Skip to main content
NCBI home page
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 1994;12(10):1012–1016. doi: 10.1038/nbt1094-1012

A High Capacity Microbial Screen for Inhibitors of Human Rhinovirus Protease 3C

J Owen McCall 1,✉, Sunil Kadam 1, Leonard Katz 1
PMCID: PMC7097487  PMID: 7765405

Abstract

We have developed a high capacity screen for compounds that inhibit the 3C protease of human rhinovirus–1b. The assay uses a recombinant strain of Escherichia coli expressing both the protease and a tetracycline resistance–conferring protein modified to contain the minimal protease cleavage site. Cultures growing in microtiter plates containing tetracycline are treated with potential inhibitors and simultaneously monitored for change in growth over time using an oxygen probe. Most of the cultures, not containing an inhibitor of the 3C protease, show reduced growth due to cleavage of the essential gene product; normal growth is seen only in the infrequent culture that contains an inhibitor. In the present example, we have used the tetA gene of plasmid pACYC184 as the modified gene. The system has been validated using inhibitors of protease 3C, and has been used to identify three new inhibitors of the enzyme, active in the micromolar range.

References

  • 1.Scharpe S, De Meester I, Hendriks D, Vanhoof G, van Sande M, Vriend G. Proteases and their inhibitors: today and tomorrow. Biochimie. 1991;73:121–126. doi: 10.1016/0300-9084(91)90084-E. [DOI] [PubMed] [Google Scholar]
  • 2.Powers JCB, Bengali ZH. Conference report—Elastase inhibitors for the treatment of emphysema—Approaches to synthesis and biological evaluation. J. Enzyme Inhibition. 1987;1:311–319. doi: 10.3109/14756368709020128. [DOI] [PubMed] [Google Scholar]
  • 3.Buo L, Aasen AO, Karlsrud TS, Johansen HT, Sivertsen SM. The role of proteases in growth, invasion, and spread of cancer cells. Tidsskr Nor. Laegeforen. 1990;110:3753–3756. [PubMed] [Google Scholar]
  • 4.Douglas LJ. Candida proteinases and candidosis. Crit. Rev. Biotechnol. 1988;8:121–129. doi: 10.3109/07388558809150541. [DOI] [PubMed] [Google Scholar]
  • 5.Lozitsky VP, Fedchuk AS, Puzis LE, Buiko VP, Girlya YI, Bubnov VV. Participation of the proteolysis system in realization of influenza virus virulence and development of the infectious process: Antiviral effect of protease inhibitors. Vopr. Virusol. 1987;32:413–419. [PubMed] [Google Scholar]
  • 6.Walpole CSJ, Wrigglesworth R. Enzyme inhibitors in medicine. Nat. Prod. Rep. 1989;6:311–346. doi: 10.1039/np9890600311. [DOI] [PubMed] [Google Scholar]
  • 7.Larson HE, Reed SE, Tyrrell DAJ. Isolation of rhinoviruses and coronaviruses from 38 colds in adults. J. Med. Virol. 1980;5:221–229. doi: 10.1002/jmv.1890050306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Fox JP. Is a rhinovirus vaccine possible? Amer. J. Epidemiol. 1976;103:345–354. doi: 10.1093/oxfordjournals.aje.a112233. [DOI] [PubMed] [Google Scholar]
  • 9.Krausslich H-G, Wimmer E. Viral proteinases. Ann. Rev. Bio-chem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
  • 10.Taylor, A., Brown, D.P., Kadam, S. et al. 1992. High-level expression and purification of mature HIV-1 protease in Escherichia coli under control of the araBAD promoter. Appl. Microbiol. Biotechnol. 37: 205–210. [DOI] [PubMed]
  • 11.Chang ACY, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic miniplasmid. J. Bacteriol. 1978;134:1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Block TM, Grafstrom RH. Novel bacteriological assay for detection of potential antiviral agents. Antimicrob. Agents Chemother. 1990;34:2337–2341. doi: 10.1128/AAC.34.12.2337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Cordingley MG, Register RB, Callahan PL, Garsky VM, Colonno RJ. Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli. J. Virology. 1989;63:5037–5045. doi: 10.1128/jvi.63.12.5037-5045.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Cordingley MG, Callahan PL, Sardana VV, Garsky VM, Colonno RJ. Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro. J. Biol. Chem. 1990;265:9062–9065. [PubMed] [Google Scholar]
  • 15.Goldberg AL. Degradation of abnormal proteins in Escherichia coli. Proc. Natl. Acad. Sci. USA. 1972;69:422–426. doi: 10.1073/pnas.69.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.O'Rourke SV, Tanner JE, Stitt DT. A method to identify methicillin resistant Staphylococcus aureus within 4 hours. Abstr. Ann. Meet. Am. Soc. Microbiol. 1992;92:462. [Google Scholar]
  • 17.Payne JW, Gilvarg C. Size restriction on peptide utilization in Escherichia coli. J. Biol. Chem. 1968;243:6291–6299. [PubMed] [Google Scholar]
  • 18.Merlin TL, Davis GE, Anderson WL, Moyzis RK, Griffith JK. Aminoglycoside uptake increased by tet gene expression. Antimicrob. Agents Chemother. 1989;33:1549–1552. doi: 10.1128/AAC.33.9.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Robenson DJ, Strobel GA. Deoxyradicinin, a novel phytotoxin from Altenaria helianthi. Phytochemistry. 1982;21:2359–2362. doi: 10.1016/0031-9422(82)85205-9. [DOI] [Google Scholar]
  • 20.Raistrick H, Smith G. Antibacterial substances from mould. I. Citrinin, a metabolic product from Penicillium citrinum. Thorn. Chemistry and Industry. 1941;1941:828–830. [Google Scholar]
  • 21.Johnson LE, Dietz A. Kalafungin, a new antibiotic produced by Streptomyces tanashiensis strain Kala. Appl. Microbiol. 1968;16:1815–1821. doi: 10.1128/am.16.12.1815-1821.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Bergy ME. Kalafungin, a new broad spectrum antibiotic. Isolation and characterization. J. Antibiot. 1968;21:454–457. doi: 10.7164/antibiotics.21.454. [DOI] [PubMed] [Google Scholar]
  • 23.Singh Sheo B., Cordingley Michael G., Ball Richard G., Smith Jack L., Dombrowski Anne W., Goetz Michael A. Structure of stereochemistry of thysanone: a novel human rhinovirus 3C-protease inhibitor from Thysanophora penicilloides. Tetrahedron Letters. 1991;32(39):5279–5282. doi: 10.1016/S0040-4039(00)92364-5. [DOI] [Google Scholar]
  • 24.Beynon RJ, Bond JS. Proteolytic Enzymes: A Practical Approach. 1989. [Google Scholar]
  • 25.Jacobs FA, Romeyer FM, Beauchemin M, Brousseau R. Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli. Gene. 1989;83:95–103. doi: 10.1016/0378-1119(89)90407-1. [DOI] [PubMed] [Google Scholar]
  • 26.Bertani G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Excherichia coli. J. Bacteriol. 1951;62:293–300. doi: 10.1128/jb.62.3.293-300.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zelenetz AD. Construction of complex directional complementary DNA libraries in SfiI. Methods Enzymol. 1992;216:517–530. doi: 10.1016/0076-6879(92)16047-N. [DOI] [PubMed] [Google Scholar]
  • 28.Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning—A Laboratory Manual. 1989. [Google Scholar]
  • 29.Hughes PJ, North C, Jellis CH, Minor PD, Stanway G. The nucleotide sequence of human rhinovirus IB: molecular relationships within the rhinovirus genus. J. Gen. Virol. 1988;69:49–58. doi: 10.1099/0022-1317-69-1-49. [DOI] [PubMed] [Google Scholar]
  • 30.Merrifield RB. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963;85:2149–2154. doi: 10.1021/ja00897a025. [DOI] [Google Scholar]
  • 31.Watts RJ, Crosby GA. Spectroscopic characterization of complexes of ruthenium (II) and indium (III) with 4,4′-diphenyl–2,2′-bipyridine and 4,7-diphenyl–1 ,10-phenanthroline. J. Am. Chem. Soc. 1971;93:3184–3188. doi: 10.1021/ja00742a016. [DOI] [Google Scholar]
  • 32.Wilcox G. The interaction of L-arabinose and D-fucose with AraC protein. J. Biol. Chem. 1974;249:6892–6894. [PubMed] [Google Scholar]

Articles from Bio/Technology (Nature Publishing Company) are provided here courtesy of Nature Publishing Group

RESOURCES