yeast. Proc Natl Acad Sci USA 2017; 114(3): E376-85.
[http://dx.doi.org/10.1073/pnas.1619735114] [PMID: 28049830]
[33]
Benko Z, Liang D, Li G, et al. A fission yeast cell-based system for multidrug resistant HIV-1 proteases. Cell Biosci 2017; 7: 5.
[http://dx.doi.org/10.1186/s13578-016-0131-5] [PMID: 28096973]
[34]
Denny PW, Steel PG. Yeast as a potential vehicle for neglected tropical disease drug discovery. J Biomol Screen 2015; 20(1): 56-63.
[http://dx.doi.org/10.1177/1087057114546552] [PMID: 25121554]
[35]
Benko Z, Elder RT, Liang D, Zhao RY. Fission yeast as a HTS platform for molecular probes of HIV-1 Vpr-induced cell death. Int J High Throughput Screen 2010; 2010(1): 151-62.
[36]
Zhao Y, Cao J, O’Gorman MRG, Yu M, Yogev R. Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe. J Virol 1996; 70(9): 5821-6.
[PMID: 8709199]
[37]
Zhao Y, Yu M, Chen M, Elder RT, Yamamoto A, Cao J. Pleiotropic effects of HIV-1 protein R (Vpr) on morphogenesis and cell survival in fission yeast and antagonism by pentoxifylline. Virology 1998; 246(2): 266-76.
[http://dx.doi.org/10.1006/viro.1998.9208] [PMID: 9657945]
[38]
Benko Z, Elder RT, Li G, Liang D, Zhao RY. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe. PLoS One 2016; 11(3)e0151286
[http://dx.doi.org/10.1371/journal.pone.0151286] [PMID: 26982200]
[39]
Zhao Y, Elder RT, Chen M, Cao J. Fission yeast expression vectors adapted for positive identification of gene insertion and green fluorescent protein fusion. Biotechniques 1998; 25(3): 438-40,42,44.
[http://dx.doi.org/10.2144/98253st06]
[40]
Maundrell K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem 1990; 265(19): 10857-64.
[PMID: 2358444]
[41]
Chen M, Elder RT, Yu M, et al. Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast. J Virol 1999; 73(4): 3236-45.
[PMID: 10074177]
[42]
Moreno S, Klar A, Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 1991; 194: 795-823.
[http://dx.doi.org/10.1016/0076-6879(91)94059-L] [PMID: 2005825]
[43]
Benko Z, Zhao RY. Zeocin for selection of bleMX6 resistance in fission yeast. Biotechniques 2011; 51(1): 57-60.
[http://dx.doi.org/10.2144/000113706] [PMID: 21781055]
[44]
Suga M, Isobe M, Hatakeyama T. Cryopreservation of competent intact yeast cells for efficient electroporation. Yeast 2000; 16(10): 889-96.
[http://dx.doi.org/10.1002/1097-0061(200007)16:10<889:AID-YEA582>3.0.CO;2-R] [PMID: 10870100]
[45]
Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 1999; 4(2): 67-73.
[http://dx.doi.org/10.1177/108705719900400206] [PMID: 10838414]
[46]
Zhang J, Fu Y, Liang D, Zhao RY, Lakowicz JR. Enhanced fluorescence images for labeled cells on silver island films. Langmuir 2008; 24(21): 12452-7.
[http://dx.doi.org/10.1021/la801749f] [PMID: 18837523]
[47]
Chetty S, Bhakat S, Martin AJ, Soliman ME. Multi-drug resistance profile of PR20 HIV-1 protease is attributed to distorted conformational and drug binding landscape: molecular dynamics insights. J Biomol Struct Dyn 2016; 34(1): 135-51.
[http://dx.doi.org/10.1080/07391102.2015.1018326] [PMID: 25671669]
[48]
Broglia R, Levy Y, Tiana G. HIV-1 protease folding and the design of drugs which do not create resistance. Curr Opin Struct Biol 2008; 18(1): 60-6.
[http://dx.doi.org/10.1016/j.sbi.2007.10.004] [PMID: 18160276]
[49]
Thaisrivongs S, Tomich PK, Watenpaugh KD, et al. Structure-based design of HIV protease inhibitors: 4-hydroxycoumarins and 4-hydroxy-2-pyrones as non-peptidic inhibitors. J Med Chem 1994; 37(20): 3200-4.
[http://dx.doi.org/10.1021/jm00046a002] [PMID: 7932546]
[50]
Hertogs K, Bloor S, Kemp SD, et al. Phenotypic and genotypic analysis of clinical HIV-1 isolates reveals extensive protease inhibitor cross-resistance: a survey of over 6000 samples. AIDS 2000; 14(9): 1203-10.
[http://dx.doi.org/10.1097/00002030-200006160-00018] [PMID: 10894285]
[51]
Thaisrivongs S, Strohbach JW. Structure-based discovery of Tipranavir disodium (PNU-140690E): a potent, orally bioavailable, nonpeptidic HIV protease inhibitor. Biopolymers 1999; 51(1): 51-8.
[http://dx.doi.org/10.1002/(SICI)1097-0282(1999)51:1<51:AID-BIP6>3.0.CO;2-U] [PMID: 10380352]


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 17
ISSUE: 6
Year: 2019
Published on: 03 January, 2020
Page: [429 - 440]
Pages: 12
DOI: 10.2174/1570162X17666191128102839
Price: $65

Article Metrics

PDF: 17
HTML: 5