Visser, E. J. | bioRxiv Science

bioRxiv2024

Molecular glues of the regulatory ChREBP/14-3-3 complex protect beta cells from glucolipotoxicity.

The Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor (TF) with two major splice isoforms ( and {beta}). In chronic hyperglycemia and glucolipotoxicity, ChREBP-mediated ChREBP{beta} expression surges, leading to insulin-secreting {beta}-cell dedifferentiation and death. 14-3-3 binding to ChREBP results in cytoplasmic retention and suppression of transcriptional activity. Thus, small molecule-mediated stabilization of this protein-protein interaction (PPI) may be of therapeutic value. Here, we show that structure-based optimizations of a molecular glue compound led to potent ChREBP/14-3-3 PPI stabilizers with cellular activity. In primary human {beta}-cells, the most active compound retained ChREBP in the cytoplasm, and efficiently protected {beta}-cells from glucolipotoxicity while maintaining {beta}-cell identity. This study may thus not only provide the basis for the development of a unique class of compounds for the treatment of Type 2 Diabetes but also showcases an alternative molecular glue approach for achieving small molecule control of notoriously difficult to target TFs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/580675v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@18a17c8org.highwire.dtl.DTLVardef@11f6c0forg.highwire.dtl.DTLVardef@1ef4e63org.highwire.dtl.DTLVardef@34799_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

A Systematic Approach to the Discovery of Protein-Protein Inter-action Stabilizers

Protein-protein interactions (PPIs) are responsible for the proper function of biological processes and, when dysregulated, commonly lead to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for screening small molecules in a quantitative, high-throughput manner. We explore the scope of the disulfide tethering technology for the discovery of selective fragments as starting points for the development of potent small molecule PPI stabilizers and molecular glues using the hub protein 14-3-3{sigma}. The complexes with 5 biologically and structurally diverse phospho-peptides, derived from the 14-3-3 client proteins ER, FOXO1, C-RAF, USP8, and SOS1, were screened for hit identification. Stabilizing fragments could be found for 4/5 client complexes with a diversified hit-rate and stabilizing efficacy for the different 14-3-3/client phospho-peptides. Extensive structural elucidation revealed the ability and adaptivity of the peptide to make productive interactions with the tethered fragments as key criterion for cooperative complex formation. We validated eight fragment stabilizers, six of which showed selectivity for one phospho-peptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious of these fragments increased 14-3-3{sigma}/C-RAF phospho-peptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3{sigma} provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.

biochemistry↗