RESUMO
Methods to direct the degradation of protein targets with proximity-inducing molecules that coopt the cellular degradation machinery are advancing in leaps and bounds, and diverse modalities are emerging. The most used and well-studied approach is to hijack E3 ligases of the ubiquitin-proteasome system. E3 ligases use specific molecular recognition to determine which proteins in the cell are ubiquitinated and degraded. This review focuses on the structural determinants of E3 ligase recruitment of natural substrates and neo-substrates obtained through monovalent molecular glues and bivalent proteolysis-targeting chimeras. We use structures to illustrate the different types of substrate recognition and assess the basis for neo-protein-protein interactions in ternary complex structures. The emerging structural and mechanistic complexity is reflective of the diverse physiological roles of protein ubiquitination. This molecular insight is also guiding the application of structure-based design approaches to the development of new and existing degraders as chemical tools and therapeutics.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Proteínas/metabolismo , Proteólise , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
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.
Assuntos
Desenho de Fármacos , Proteólise , Especificidade por Substrato , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas que Contêm Bromodomínio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Quimera de Direcionamento de Proteólise , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
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.
Assuntos
Proteínas de Transporte , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Proteínas de Transporte/metabolismoRESUMO
Proteolysis targeting chimeras (PROTACs) are heterobifunctional small-molecule degraders made of a linker connecting a target-binding moiety to a ubiquitin E3 ligase-binding moiety. The linker unit is known to influence the physicochemical and pharmacokinetic properties of PROTACs, as well as the properties of ternary complexes, in turn impacting on their degradation activity in cells and in vivo. Our LRRK2 PROTAC XL01126, bearing a trans-cyclohexyl group in the linker, is a better and more cooperative degrader than its corresponding cis- analogue despite its much weaker binary binding affinities. Here, we investigate how this subtle stereocenter alteration in the linker affects the ligand binding affinity to the E3 ligase VHL. We designed a series of molecular matched pairs, truncating from the full PROTACs down to the VHL ligand, and find that across the series the trans-cyclohexyl compounds showed consistently weaker VHL-binding affinity compared to the cis- counterparts. High-resolution co-crystal structures revealed that the trans linker exhibits a rigid stick-out conformation, while the cis linker collapses into a folded-back conformation featuring a network of intramolecular contacts and long-range interactions with VHL. These observations are noteworthy as they reveal how a single stereochemical inversion within a PROTAC linker impacts conformational rigidity and binding mode, in turn fine-tuning differentiated propensity to binary and ternary complex formation, and ultimately cellular degradation activity.
Assuntos
Proteína Supressora de Tumor Von Hippel-Lindau , Humanos , Sítios de Ligação , Ligantes , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteólise/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/química , Cicloexanos/químicaRESUMO
Dynamic combinatorial chemistry (DCC) leverages a reversible reaction to generate compound libraries from constituting building blocks under thermodynamic control. The position of this equilibrium can be biased by addition of a target macromolecule towards enrichment of bound ligands. While DCC has been applied to select ligands for a single target protein, its application to identifying chimeric molecules inducing proximity between two proteins is unprecedented. In this proof-of-concept study, we develop a DCC approach to select bifunctional proteolysis targeting chimeras (PROTACs) based on their ability to stabilize the ternary complex. We focus on VHL-targeting Homo-PROTACs as model system, and show that the formation of a VHL2 : Homo-PROTAC ternary complex reversibly assembled using thiol-disulfide exchange chemistry leads to amplification of potent VHL Homo-PROTACs with degradation activities which correlated well with their biophysical ability to dimerize VHL. Ternary complex templated dynamic combinatorial libraries allowed identification of novel Homo-PROTAC degraders. We anticipate future applications of ternary-complex directed DCC to early PROTAC screenings and expansion to other proximity-inducing modalities beyond PROTACs.
Assuntos
Técnicas de Química Combinatória , Proteína Supressora de Tumor Von Hippel-Lindau , Humanos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteólise , Ligantes , Termodinâmica , Quimera de Direcionamento de ProteóliseRESUMO
Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhance degradation. Here, we designed trivalent PROTACs consisting of a bivalent bromo and extra terminal (BET) inhibitor and an E3 ligand tethered via a branched linker. We identified von Hippel-Lindau (VHL)-based SIM1 as a low picomolar BET degrader with preference for bromodomain containing 2 (BRD2). Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anticancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL, exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Humanos , ProteóliseRESUMO
The von Hippel-Lindau (VHL) Cullin RING E3 ligase is an essential enzyme in the ubiquitin-proteasome system that recruits substrates such as the hypoxia inducible factor for ubiquitination and subsequent proteasomal degradation. The ubiquitin-proteasome pathway can be hijacked toward non-native neo-substrate proteins using proteolysis targeting chimeras (PROTACs), bifunctional molecules designed to simultaneously bind to an E3 ligase and a target protein to induce target ubiquitination and degradation. The availability of high-quality small-molecule ligands with good binding affinity for E3 ligases is fundamental for PROTAC development. Lack of good E3 ligase ligands as starting points to develop PROTAC degraders was initially a stumbling block to the development of the field. Herein, the journey towards the design of small-molecule ligands binding to VHL is presented. We cover the structure-based design of VHL ligands, their application as inhibitors in their own right, and their implementation into rationally designed, potent PROTAC degraders of various target proteins. We highlight the key findings and learnings that have provided strong foundations for the remarkable development of targeted protein degradation, and that offer a blueprint for designing new ligands for E3 ligases beyond VHL.
Assuntos
Complexo de Endopeptidases do Proteassoma , Proteína Supressora de Tumor Von Hippel-Lindau , Proteínas Culina , Ligantes , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Von Hippel-Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2VHL). CRL2VHL plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHL:HIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought.
Assuntos
Ciclopropanos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Hipóxia/metabolismo , Proteômica/métodos , Pirrolidinas/farmacologia , Tiazóis/farmacologia , Proteína Supressora de Tumor Von Hippel-Lindau/antagonistas & inibidores , Doença de von Hippel-Lindau/patologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/metabolismoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is one of the most promising targets for Parkinson's disease. LRRK2-targeting strategies have primarily focused on type 1 kinase inhibitors, which, however, have limitations as the inhibited protein can interfere with natural mechanisms, which could lead to undesirable side effects. Herein, we report the development of LRRK2 proteolysis targeting chimeras (PROTACs), culminating in the discovery of degrader XL01126, as an alternative LRRK2-targeting strategy. Initial designs and screens of PROTACs based on ligands for E3 ligases von Hippel-Lindau (VHL), Cereblon (CRBN), and cellular inhibitor of apoptosis (cIAP) identified the best degraders containing thioether-conjugated VHL ligand VH101. A second round of medicinal chemistry exploration led to qualifying XL01126 as a fast and potent degrader of LRRK2 in multiple cell lines, with DC50 values within 15-72 nM, Dmax values ranging from 82 to 90%, and degradation half-lives spanning from 0.6 to 2.4 h. XL01126 exhibits high cell permeability and forms a positively cooperative ternary complex with VHL and LRRK2 (α = 5.7), which compensates for a substantial loss of binary binding affinities to VHL and LRRK2, underscoring its strong degradation performance in cells. Remarkably, XL01126 is orally bioavailable (F = 15%) and can penetrate the blood-brain barrier after either oral or parenteral dosing in mice. Taken together, these experiments qualify XL01126 as a suitable degrader probe to study the noncatalytic and scaffolding functions of LRRK2 in vitro and in vivo and offer an attractive starting point for future drug development.
Assuntos
Barreira Hematoencefálica , Ubiquitina-Proteína Ligases , Animais , Camundongos , Barreira Hematoencefálica/metabolismo , Leucina , Ligantes , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Sulfetos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Epigenetic modulators perform critical functions in gene expression for rapid adaption to external stimuli and are prevalent in all higher-order organisms. The establishment of a link between dysregulation of epigenetic processes and disease pathogenesis, particularly in cancer, has led to much interest in identifying drug targets. This prompted the development of small molecule inhibitors, primarily in haematological malignancies. While there have been epigenetic-targeting drugs to receive FDA approval for the treatment of cancers, many suffer from limited applicability, toxicity and the onset of drug resistance, as our understanding of the biology remains incomplete. The recent advent of genome-wide RNAi and CRISPR screens has shed new light on loss of specific proteins causing vulnerabilities of specific cancer types, highlighting the potential for exploiting synthetic lethality as a therapeutic approach. However, small molecule inhibitors have largely been unable to recapitulate phenotypic effects observed using genome-wide knockdown approaches. This mechanistic disconnect and gap are set to be addressed by targeted protein degradation. Degraders such as PROTACs targeting epigenetic proteins recapitulate CRISPR mediated genetic knockdown at the post-translational level and therefore can better exploit target druggability. Here, we review the current landscape of epigenetic drug discovery, the rationale behind and progress made in the development of PROTAC degraders, and look at future perspectives for the field.
Assuntos
Epigênese Genética , Neoplasias , Humanos , Descoberta de Drogas , Neoplasias/tratamento farmacológico , ProteóliseRESUMO
Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Estrutura Molecular , Proteínas Nucleares/metabolismoRESUMO
In the version of this article originally published, several lines of text in the last paragraph of the right column on page 1 of the PDF were transposed into the bottom paragraph of the left column. The affected text of the left column should read "The ATP-dependent activities of the BAF (SWI/SNF) chromatin remodeling complexes affect the positioning of nucleosomes on DNA and thereby many cellular processes related to chromatin structure, including transcription, DNA repair and decatenation of chromosomes during mitosis12,13." The affected text of the right column should read "SMARCA2/4BD inhibitors are thus precluded from use for the treatment of SMARCA4 mutant cancers but could provide attractive ligands for PROTAC conjugation. Small molecules binding to other bromodomains have been successfully converted into PROTACs by conjugating them with structures capable of binding to the E3 ligases von Hippel-Lindau (VHL) or cereblon5,6,10,11,25,26,27." The errors have been corrected in the PDF version of the paper.
RESUMO
Nuclear magnetic resonance (NMR) is an effective, commonly used experimental approach to screen small organic molecules against a protein target. A very popular method consists of monitoring the changes of the NMR chemical shifts of the protein nuclei upon addition of the small molecule to the free protein. Multidimensional NMR experiments allow the interacting residues to be mapped along the protein sequence. A significant amount of human effort goes into manually tracking the chemical shift variations, especially when many signals exhibit chemical shift changes and when many ligands are tested. Some computational approaches to automate the procedure are available, but none of them as a web server. Furthermore, some methods require the adoption of a fairly specific experimental setup, such as recording a series of spectra at increasing small molecule:protein ratios. In this work, we developed a tool requesting a minimal amount of experimental data from the user, implemented it as an open-source program, and made it available as a web application. Our tool compares two spectra, one of the free protein and one of the small molecule:protein mixture, based on the corresponding peak lists. The performance of the tool in terms of correct identification of the protein-binding regions has been evaluated on different protein targets, using experimental data from interaction studies already available in the literature. For a total of 16 systems, our tool achieved between 79% and 100% correct assignments, properly identifying the protein regions involved in the interaction.
Assuntos
Algoritmos , Proteínas , Sequência de Aminoácidos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/químicaRESUMO
Developing stereoselective synthetic routes that are efficient and cost-effective allows easy access to biologically active molecules. Our previous syntheses of allele-selective bumped inhibitors of the Bromo and Extra-Terminal (BET) domain proteins, Brd2, Brd3, Brd4 and BrdT, required a wasteful, late-stage alkylation step and expensive chiral separation. To circumvent these limitations, we developed a route based on stereocontrolled alkylation of an N-Pf protected aspartic acid derivative that was used in a divergent, racemisation-free protocol to yield structurally diverse and enantiopure triazolodiazepines. With this approach, we synthesized bumped thienodiazepine-based BET inhibitor, ET-JQ1-OMe, in five steps and 99% ee without the need for chiral chromatography. Exquisite selectivity of ET-JQ1-OMe for Leu-Ala and Leu-Val mutants over wild-type bromodomain was established by isothermal titration calorimetry and X-ray crystallography. Our new approach provides unambiguous chemical evidence for the absolute stereochemistry of the active, allele-specific BET inhibitors and a viable route that will open wider access to this compound class.
Assuntos
Proteínas NuclearesRESUMO
Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co-crystal structure of macroPROTAC-1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12-fold loss of binary binding affinity for Brd4, macroPROTAC-1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.
Assuntos
Desenho de Fármacos , Compostos Macrocíclicos , Compostos Macrocíclicos/química , Estrutura Molecular , Conformação ProteicaRESUMO
Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase-PROTAC-target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4BD2). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4BD2. Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.
Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Cristalografia por Raios X , Dipeptídeos/química , Dipeptídeos/farmacologia , Elonguina , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Termodinâmica , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Pluripotent stem cells (PSCs) hold great clinical potential, as they possess the capacity to differentiate into fully specialised tissues such as pancreas, liver, neurons and cardiac muscle. However, the molecular mechanisms that coordinate pluripotent exit with lineage specification remain poorly understood. To address this question, we perform a small molecule screen to systematically identify novel regulators of the Smad2 signalling network, a key determinant of PSC fate. We reveal an essential function for BET family bromodomain proteins in Smad2 activation, distinct from the role of Brd4 in pluripotency maintenance. Mechanistically, BET proteins specifically engage Nodal gene regulatory elements (NREs) to promote Nodal signalling and Smad2 developmental responses. In pluripotent cells, Brd2-Brd4 occupy NREs, but only Brd4 is required for pluripotency gene expression. Brd4 downregulation facilitates pluripotent exit and drives enhanced Brd2 NRE occupancy, thereby unveiling a specific function for Brd2 in differentiative Nodal-Smad2 signalling. Therefore, distinct BET functionalities and Brd4-Brd2 isoform switching at NREs coordinate pluripotent exit with lineage specification.
Assuntos
Diferenciação Celular , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Smad2/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem da Célula , Humanos , Camundongos , Proteínas/metabolismo , Transdução de SinaisRESUMO
The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.