bioRxiv Science⌕ Search

bioRxiv · 10.1101/2023.10.31.565018

Covalent Degrader of the Oncogenic Transcription Factor β-Catenin

Abstract

{beta}-catenin (CTNNB1) is an oncogenic transcription factor that is important in cell-cell adhesion and transcription of cell proliferation and survival genes that drives the pathogenesis of many different types of cancers. However, direct pharmacological targeting of CTNNB1 has remained challenging deeming this transcription factor as "undruggable." Here, we have performed a screen with a library of cysteine-reactive covalent ligands to identify a monovalent degrader EN83 that depletes CTNNB1 in a ubiquitin-proteasome-dependent manner. We show that EN83 directly and covalently targets CTNNB1 through targeting four distinct cysteines within the armadillo repeat domain--C439, C466, C520, and C619--leading to a destabilization of CTNNB1. Using covalent chemoproteomic approaches, we show that EN83 directly engages CTNNB1 in cells with a moderate degree of selectivity. We further demonstrate that direct covalent targeting of three of these four cysteines--C466, C520, and C619--in cells contributes to CTNNB1 degradation in cells. We also demonstrate that EN83 can be further optimized to yield more potent CTNNB1 binders and degraders. Our results show that chemoproteomic approaches can be used to covalently target and degrade challenging transcription factors like CTNNB1 through a destabilization-mediated degradation.

Source connections

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Gowans, F. A., Forte, N., Hatcher, J., Wang, Y., Poblano, B. E. A., Wertz, I. E., Nomura, D. K.. 2023-11-01. Covalent Degrader of the Oncogenic Transcription Factor β-Catenin. https://doi.org/10.1101/2023.10.31.565018

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

Deep reinforcement learning-driven discovery of a MsbA-targeted small-molecule antibiotic for the treatment of Acinetobacter baumannii infection

Antibiotics with new mechanisms are highly pursued to address the threat of infections caused by drug-resistant Gram-negative bacteria. Targeting MsbA, a key protein of the lipopolysaccharide biosynthesis pathway, represents a promising strategy to discover new classes of antibiotics. However, currently available MsbA-targeted molecules either lack sufficient potency or have unfavorable properties, necessitating expansion of chemical space. In this study, we chose the most promising cerastecin Cpd 4 as the template, and used two Artificial Intelligence (AI)-based tools, i.e. Link-INVENT and AutoMolDesigner for molecular design, performed chemical derivatization and antibacterial activity evaluation, which led to the discovery of Y-11 (MIC for A. baumannii: 0.5 g/mL). Encouragingly, Y-11 showed equivalent potency to Cpd4 for carbapenem-resistant A. baumannii, and less cytotoxicity and hemolysis as well as lower spontaneous resistance frequency. In vivo efficacy study demonstrated that Y-11 could effectively reduce bacterial loads in the mice infected by A. baumannii. The following mechanism study including molecular dynamics simulation, biochemical assay, and transmission electron microscope (TEM) analysis suggested that Y-11 inhibited the transport of lipooligosaccharide and impaired the formation of outer membrane, probably by competitively binding to the substrate binding site of MsbA and modulating ATPase activity. Taken together, we have discovered a MsbA-targeted small molecule Y-11 via AI-driven drug design, which provides a foundation for future antibiotic development.

biochemistry↗

Dynamic architecture of the Rixosome reveals mechanism of activation and ITS2 processing

Eukaryotic ribosome assembly requires the coordinated processing and extensive remodeling of pre-rRNAs. During late nuclear maturation of the 60S subunit, sequential removal of the internal transcribed spacer 2 (ITS2) is initiated by endonucleolytic cleavage at site C2 by the conserved Las1 nuclease. Las1 acts together with the kinase Grc3 and the Rix1 complex to form the Rixosome, which also functions in transcriptional regulation. However, the assembly of the Rixosome, its recruitment to pre-ribosomes, and its activation for ITS2 cleavage remain unclear. Here, we present cryo-EM structures of the human LAS1 complex, two structures of the isolated Rixosome and nine transition states of Rix1-bound pre-60S particles from Schizosaccharomyces pombe. These structures reveal a dynamic Rixosome architecture in which the heterotetrameric Las1 complex engages one or two copies of the Rix1 complex. Rix1 binding is highly flexible in the human Rixosome but rigid in the yeast complex. The isolated yeast Rixosome remains inactive, but binding to the pre-60S particle triggers a structural rearrangement that allows for substrate engagement and activation of the nuclease. Together, our results define the dynamic architecture of the Rixosome and provide a structural framework for ITS2 processing during nuclear maturation of the eukaryotic 60S ribosomal subunit.

biochemistry↗

SGFP-Grid Split GFP Graphene Grids

Affinity graphene grids provide a promising approach for selective protein capture in cryo-EM. Here, we introduce a split-GFP graphene grid platform(SGFP-G), in which graphene-conjugated GFP 1-10 selectively captures GFP11 tagged proteins from low concentration samples or cell lysates. This platform enables rapid assessment of target protein enrichment and particle distribution before vitrification via fluorescence imaging, while the grid design positions captured proteins away from the graphene surface and air-water interface. We also introduce a unique strategy to minimize nonspecific protein adsorption, thereby improving the selective enrichment of target proteins on this grid. Using GFP11-tagged apoferritin, we demonstrate fluorescence guided protein capture and obtain a 2.58 [A] cryoEM reconstruction, establishing SGFP-G as an affinity grid platform for high resolution structural studies with reduced sample requirements.

biochemistry↗