Cereblon versus VHL:Hijacking E3 ligases against each other using PROTACs

<div> <div> <div> <p>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. </p> </div> </div> </div

Rapid and reversible knockdown of endogenously tagged endosomal proteins via an optimized HaloPROTAC degrader

Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC₅₀ of 3–10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (<i>D</i>_max of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function

DEGRONOPEDIA:a web server for proteome-wide inspection of degrons

E3 ubiquitin ligases recognize substrates through their short linear motifs termed degrons. While degron-signaling has been a subject of extensive study, resources for its systematic screening are limited. To bridge this gap, we developed DEGRONOPEDIA, a web server that searches for degrons and maps them to nearby residues that can undergo ubiquitination and disordered regions, which may act as protein unfolding seeds. Along with an evolutionary assessment of degron conservation, the server also reports on post-translational modifications and mutations that may modulate degron availability. Acknowledging the prevalence of degrons at protein termini, DEGRONOPEDIA incorporates machine learning to assess N-/C-terminal stability, supplemented by simulations of proteolysis to identify degrons in newly formed termini. An experimental validation of a predicted C-terminal destabilizing motif, coupled with the confirmation of a post-proteolytic degron in another case, exemplifies its practical application. DEGRONOPEDIA can be freely accessed at degronopedia.com

DEGRONOPEDIA:a web server for proteome-wide inspection of degrons

E3 ubiquitin ligases recognize substrates through their short linear motifs termed degrons. While degron-signaling has been a subject of extensive study, resources for its systematic screening are limited. To bridge this gap, we developed DEGRONOPEDIA, a web server that searches for degrons and maps them to nearby residues that can undergo ubiquitination and disordered regions, which may act as protein unfolding seeds. Along with an evolutionary assessment of degron conservation, the server also reports on post-translational modifications and mutations that may modulate degron availability. Acknowledging the prevalence of degrons at protein termini, DEGRONOPEDIA incorporates machine learning to assess N-/C-terminal stability, supplemented by simulations of proteolysis to identify degrons in newly formed termini. An experimental validation of a predicted C-terminal destabilizing motif, coupled with the confirmation of a post-proteolytic degron in another case, exemplifies its practical application. DEGRONOPEDIA can be freely accessed at degronopedia.com

Borderline Personality in Patients with Poly-Diagnoses Treated for a Bipolar Disorder

Some patients with dysphoria, explosive behaviour, or suicidal ideation, may receive a diagnosis of, and treatment for Bipolar Disorder (BD) and, not infrequently. The coexistence of these two diagnoses has been explained in different ways. Some authors include the BPD in the bipolar spectrum; others are sceptical about the existence of real comorbidity, suggesting a misdiagnosis. This study aimed to assess the personality of this group of poly-diagnosed patients (PolyD) and hypothesised they had a pathological borderline organisation. Via the administration of the Schedler Westen Assessment Procedure (SWAP-200), we compared PolyD patients with those suffering from BPD or BD only. We performed two different MANCOVAs to test PolyD, BPD and BD patients’ differences in PD-factors, Q-traits and age. The sample comprised 45 patients (Mean age=43.3, SD=15.7; Females 57.7%, N=26). BD patients (N=15) did not present any personality disorder, they had a higher functioning and Obsessive Q-traits, and a lower Histrionic PD-factor than both PolyD (N=20) and BPD (N=10) patients. Compared to PolyD patients, BD had inferior PD-Borderline, PD-Antisocial factor and Dependent-Masochistic Q-traits, but there were no other differences with BPD patients. PolyD did not differ from BPD patients in any of the PD-factors and Q-traits. Our results suggest that PolyD patients are different from BD patients and propose to consider the pathological borderline personality as a central core of their disease

The bromodomain and extra-terminal domain degrader MZ1 exhibits preclinical anti-tumoral activity in diffuse large B-cell lymphoma of the activated B cell-like type

AIM: Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that play a fundamental role in transcription regulation. Preclinical and early clinical evidence sustain BET targeting as an anti-cancer approach. BET degraders are chimeric compounds comprising of a BET inhibitor, which allows the binding to BET bromodomains, linked to a small molecule, binder for an E3 ubiquitin ligase complex, triggering BET proteins degradation via the proteasome. These degraders, called proteolysis-targeting chimeras (PROTACs), can exhibit greater target specificity compared to BET inhibitors and overcome some of their limitations, such as the upregulation of the BET proteins themselves. Here are presented data on the anti-tumor activity and the mechanism of action of the BET degrader MZ1 in diffuse large B cell lymphoma (DLBCL) of the activated B-cell like (ABC, ABC DLBCL), using a BET inhibitor as a comparison. METHODS: Established lymphoma cell lines were exposed for 72 h to increasing doses of the compounds. Cell proliferation was evaluated by using an 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assay. Fluorescent-Activated Cell Sorter (FACS) analysis was performed to measure apoptotic activation and RNA sequencing (RNA-Seq) to study the transcriptional changes induced by the compounds. RESULTS: MZ1, and not its negative control epimer cisMZ1, was very active with a median half maximal inhibitory concentration (IC(50)) of 49 nmol/L. MZ1 was more in vitro active than the BET inhibitor birabresib (OTX015). Importantly, MZ1 induced cell death in all the ABC DLBCL cell lines, while the BET inhibitor was cytotoxic only in a fraction of them. BET degrader and inhibitor shared partially similar changes at transcriptome level but the MZ1 effect was stronger and overlapped with that caused cyclin-dependent kinase 9 (CDK9) inhibition. CONCLUSIONS: The BET degrader MZ1 had strong cytotoxic activity in all the ABC DLBCL cell lines that were tested, and, at least in vitro, it elicited more profound effects than BET inhibitors, and encourages further investigations

Iterative design and optimization of initially inactive Proteolysis Targeting Chimeras (PROTACs) identify VZ185 as a potent, fast and selective von Hippel-Lindau (VHL)-based dual degrader probe of BRD9 and BRD7

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers, and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a potent, fast and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown, and provide a roadmap for PROTAC development against seemingly incompatible target-ligase combinations