Discoveryof Noninhibitory Macrocyclic Ligands forProtein Tyrosine Phosphatase 1B Using a Function-Based, IterativeScreening Strategy

Chemically induced proximity is a powerful modality for manipulating protein function. Most of the effort in this field has focused on targeted protein degradation, but recruitment of other types of post-translational modification enzymes to a target protein is also of interest. To construct such reagents, one would ideally like to have ligands that engage the enzyme without inhibiting its activity. In this study, we describe a screening platform for the discovery of noninhibitory macrocyclic ligands for a protein tyrosine phosphatase, using PTP1B as an exemplary model target. This workflow involves sequential screens of small libraries of bead-displayed macrocycles in which only one position of the macrocycle is varied in each round of screening while the others are held as invariant placeholders. The beads co-display a high KM substrate for the phosphatase, allowing ligand-dependent recruitment of the enzyme to the bead surface to be coupled to dephosphorylation of the co-displayed substrate. This is detected by staining with a labeled anti-phosphotyrosine antibody. Finally, we demonstrate that the same general approach can be applied to proteins lacking enzymatic activity by screening against biotin ligase-target protein fusions and employing a proximity labeling-like assay to register screening hits.