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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
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Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target the E-box DNA Site.

Ichiro Inamoto1, Inder Sheoran1, Serban C Popa1

  • 1Department of Chemistry, University of Toronto, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada.

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We engineered a novel protein drug, MEF, to target the Myc proto-oncoprotein. MEF demonstrates enhanced binding and specificity for E-box DNA, showing promise for cancer therapy.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • Protein-based therapeutics are emerging as a new class of drugs targeting oncogenic proteins.
  • The proto-oncoprotein Myc plays a critical role in cancer development.
  • Targeting Myc's interaction with DNA via the E-box motif is a promising therapeutic strategy.

Purpose of the Study:

  • To design and develop a novel protein therapeutic, MEF, targeting the Myc-Max complex.
  • To enhance the DNA-binding affinity and specificity of a previously developed inhibitor.
  • To leverage a combination of rational design and directed evolution for protein drug development.

Main Methods:

  • Rational design of a hybrid protein (ME47) combining Max basic region and E47 helix-loop-helix.
  • Phage-assisted continuous evolution (PACE) to introduce stabilizing mutations.
  • Protein engineering to enhance dimerization and DNA-binding activity, creating the MEF protein.

Main Results:

  • MEF exhibits a 2-fold increase in binding affinity to the E-box motif compared to ME47.
  • MEF shows a 4-fold improvement in specificity for E-box over non-specific DNA sequences.
  • Mutations identified via PACE enhanced the stability of the protein.

Conclusions:

  • The engineered protein MEF demonstrates superior DNA-binding properties for targeting Myc.
  • The combined approach of rational design and PACE is effective for developing robust protein therapeutics.
  • MEF represents a promising candidate for next-generation cancer therapies targeting Myc.