A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations

By B. Dickinson, M. Packer, A. Badran, David R. Liu. 2014

97 citations recorded by the source corpus.

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. Phage-assisted continuous evolution (PACE) has the potential to rapidly evolve drug-resistant mutations. Here, Dickinson et al.present a protease PACE system that identifies clinically relevant mutations conferring resistance to protease inhibitors in only a few days of continuous evolution.

Subfields: Hepatology, Molecular Biology, Radiology, Nuclear Medicine and Imaging

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Citations in the corpus

• In Vivo Continuous Directed Evolution (2014, unknown): “This system has been further modified to enhance its ability to serve as a basis for alternative selection schemes, and ongoing studies use PACE to mediate the continuous directed evolution of DNA-binding domains, protein-protein interactions, genome modification enzymes, proteases, and other enzyme”
• Simultaneous selection and counter‐selection for the directed evolution of proteases in E. coli using a cytoplasmic anchoring strategy (2016, background): “, 2012), translation (Dickinson et al., 2014), or non-toxicity (O’Loughlin et al.”