Discovery of Noninhibitory Macrocyclic Ligands for Protein Tyrosine Phosphatase 1B Using a Function-Based, Iterative...

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Chemically induced proximity is a key technique for manipulating protein function.
• While targeted protein degradation is common, recruiting other enzymes like post-translational modification enzymes is also valuable.
• Developing ligands that engage enzymes without inhibiting them is crucial for creating effective reagents.

Purpose of the Study:

• To establish a screening platform for identifying non-inhibitory macrocyclic ligands.
• To use protein tyrosine phosphatase 1B (PTP1B) as a model target for ligand discovery.
• To demonstrate the platform's applicability to non-enzymatic proteins.

Main Methods:

• Sequential screening of bead-displayed macrocycle libraries with one varied position per round.
• Co-display of a high K_M substrate on beads for enzyme recruitment and dephosphorylation.
• Detection of dephosphorylation via staining with a labeled anti-phosphotyrosine antibody.
• Adaptation of the platform for non-enzymatic targets using biotin ligase-target protein fusions and proximity labeling assays.

Main Results:

• Successfully developed a screening platform for non-inhibitory macrocyclic ligands.
• Identified ligands for the protein tyrosine phosphatase PTP1B.
• Demonstrated the platform's versatility by applying it to proteins lacking enzymatic activity.

Conclusions:

• The described screening platform is effective for discovering non-inhibitory ligands for enzymes.
• This approach can be extended to target proteins lacking enzymatic activity.
• The methodology facilitates the development of novel chemical biology tools for protein manipulation.