Dataset Information

BRD4 degradation via stabilization of a native interaction with DCAF16

ABSTRACT: Targeted protein degradation is a modality based on small molecules that induce proximity between a protein of interest (POI) and an E3 ubiquitin ligase, thus prompting POI ubiquitination and degradation. To expand the reach of TPD, current research is geared to unlock novel E3s. To that end, the transcriptional co-activator protein BRD4 has emerged as a frequently used POI. Derivatization of known BRD4 ligands with structurally diverse substituents has yielded a suite of potent BRD4 degraders, thus generating the notion that BRD4 is particularly amenable to TPD. Here, we mechanistically characterize two BRD4 degraders via orthogonal CRISPR screens. Despite their structural dissimilarity, both degraders functionally converge on a unifying mechanism of action that involves the CRL4DCAF16 ligase complex. Using recombinant reconstitution, as well as biophysical and structural elucidation, we demonstrate that BRD4 has an intrinsic activity for DCAF16, which is further enhanced by both compounds. We uncover a novel mode of molecular recognition based on multivalent "gluing" of BRD4 onto DCAF16 that requires both bromodomains of BRD4. Our data thus imply a substantial conceptual overlap between PROTACs and molecular glue degraders, and highlight multivalency as a novel concept in the design of proximity-inducing drugs.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Andrea Rukavina

LAB HEAD: Georg Winter

PROVIDER: PXD040570 | Pride | 2024-01-08

REPOSITORIES: Pride

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Publications

Targeted protein degradation via intramolecular bivalent glues.

Hsia Oliver O Hinterndorfer Matthias M Cowan Angus D AD Iso Kentaro K Ishida Tasuku T Sundaramoorthy Ramasubramanian R Nakasone Mark A MA Imrichova Hana H Schätz Caroline C Rukavina Andrea A Husnjak Koraljka K Wegner Martin M Correa-Sáez Alejandro A Craigon Conner C Casement Ryan R Maniaci Chiara C Testa Andrea A Kaulich Manuel M Dikic Ivan I Winter Georg E GE Ciulli Alessio A

Nature 20240221 8002

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target<sup>1-4</sup>. Here, using orthogo ...[more]

PMID: 38383787

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Project description:The histone acetyl-reader BRD4 is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1. We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression, and pharmacologic inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin-associated suggests a direct role for nuclear metabolism in the control of gene expression. BRD4 is an important chromatin regulator with roles in gene regulation, DNA damage, cell proliferation and cancer progression1-4. The protein is recruited to distinct genomic loci by the interaction of its tandem bromodomains with acetylated lysines on histones and other nuclear proteins5. There, BRD4 acts as a transcriptional activator by P-TEFb-mediated stimulation of transcriptional elongation6. The activating function of BRD4 on key driver oncogenes like MYC have made this epigenetic enzyme an important therapeutic target in both BRD4 translocated and BRD4 wild-type cancers3,7-12, and at least seven bromodomain inhibitors have reached the clinical stage13. Genome-wide studies have identified the role of BRD4-induced epigenetic heterogeneity in cancer cell resistance14, and factors defining BRD4 inhibitor response15,16. However, despite its clinical importance and the broad role of BRD4 in chromatin organization, surprisingly little is known about factors that are directly required for BRD4 function. To systematically expand the list of known BRD4 interactors5 and to characterize proteins directly required for BRD4 function, we developed a strategy of two complementary screens for genetic and physical partners of BRD4. The two approaches converge on a single factor, methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). Our description of a transcriptional role for this C-1-tetrahydrofolate synthase highlights a direct connection between nuclear folate metabolism and cancer regulation.

Project description:Targeted protein degradation (TPD) has emerged as a powerful approach for removing (rather than inhibiting) proteins implicated in diseases. A key step in TPD is the formation of an induced proximity complex where a degrader molecule recruits an E3 ligase to the protein of interest (POI), facilitating the transfer of ubiquitin to the POI and initiating the proteasomal degradation process. Here, we address three critical aspects of the TPD process using atomistic simulations: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) degradation efficiency via the full Cullin Ring Ligase (CRL) macromolecular assembly. We combine experimental biophysical data---in this case hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues)---with molecular dynamics (MD) simulations aided by enhanced sampling techniques to accurately predict ternary complex structures at atomic resolution. We demonstrate improved efficiency, accuracy, and reliability in the ternary structure predictions of the bromodomain of the cancer target SMARCA2 with the E3 ligase VHL mediated by three different degrader molecules. The simulations accurately reproduce X-ray crystal structures -- including a new structure that we determined in this work (PDB ID: 7S4E) -- with root mean square deviations (RMSD) of 1.1 to 1.6 \r{A}. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex. Snapshots from these simulations are used as seeds for additional simulations, where we perform 7.1 milliseconds of aggregate simulation time using Folding@home. The detailed free energy surface captures the crystal structure conformation within a low-energy basin and is consistent with solution-phase experimental data (HDX-MS and SAXS). Finally, we graft a structural ensemble of the ternary complexes onto the full CRL and perform enhanced sampling simulations, which suggest that differences in degradation efficiency may be related to the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin. Several of the predicted ubiquitinated lysine residues have been validated through a ubiquitin mapping proteomics experiment. DOI 10.1038/s41467-022-33575-4

Dataset Information

BRD4 degradation via stabilization of a native interaction with DCAF16

Publications

Targeted protein degradation via intramolecular bivalent glues.

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