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High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines
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Elucidating the cellular determinants of targeted membrane protein degradation by lysosome-targeting chimeras

Green Ahn1,2, Nicholas M Riley1,2, Roarke A Kamber3

  • 1Department of Chemistry, Stanford University, Stanford, CA 94305, USA.

Science (New York, N.Y.)
|October 19, 2023
PubMed

Insights

Disrupting retromer genes enhances lysosome-targeting chimera (LYTAC) degradation of membrane proteins by limiting LYTAC recycling. Neddylated cullin-3 and M6P biosynthesis inhibition further optimize LYTAC efficacy for therapeutic development.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Drug Discovery

Background:

  • Targeted protein degradation offers advantages over inhibition for therapeutics.
  • Lysosome-targeting chimeras (LYTACs) use receptors like CI-M6PR to deliver extracellular proteins to lysosomes.

Purpose of the Study:

  • To identify modulators of LYTAC-mediated membrane protein degradation using a genome-wide CRISPR knockout screen.
  • To elucidate mechanisms governing LYTAC trafficking and efficacy.

Main Methods:

  • Genome-wide CRISPR knockout screening in human cells.
  • Analysis of LYTAC recycling, lysosomal maturation, and receptor occupancy.
  • Investigating the role of retromer, cullin-3, and M6P biosynthesis.

Main Results:

  • Disrupting retromer genes enhanced target degradation by reducing LYTAC recycling.
  • Neddylated cullin-3 was identified as a facilitator of LYTAC-complex lysosomal maturation and a predictor of LYTAC efficacy.
  • Inhibiting M6P biosynthesis increased LYTAC-target complex internalization due to reduced CI-M6PR occupancy.

Conclusions:

  • Retromer disruption and modulation of cullin-3 activity are key strategies to improve LYTAC-mediated protein degradation.
  • Targeting M6P biosynthesis can enhance LYTAC internalization and efficacy.
  • Findings provide insights for designing next-generation LYTACs and understanding receptor trafficking.

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