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

Dickinson BC, Packer MS, Badran AH, Liu DR - Nat Commun (2014)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, USA.

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.

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HCV PA-RNAP response to protease inhibitors in E. coli cellsHost cells expressing the HCV PA-RNAP were incubated with HCV protease inhibitors danoprevir (a) or asunaprevir (b) for 90 min, followed by inoculation with HCV protease encoding phage. After 3 h, luminescence assays were used to quantify relative gene activation resulting from the PA-RNAP. Luminescence experiments were performed in triplicate with error bars depicting the standard deviation.
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Figure 3: HCV PA-RNAP response to protease inhibitors in E. coli cellsHost cells expressing the HCV PA-RNAP were incubated with HCV protease inhibitors danoprevir (a) or asunaprevir (b) for 90 min, followed by inoculation with HCV protease encoding phage. After 3 h, luminescence assays were used to quantify relative gene activation resulting from the PA-RNAP. Luminescence experiments were performed in triplicate with error bars depicting the standard deviation.

Mentions: As an initial application of protease PACE, we continuously evolved protease enzymes to rapidly assess the drug resistance susceptibility of small-molecule protease inhibitors. Several HCV protease inhibitors are in late-stage clinical trials or are awaiting FDA approval33,34. For some HCV protease inhibitor drug candidates, clinically isolated drug resistance mutations are known20. First we tested whether small-molecule HCV protease inhibitors can modulate protease activity in the protease PACE system. We observed that the incubation of host cells with either danoprevir (IC50 = ~0.3 nM)35 or asunaprevir (IC50 = ~1.0 nM)36, two second-generation HCV protease inhibitors, inhibited the cellular gene expression arising from the activity of HCV protease on the HCV PA-RNAP in a dose-dependent manner (Fig. 3). These observations suggest that protease inhibitors can create selection pressure during PACE favoring the evolution of protease mutants that retain their ability to cleave a cognate substrate despite the presence of the drug candidates.


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

Dickinson BC, Packer MS, Badran AH, Liu DR - Nat Commun (2014)

HCV PA-RNAP response to protease inhibitors in E. coli cellsHost cells expressing the HCV PA-RNAP were incubated with HCV protease inhibitors danoprevir (a) or asunaprevir (b) for 90 min, followed by inoculation with HCV protease encoding phage. After 3 h, luminescence assays were used to quantify relative gene activation resulting from the PA-RNAP. Luminescence experiments were performed in triplicate with error bars depicting the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4215169&req=5

Figure 3: HCV PA-RNAP response to protease inhibitors in E. coli cellsHost cells expressing the HCV PA-RNAP were incubated with HCV protease inhibitors danoprevir (a) or asunaprevir (b) for 90 min, followed by inoculation with HCV protease encoding phage. After 3 h, luminescence assays were used to quantify relative gene activation resulting from the PA-RNAP. Luminescence experiments were performed in triplicate with error bars depicting the standard deviation.
Mentions: As an initial application of protease PACE, we continuously evolved protease enzymes to rapidly assess the drug resistance susceptibility of small-molecule protease inhibitors. Several HCV protease inhibitors are in late-stage clinical trials or are awaiting FDA approval33,34. For some HCV protease inhibitor drug candidates, clinically isolated drug resistance mutations are known20. First we tested whether small-molecule HCV protease inhibitors can modulate protease activity in the protease PACE system. We observed that the incubation of host cells with either danoprevir (IC50 = ~0.3 nM)35 or asunaprevir (IC50 = ~1.0 nM)36, two second-generation HCV protease inhibitors, inhibited the cellular gene expression arising from the activity of HCV protease on the HCV PA-RNAP in a dose-dependent manner (Fig. 3). These observations suggest that protease inhibitors can create selection pressure during PACE favoring the evolution of protease mutants that retain their ability to cleave a cognate substrate despite the presence of the drug candidates.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, USA.

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.

Show MeSH
Related in: MedlinePlus