title: A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations.
abstract: The laboratory evolution of protease enzymes has the potential to generate proteases with therapeutically relevant specificities and to assess the vulnerability of protease inhibitor drug candidates to the evolution of drug resistance. Here we describe a system for the continuous directed evolution of proteases using phage-assisted continuous evolution (PACE) that links the proteolysis of a target peptide to phage propagation through a protease-activated RNA polymerase (PA-RNAP). We use protease PACE in the presence of danoprevir or asunaprevir, two hepatitis C virus (HCV) protease inhibitor drug candidates in clinical trials, to continuously evolve HCV protease variants that exhibit up to 30-fold drug resistance in only 1 to 3 days of PACE. The predominant mutations evolved during PACE are mutations observed to arise in human patients treated with danoprevir or asunaprevir, demonstrating that protease PACE can rapidly identify the vulnerabilities of drug candidates to the evolution of clinically relevant drug resistance.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, USA.
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Dickinson
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Packer
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, USA.
authors.forename: Ahmed H
authors.initials: AH
authors.lastname: Badran
authors.affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, USA [2] Howard Hughes Medical Institute, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D017104:Bacteriophage M13; D003521:Cyclopropanes; D024882:Drug Resistance, Viral / Q000235:genetics*; D019143:Evolution, Molecular; D015870:Gene Expression; D005821:Genetic Techniques; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D054833:Isoindoles; D007546:Isoquinolines; D007769:Lactams; D047029:Lactams, Macrocyclic; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics*; D011392:Proline / Q000031:analogs & derivatives; D011480:Protease Inhibitors; D013449:Sulfonamides; D014764:Viral Proteins / Q000235:genetics
chemical_list: D003521:Cyclopropanes; D054833:Isoindoles; D007546:Isoquinolines; D007769:Lactams; D047029:Lactams, Macrocyclic; D011480:Protease Inhibitors; D013449:Sulfonamides; D014764:Viral Proteins; C553752:danoprevir; D011392:Proline; D010447:Peptide Hydrolases; C571889:asunaprevir