ABSTRACT
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 target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.
Subject(s)
Drug Design , Proteolysis , Substrate Specificity , Ubiquitin-Protein Ligases , Ubiquitination , Bromodomain Containing Proteins/metabolism , Cell Cycle Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis Targeting Chimera , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Protein Binding , Protein DomainsABSTRACT
Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined 'functional hotspots'. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.
Subject(s)
Carrier Proteins , Ubiquitin-Protein Ligases , Ubiquitin-Protein Ligases/metabolism , Proteolysis , Carrier Proteins/metabolismABSTRACT
The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.
Subject(s)
Proteins , Ubiquitin-Protein Ligases , Ubiquitin-Protein Ligases/metabolism , Phosphotyrosine , Ligands , src Homology DomainsABSTRACT
Hypoxia-inducible factor-1α (HIF-1α) constitutes the principal mediator of cellular adaptation to hypoxia in humans. The HIF-1α protein level and activity are tightly regulated by the ubiquitin E3 ligase von Hippel-Lindau (VHL). Here, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors. Our iterative and combinatorial strategy focused on chemical variability at the phenylene unit and encompassed further points of diversity. The exploitation of tailored phenylene fragments and the stereoselective installation of the benzylic methyl group provided potent VHL ligands. Three high-resolution structures of VHL-ligand complexes were determined, and bioactive conformations of these ligands were explored. The most potent inhibitor (30) exhibited dissociation constants lower than 40 nM, independently determined by fluorescence polarization and surface plasmon resonance and an enhanced cellular potency, as evidenced by its superior ability to induce HIF-1α transcriptional activity. Our work is anticipated to inspire future efforts toward HIF-1α stabilizers and new ligands for proteolysis-targeting chimera (PROTAC) degraders.
Subject(s)
Ubiquitin-Protein Ligases , Von Hippel-Lindau Tumor Suppressor Protein , Humans , Ubiquitin-Protein Ligases/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Ligands , Hypoxia-Inducible Factor 1, alpha Subunit , Ubiquitin/metabolism , HypoxiaABSTRACT
The rapid and ever-growing advancements from within the field of proteolysis-targeting chimeras (PROTAC)-induced protein degradation have driven considerable development to gain a deeper understanding of their mode of action. The ternary complex formed by PROTACs with their target protein and E3 ubiquitin ligase is the key species in their substoichiometric catalytic mechanism. Here, we describe the theoretical framework that underpins ternary complexes, including a current understanding of the three-component binding model, cooperativity, hook effect and structural considerations. We discuss in detail the biophysical methods used to interrogate ternary complex formation in vitro, including X-ray crystallography, AlphaLISA, FRET, FP, ITC and SPR. Finally, we provide detailed ITC methods and discuss approaches to assess binary and ternary target engagement, target ubiquitination and degradation that can be used to obtain a more holistic understanding of the mode of action within a cellular environment.