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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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Continuous evolution of drug resistance in HCV protease(a, b) PACE condition timeline for evolution in the presence of danoprevir (a) or asunaprevir (b). The blue arrows indicate arabinose-induced enhanced mutagenesis, and the red arrow shows the timing and dosing of HCV protease inhibitors. (c) High-throughput sequencing data from phage populations in replicate lagoons (L1 and L2) subjected to danoprevir treatment at 28 h, asunaprevir treatment at 75 h, and no drug at 72 h. All mutations with frequencies more than 1% above the allele-specific error rate are shown. (d) In vitro analysis of danoprevir inhibition of mutant HCV proteases that evolved during PACE. (e) In vitro analysis of asunaprevir inhibition of mutant HCV proteases that evolved during PACE. For (d) and (e), evolved HCV protease variants were expressed and purified, then assayed using an internally quenched fluorescent-substrate (Anaspec). In vitro analyses were performed in triplicate with error bars calculated as the standard deviation.
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Figure 4: Continuous evolution of drug resistance in HCV protease(a, b) PACE condition timeline for evolution in the presence of danoprevir (a) or asunaprevir (b). The blue arrows indicate arabinose-induced enhanced mutagenesis, and the red arrow shows the timing and dosing of HCV protease inhibitors. (c) High-throughput sequencing data from phage populations in replicate lagoons (L1 and L2) subjected to danoprevir treatment at 28 h, asunaprevir treatment at 75 h, and no drug at 72 h. All mutations with frequencies more than 1% above the allele-specific error rate are shown. (d) In vitro analysis of danoprevir inhibition of mutant HCV proteases that evolved during PACE. (e) In vitro analysis of asunaprevir inhibition of mutant HCV proteases that evolved during PACE. For (d) and (e), evolved HCV protease variants were expressed and purified, then assayed using an internally quenched fluorescent-substrate (Anaspec). In vitro analyses were performed in triplicate with error bars calculated as the standard deviation.

Mentions: Phage populations at 6 and 28 h from replicate lagoons were analyzed by high-throughput DNA sequencing. No mutations were substantially enriched in the control lagoons propagated in the absence of any drug candidate (Fig. 4c). In contrast, several mutations rapidly evolved in both replicate lagoons in the presence of danoprevir. Mutations at position D168 were predominant among these mutations. By 28 h, lagoon 1 with danoprevir contained 38.8% D168E, 8.3% D168Y, 2.1% D168A, and 1.1% D168V, while lagoon 2 with danoprevir contained 40.3% D168E and 10.7% D168Y (Fig. 4c). Other genetic differences between the SPs of these two replicate populations, such as R130C (5.1% in lagoon 1, undetectable in lagoon 2) and T72I (10.8% in lagoon 2, undetectable in lagoon 1), suggest that cross-contamination did not lead to the observed protease variants in these experiments. These findings reveal that the presence of danoprevir caused the population of continuously evolving proteases to rapidly acquire mutations at D168.


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)

Continuous evolution of drug resistance in HCV protease(a, b) PACE condition timeline for evolution in the presence of danoprevir (a) or asunaprevir (b). The blue arrows indicate arabinose-induced enhanced mutagenesis, and the red arrow shows the timing and dosing of HCV protease inhibitors. (c) High-throughput sequencing data from phage populations in replicate lagoons (L1 and L2) subjected to danoprevir treatment at 28 h, asunaprevir treatment at 75 h, and no drug at 72 h. All mutations with frequencies more than 1% above the allele-specific error rate are shown. (d) In vitro analysis of danoprevir inhibition of mutant HCV proteases that evolved during PACE. (e) In vitro analysis of asunaprevir inhibition of mutant HCV proteases that evolved during PACE. For (d) and (e), evolved HCV protease variants were expressed and purified, then assayed using an internally quenched fluorescent-substrate (Anaspec). In vitro analyses were performed in triplicate with error bars calculated as the standard deviation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215169&req=5

Figure 4: Continuous evolution of drug resistance in HCV protease(a, b) PACE condition timeline for evolution in the presence of danoprevir (a) or asunaprevir (b). The blue arrows indicate arabinose-induced enhanced mutagenesis, and the red arrow shows the timing and dosing of HCV protease inhibitors. (c) High-throughput sequencing data from phage populations in replicate lagoons (L1 and L2) subjected to danoprevir treatment at 28 h, asunaprevir treatment at 75 h, and no drug at 72 h. All mutations with frequencies more than 1% above the allele-specific error rate are shown. (d) In vitro analysis of danoprevir inhibition of mutant HCV proteases that evolved during PACE. (e) In vitro analysis of asunaprevir inhibition of mutant HCV proteases that evolved during PACE. For (d) and (e), evolved HCV protease variants were expressed and purified, then assayed using an internally quenched fluorescent-substrate (Anaspec). In vitro analyses were performed in triplicate with error bars calculated as the standard deviation.
Mentions: Phage populations at 6 and 28 h from replicate lagoons were analyzed by high-throughput DNA sequencing. No mutations were substantially enriched in the control lagoons propagated in the absence of any drug candidate (Fig. 4c). In contrast, several mutations rapidly evolved in both replicate lagoons in the presence of danoprevir. Mutations at position D168 were predominant among these mutations. By 28 h, lagoon 1 with danoprevir contained 38.8% D168E, 8.3% D168Y, 2.1% D168A, and 1.1% D168V, while lagoon 2 with danoprevir contained 40.3% D168E and 10.7% D168Y (Fig. 4c). Other genetic differences between the SPs of these two replicate populations, such as R130C (5.1% in lagoon 1, undetectable in lagoon 2) and T72I (10.8% in lagoon 2, undetectable in lagoon 1), suggest that cross-contamination did not lead to the observed protease variants in these experiments. These findings reveal that the presence of danoprevir caused the population of continuously evolving proteases to rapidly acquire mutations at D168.

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