Proteolysis targeting chimeric (PROTAC) molecules that hijack the cellular protein ubiquitination machinery to degrade proteins of interest (POIs) have offered exciting opportunities for use as research tools for biological inquiry and as therapeutic agents. However, ligand identification for the POI remains challenging, especially for proteins without active sites or ligandable pockets. Unlike small molecule ligands, antibodies or nanobodies (Nbs) recognize protein antigens with exceptional specificity and remarkable affinity through the features on protein surfaces, and could be employed as the POI-targeting ligand in a PROTAC. However, their use as ligands is limited by the challenge of delivering the protein-based molecules to intracellular POIs. Here, we devised a strategy to utilize a Nb as ligand for targeted protein degradation of a transcription factor. By appending a Nb to a cell-permeant miniature protein and the catalytic domain of an E3 recruiting protein,<sub> </sub>we demonstrated the proteasomal degradation of BCL11A, the most important transcriptional regulator of hemoglobin in humans. BCL11A controls the switch from fetal to adult hemoglobin, and its loss (through gene deletion and silencing) is a clinical strategy to ameliorate the severity of sickle cell disease and β-thalassemia. Our cell permeable nanobody-based PROTAC provides a mean of rapid induction of fetal hemoglobin through reversible, temporal modulation of BCL11A. Additionally, its development provides a new paradigm for the targeting of therapeutically relevant intracellular proteins that were previously inaccessible to PROTACs.

Cell permeable nanobody-based degraders for targeted protein degradation

ACS Spring 2022

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