2. Academic Validation
  3. Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening

Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening

  • J Med Chem. 2022 Feb 24;65(4):3266-3305. doi: 10.1021/acs.jmedchem.1c01834.
Sang-Woo Lee 1,  Kim Tai Tran 1 2,  Ruben Vazquez-Uribe 1,  Charlotte Held Gotfredsen 3,  Mads Hartvig Clausen 4,  Blanca Lopez Mendez 5,  Guillermo Montoya 5,  Anders Bach 2,  Morten Otto Alexander Sommer 1
Affiliations

Affiliations

  • 1 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
  • 2 Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
  • 3 Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
  • 4 Center for Nanomedicine and Theranostics, Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
  • 5 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
PMID: 35142491 DOI: 10.1021/acs.jmedchem.1c01834
Abstract

CRISPR/Cas9 has revolutionized several areas of life science; however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity; however, in vivo delivery of such proteins is problematic. Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system. Extensive structure-activity relationship studies, which involved a deconstruction-reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Based on NMR and electrophoretic mobility shift assays, we propose that the inhibitory action of these compounds likely results from direct binding to apo-Cas9, preventing Cas9:gRNA complex formation. These molecules may find use as Cas9 modulators in various applications.

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