From Thalidomide to Rational Molecular Glue Design for Targeted Protein Degradation.

Thalidomide and its derivatives are powerful cancer therapeutics that are among the best-understood molecular glue degraders (MGDs). These drugs selectively reprogram the E3 ubiquitin ligase cereblon (CRBN) to commit target proteins for degradation by the ubiquitin-proteasome system. MGDs create novel recognition interfaces on the surface of the E3 ligase that engage in induced protein-protein interactions with neosubstrates. Molecular insight into their mechanism of action opens exciting opportunities to engage a plethora of targets through a specific recognition motif, the G-loop. Our analysis shows that current CRBN-based MGDs can in principle recognize over 2,500 proteins in the human proteome that contain a G-loop. We review recent advances in tuning the specificity between CRBN and its MGD-induced neosubstrates and deduce a set of simple rules that govern these interactions. We conclude that rational MGD design efforts will enable selective degradation of many more proteins, expanding this therapeutic modality to more disease areas.

Cereblon influences the timing of muscle differentiation in Ciona tadpoles.

Thalidomide has a dark history as a teratogen, but in recent years, its derivates have been shown to function as potent chemotherapeutic agents. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here, we employ Ciona, a simple invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Mrf and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation.

The emerging role of targeted protein degradation to treat and study cancer.

The evolution of cancer treatment has provided increasingly targeted strategies both in the upfront and relapsed disease settings. Small-molecule inhibitors and immunotherapy have risen to prominence with chimeric antigen receptor T-cells, checkpoint inhibitors, kinase inhibitors, and monoclonal antibody therapies being deployed across a range of solid organ and haematological malignancies. However, novel approaches are required to target transcription factors and oncogenic fusion proteins that are central to cancer biology and have generally eluded successful drug development. Thalidomide analogues causing protein degradation have been a cornerstone of treatment in multiple myeloma, but a lack of in-depth mechanistic understanding initially limited progress in the field. When the protein cereblon (CRBN) was found to mediate thalidomide analogues' action and CRBN's neo-targets were identified, existing and novel drug development accelerated, with applications outside multiple myeloma, including non-Hodgkin's lymphoma, myelodysplastic syndrome, and acute leukaemias. Critically, transcription factors were the first canonical targets described. In addition to broadening the application of protein-degrading drugs, resistance mechanisms are being overcome and targeted protein degradation is widening the scope of druggable proteins against which existing approaches have been ineffective. Examples of targeted protein degraders include molecular glues and proteolysis targeting chimeras (PROTACs): heterobifunctional molecules that bind to proteins of interest and cause proximity-induced ubiquitination and proteasomal degradation via a linked E3 ligase. Twenty years since their inception, PROTACs have begun progressing through clinical trials, with early success in targeting the oestrogen receptor and androgen receptor in breast and prostate cancer respectively. This review explores important developments in targeted protein degradation to both treat and study cancer. It also considers the potential advantages and challenges in the translational aspects of developing new treatments. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

IKZF2 Degradation: It's Time to Take into Account it When Designing Cereblon-Based PROTACs.

Proteolysis-targeting chimera (PROTAC) has become a very important means of protein degradation and a new way of disease treatment. In particular, PROTACs constructed with ligands for E3 ligase cereblon account for more than 90 % of the PROTACs currently in clinical research. Notably, CRBN ligands themselves are a class of molecular glue compounds capable of degrading neo-substrate proteins. Compared to the target proteins degradation, the degradation of neo-substrates, especially IKZF2, has not received enough attention. Therefore, this review summarizes the currently published IKZF2 degraders derived from articles and patents, which are conducive to the design of PROTACs with desired IKZF2 degradation from the perspective of medicinal chemistry.

Revisiting the antiangiogenic mechanisms of fluorinated thalidomide derivatives.

Introduction of fluorine into bioactive molecules has attracted much attention in drug development. For example, tetrafluorination of the phthalimide moiety of immunomodulatory drugs (IMiDs) has a strong beneficial effect on the ability to inhibit angiogenesis. The neomorphic activity of E3 ligase complexes is induced by the binding of IMiDs to cereblon. We investigated that a set of eight thalidomide analogs, comprising non- and tetrafluorinated counterparts, did not induce the degradation of neomorphic substrates (IKZF3, GSPT1, CK1α, SALL4). Hence, the antiangiogenic activity of fluorinated IMiDs was not triggered by neosubstrate degradation features. A fluorine scanning of non-traditional IMiDs of the benzamido glutarimide chemotype was performed. By measuring the endothelial cell tube formation, no angiogenesis inhibitors were identified, confirming the narrow structure-activity window of IMiD-induced antiangiogenesis.

Pyrvinium doubles against WNT-driven cancer.

The WNT-ß-catenin signaling pathway has a major role in regulating cell proliferation and differentiation. Aberrant activation of the pathway contributes to various human cancer types. Because casein kinase CK1α-initiated phosphorylation of ß-catenin is a key first step to restrain WNT signaling, effective restoration of CK1α activity represents an innovative strategy to combat WNT-driven cancer. A recent study in JBC reveals the anthelmintic pyrvinium directly binds to CK1α as an activator and also stabilizes CK1α protein, doubling against WNT-driven cancer activity.

Synthesis and Evaluation of Cereblon-Recruiting HaloPROTACs.

Target validation is key to the development of protein degrading molecules such as proteolysis-targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin-proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3-catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN-pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP-HaloTag fusion with higher activities than VHL-engaging HaloPROTACs at sub-micromolar concentrations of the compound. The CRBN-engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.

A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity.

Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.

A Modular Chemistry Platform for the Development of a Cereblon E3 Ligase-Based Partial PROTAC Library.

Proteolysis targeting chimeras (PROTACs) are a promising therapeutic strategy to selectively promote the degradation of protein targets by exploiting the ubiquitin-proteasome system. Among the limited number of E3 ligase ligands discovered for the PROTAC technology, ligands of cereblon (CRBN) E3 ligase, such as pomalidomide, thalidomide, or lenalidomide, are the most frequently used for the development of PROTACs. Our group previously reported that a phenyl group could be tolerated on the C4-position of lenalidomide as the ligand of CRBN to develop PROTACs. Herein, we report a modular chemistry platform for the efficient attachment of various ortho-, meta-, and para-substituted phenyls to the C4-position of the lenalidomide via Suzuki cross-coupling reaction, which allows the systematic investigation of the linker effect for the development of PROTACs against any target. We examined the substrate scope by preparing twelve lenalidomide-derived CRBN E3 ligase ligands with different linkers.

Novel, thalidomide-like, non-cereblon binding drug tetrafluorobornylphthalimide mitigates inflammation and brain injury.

BACKGROUND: Quelling microglial-induced excessive neuroinflammation is a potential treatment strategy across neurological disorders, including traumatic brain injury (TBI), and can be achieved by thalidomide-like drugs albeit this approved drug class is compromised by potential teratogenicity. Tetrafluorobornylphthalimide (TFBP) and tetrafluoronorbornylphthalimide (TFNBP) were generated to retain the core phthalimide structure of thalidomide immunomodulatory imide drug (IMiD) class. However, the classical glutarimide ring was replaced by a bridged ring structure. TFBP/TFNBP were hence designed to retain beneficial anti-inflammatory properties of IMiDs but, importantly, hinder cereblon binding that underlies the adverse action of thalidomide-like drugs. METHODS: TFBP/TFNBP were synthesized and evaluated for cereblon binding and anti-inflammatory actions in human and rodent cell cultures. Teratogenic potential was assessed in chicken embryos, and in vivo anti-inflammatory actions in rodents challenged with either lipopolysaccharide (LPS) or controlled cortical impact (CCI) moderate traumatic brain injury (TBI). Molecular modeling was performed to provide insight into drug/cereblon binding interactions. RESULTS: TFBP/TFNBP reduced markers of inflammation in mouse macrophage-like RAW264.7 cell cultures and in rodents challenged with LPS, lowering proinflammatory cytokines. Binding studies demonstrated minimal interaction with cereblon, with no resulting degradation of teratogenicity-associated transcription factor SALL4 or of teratogenicity in chicken embryo assays. To evaluate the biological relevance of its anti-inflammatory actions, two doses of TFBP were administered to mice at 1 and 24 h post-injury following CCI TBI. Compared to vehicle treatment, TFBP reduced TBI lesion size together with TBI-induction of an activated microglial phenotype, as evaluated by immunohistochemistry 2-weeks post-injury. Behavioral evaluations at 1- and 2-weeks post-injury demonstrated TFBP provided more rapid recovery of TBI-induced motor coordination and balance impairments, versus vehicle treated mice. CONCLUSION: TFBP and TFNBP represent a new class of thalidomide-like IMiDs that lower proinflammatory cytokine generation but lack binding to cereblon, the main teratogenicity-associated mechanism. This aspect makes TFBP and TFNBP potentially safer than classic IMiDs for clinical use. TFBP provides a strategy to mitigate excessive neuroinflammation associated with moderate severity TBI to, thereby, improve behavioral outcome measures and warrants further investigation in neurological disorders involving a neuroinflammatory component.

Hypnotic effect of thalidomide is independent of teratogenic ubiquitin/proteasome pathway.

Thalidomide exerts its teratogenic and immunomodulatory effects by binding to cereblon (CRBN) and thereby inhibiting/modifying the CRBN-mediated ubiquitination pathway consisting of the Cullin4-DDB1-ROC1 E3 ligase complex. The mechanism of thalidomide's classical hypnotic effect remains largely unexplored, however. Here we examined whether CRBN is involved in the hypnotic effect of thalidomide by generating mice harboring a thalidomide-resistant mutant allele of Crbn (Crbn YW/AA knock-in mice). Thalidomide increased non-REM sleep time in Crbn YW/AA knock-in homozygotes and heterozygotes to a similar degree as seen in wild-type littermates. Thalidomide similarly depressed excitatory synaptic transmission in the cortical slices obtained from wild-type and Crbn YW/AA homozygous knock-in mice without affecting GABAergic inhibition. Thalidomide induced Fos expression in vasopressin-containing neurons of the supraoptic nucleus and reduced Fos expression in the tuberomammillary nuclei. Thus, thalidomide's hypnotic effect seems to share some downstream mechanisms with general anesthetics and GABAA-activating sedatives but does not involve the teratogenic CRBN-mediated ubiquitin/proteasome pathway.

Cereblon contributes to cardiac dysfunction by degrading Cav1.2α.

AIMS: Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that was reported to target ion channel proteins. L-type voltage-dependent Ca2+ channel (LTCC) density and dysfunction is a critical player in heart failure with reduced ejection fraction (HFrEF). However, the underlying cellular mechanisms by which CRBN regulates LTCC subtype Cav1.2α during cardiac dysfunction remain unclear. Here, we explored the role of CRBN in HFrEF by investigating the direct regulatory role of CRBN in Cav1.2α activity and examining how it can serve as a target to address myocardial dysfunction. METHODS AND RESULTS: Cardiac tissues from HFrEF patients exhibited increased levels of CRBN compared with controls. In vivo and ex vivo studies demonstrated that whole-body CRBN knockout (CRBN-/-) and cardiac-specific knockout mice (Crbnfl/fl/Myh6Cre+) exhibited enhanced cardiac contractility with increased LTCC current (ICaL) compared with their respective controls, which was modulated by the direct interaction of CRBN with Cav1.2α. Mechanistically, the Lon domain of CRBN directly interacted with the N-terminal of Cav1.2α. Increasing CRBN levels enhanced the ubiquitination and proteasomal degradation of Cav1.2α and decreased ICaL. In contrast, genetic or pharmacological depletion of CRBN via TD-165, a novel PROTAC-based CRBN degrader, increased surface expression of Cav1.2α and enhanced ICaL. Low CRBN levels protected the heart against cardiomyopathy in vivo. CONCLUSION: Cereblon selectively degrades Cav1.2α, which in turn facilitates cardiac dysfunction. A targeted approach or an efficient method of reducing CRBN levels could serve as a promising strategy for HFrEF therapeutics.

Glucokinase Variant Proteins Are Resistant to Fasting-Induced Uridine Diphosphate Glucose-Dependent Degradation in Maturity-Onset Diabetes of the Young Type 2 Patients.

We previously reported that glucokinase undergoes ubiquitination and subsequent degradation, a process mediated by cereblon, particularly in the presence of uridine diphosphate glucose (UDP-glucose). In this context, we hereby present evidence showcasing the resilience of variant glucokinase proteins of maturity-onset diabetes of the young type 2 (MODY2) against degradation and, concomitantly, their influence on insulin secretion, both in cell lines and in the afflicted MODY2 patient. Hence, glucose-1-phodphate promotes UDP-glucose production by UDP-glucose pyrophosphorylase 2; consequently, UDP-glucose-dependent glucokinase degradation may occur during fasting. Next, we analyzed glucokinase variant proteins from MODY2 or persistent hyperinsulinemic hypoglycemia in infancy (PHHI). Among the eleven MODY2 glucokinase-mutated proteins tested, those with a lower glucose-binding affinity exhibited resistance to UDP-glucose-dependent degradation. Conversely, the glucokinaseA456V-mutated protein from PHHI had a higher glucose affinity and was sensitive to UDP-glucose-dependent degradation. Furthermore, in vitro studies involving UDP-glucose-dependent glucokinase variant proteins and insulin secretion during fasting in Japanese MODY2 patients revealed a strong correlation and a higher coefficient of determination. This suggests that UDP-glucose-dependent glucokinase degradation plays a significant role in the pathogenesis of glucose-homeostasis-related hereditary diseases, such as MODY2 and PHHI.

Recent Advances in the Applications of Small Molecules in the Treatment of Multiple Myeloma.

Therapy for multiple myeloma (MM), a hematologic neoplasm of plasma cells, has undergone remarkable changes over the past 25 years. Small molecules (molecular weight of less than one kDa), together with newer immunotherapies that include monoclonal antibodies, antibody-drug conjugates, and most recently, chimeric antigen receptor (CAR) T-cells, have combined to double the disease's five-year survival rate to over 50% during the past few decades. Despite these advances, the disease is still considered incurable, and its treatment continues to pose substantial challenges, since therapeutic refractoriness and patient relapse are exceedingly common. This review focuses on the current pipeline, along with the contemporary roles and future prospects for small molecules in MM therapy. While small molecules offer prospective benefits in terms of oral bioavailability, cellular penetration, simplicity of preparation, and improved cost-benefit considerations, they also pose problems of toxicity due to off-target effects. Highlighted in the discussion are recent developments in the applications of alkylating agents, immunomodulators, proteasome inhibitors, apoptosis inducers, kinesin spindle protein inhibitors, blockers of nuclear transport, and drugs that affect various kinases involved in intracellular signaling pathways. Molecular and cellular targets are described for each class of agents in relation to their roles as drivers of MM.

A New Strategy for the Old Challenge of Thalidomide: Systems Biology Prioritization of Potential Immunomodulatory Drug (IMiD)-Targeted Transcription Factors.

Several molecular mechanisms of thalidomide embryopathy (TE) have been investigated, from anti-angiogenesis to oxidative stress to cereblon binding. Recently, it was discovered that thalidomide and its analogs, named immunomodulatory drugs (IMiDs), induced the degradation of C2H2 transcription factors (TFs). This mechanism might impact the strict transcriptional regulation of the developing embryo. Hence, this study aims to evaluate the TFs altered by IMiDs, prioritizing the ones associated with embryogenesis through transcriptome and systems biology-allied analyses. This study comprises only the experimental data accessed through bioinformatics databases. First, proteins and genes reported in the literature as altered/affected by the IMiDs were annotated. A protein systems biology network was evaluated. TFs beta-catenin (CTNNB1) and SP1 play more central roles: beta-catenin is an essential protein in the network, while SP1 is a putative C2H2 candidate for IMiD-induced degradation. Separately, the differential expressions of the annotated genes were analyzed through 23 publicly available transcriptomes, presenting 8624 differentially expressed genes (2947 in two or more datasets). Seventeen C2H2 TFs were identified as related to embryonic development but not studied for IMiD exposure; these TFs are potential IMiDs degradation neosubstrates. This is the first study to suggest an integration of IMiD molecular mechanisms through C2H2 TF degradation.

Thalidomide interaction with inflammation in idiopathic pulmonary fibrosis.

The "Thalidomide tragedy" is a landmark in the history of the pharmaceutical industry. Despite limited clinical trials, there is a continuous effort to investigate thalidomide as a drug for cancer and inflammatory diseases such as rheumatoid arthritis, lepromatous leprosy, and COVID-19. This review focuses on the possibilities of targeting inflammation by repurposing thalidomide for the treatment of idiopathic pulmonary fibrosis (IPF). Articles were searched from the Scopus database, sorted, and selected articles were reviewed. The content includes the proven mechanisms of action of thalidomide relevant to IPF. Inflammation, oxidative stress, and epigenetic mechanisms are major pathogenic factors in IPF. Transforming growth factor-ß (TGF-ß) is the major biomarker of IPF. Thalidomide is an effective anti-inflammatory drug in inhibiting TGF-ß, interleukins (IL-6 and IL-1ß), and tumour necrosis factor-α (TNF-α). Thalidomide binds cereblon, a process that is involved in the proposed mechanism in specific cancers such as breast cancer, colon cancer, multiple myeloma, and lung cancer. Cereblon is involved in activating AMP-activated protein kinase (AMPK)-TGF-ß/Smad signalling, thereby attenuating fibrosis. The past few years have witnessed an improvement in the identification of biomarkers and diagnostic technologies in respiratory diseases, partly because of the COVID-19 pandemic. Hence, investment in clinical trials with a systematic plan can help repurpose thalidomide for pulmonary fibrosis.

Cereblon Regulates the Proteotoxicity of Tau by Tuning the Chaperone Activity of DNAJA1.

Protein aggregation can induce explicit neurotoxic events that trigger a number of presently untreatable neurodegenerative disorders. Chaperones, on the other hand, play a neuroprotective role because of their ability to unfold and refold abnormal proteins. The progressive nature of neurotoxic events makes it important to discover endogenous factors that affect pathologic and molecular phenotypes of neurodegeneration in animal models. Here, we identified microtubule-associated protein tau, and chaperones Hsp70 (heat shock protein 70) and DNAJA1 (DJ2) as endogenous substrates of cereblon (CRBN), a substrate-recruiting subunit of cullin4-RING-E3-ligase. This recruitment results in ubiquitin-mediated degradation of tau, Hsp70, and DJ2. Knocking out CRBN enhances the chaperone activity of DJ2, resulting in decreased phosphorylation and aggregation of tau, improved association of tau with microtubules, and reduced accumulation of pathologic tau across brain. Functionally abundant DJ2 could prevent tau aggregation induced by various factors like okadaic acid and heparin. Depletion of CRBN also decreases the activity of tau-kinases including GSK3α/ß, ERK, and p38. Intriguingly, we found a high expression of CRBN and low levels of DJ2 in neuronal tissues of 5XFAD and APP knock-in male mouse models of Alzheimer's disease. This implies that CRBN-mediated DJ2/Hsp70 pathway may be compromised in neurodegeneration. Being one of the primary pathogenic events, elevated CRBN can be a contributing factor for tauopathies. Our data provide a functional link between CRBN and DJ2/Hsp70 chaperone machinery in abolishing the cytotoxicity of aggregation-prone tau and suggest that Crbn-/- mice serve as an animal model of resistance against tauopathies for further exploration of the molecular mechanisms of neurodegeneration.

Cereblon regulates NK cell cytotoxicity and migration via Rac1 activation.

Rearrangement of the actin cytoskeleton is critical for cytotoxic and immunoregulatory functions as well as migration of natural killer (NK) cells. However, dynamic reorganization of actin is a complex process, which remains largely unknown. Here, we investigated the role of the protein Cereblon (CRBN), an E3 ubiquitin ligase complex co-receptor and the primary target of the immunomodulatory drugs, in NK cells. We observed that CRBN partially colocalizes with F-actin in chemokine-treated NK cells and is recruited to the immunological synapse, thus suggesting a role for this protein in cytoskeleton reorganization. Accordingly, silencing of CRBN in NK cells results in a reduced cytotoxicity that correlates with a defect in conjugate and lytic synapse formation. Moreover, CRBN depletion significantly impairs the ability of NK cells to migrate and reduces the enhancing effect of lenalidomide on NK cell migration. Finally, we provided evidence that CRBN is required for activation of the small GTPase Rac1, a critical mediator of cytoskeleton dynamics. Indeed, in CRBN-depleted NK cells, chemokine-mediated or target cell-mediated Rac1 activation is significantly reduced. Altogether our data identify a critical role for CRBN in regulating NK cell functions and suggest that this protein may mediate the stimulatory effect of lenalidomide on NK cells.

The role of E3 ubiquitin ligase in multiple myeloma: potential for cereblon E3 ligase modulators in the treatment of relapsed/refractory disease.

INTRODUCTION: Insights into the mechanisms of protein homeostasis and proteasomal degradation have led to new strategies of redirecting the ubiquitin-proteasome system (UPS) to reduce or eliminate proteins or survival factors key to malignant pathobiology, multiple myeloma (MM) in particular. These strategies have enabled researchers to target proteins that were previously considered difficult to modulate by pharmacological means. AREAS COVERED: This review provides a brief overview of UPS biology, particularly the role of the CRL4CRBN E3 ubiquitin ligase complex, and summarizes current strategies for co-opting the UPS, including CELMoD compounds, SNIPERs, PROTACs, and degronimids. A detailed discussion is provided on lead CELMoD compounds iberdomide and mezigdomide, which are currently being evaluated in clinical trials in patients with MM. EXPERT OPINION: Since a high proportion of patients develop drug resistance, it is vital to have novel therapeutic agents for treating relapsed patients with MM more effectively. It is encouraging that the expanding pathophysiological insight into cellular signaling pathways in MM increasingly translates into the development of novel therapeutic agents such as targeted protein degraders. This holds promise for improving outcomes in MM and beyond.

Selective Wee1 degradation by PROTAC degraders recruiting VHL and CRBN E3 ubiquitin ligases.

The Ser/Thr protein kinase Wee1 plays a regulatory role at the G2/M checkpoint by phosphorylating CDK1 when DNA is damaged to allow time for DNA to repair, disruption of which is a key approach to sensitise cancer cells to DNA-damaging therapies. The main selective inhibitor for Wee1 undergoing development in clinical trials, AZD1775, however, has been shown to have off target effects towards other protein kinases with similar potency. Here we describe the synthesis and assessment of a series of Wee1-degrading PROTACs using AZD1775 linked to either the VHL ligand VH032 or to the CRBN ligand pomalidomide using different types and lengths of linkers. The conversion of AZD1775 into a PROTAC induces selective Wee1 degradation for compounds of both series depending on the nature of the linker.