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

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.     

The rise of covalent proteolysis targeting chimeras

Targeted protein degradation offers several advantages over direct inhibition of protein activity and is gaining increasing interest in chemical biology and drug discovery. Proteolysis targeting chimeras (PROTACs) in particular are enjoying widespread application. However, PROTACs, which recruit an E3 ligase for degradation of a target protein, still suffer from certain challenges. These include a limited selection for E3 ligases on the one hand and the requirement for potent target binding on the other hand. Both issues restrict the target scope available for PROTACs. Degraders that covalently engage the target protein or the E3 ligase can potentially expand the pool of both targets and E3 ligases. Moreover, they may offer additional advantages by improving the kinetics of ternary complex formation or by endowing additional selectivity to the degrader. Here, we review the recent progress in the emerging field of covalent PROTACs.     

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously “undruggable” proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12–186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target “degradability.”     

Interaction Between Syntaxin 8 and HECTd3, a HECT Domain Ligase

Ubiquitination of proteins and their degradation within the proteasome has emerged as the major proteolytic mechanism used by mammalian cells to regulate cytosolic and nuclear protein levels. Substrate ubiquitylation is mediated by ubiquitin (Ub) ligases, also called E3 Ub ligases. HECT-E3 Ub ligases are characterized by the presence of a C-terminal HECT domain that contains the active site for Ub transfer onto substrates. Among the many E3 Ub ligases, the family homologous to E6-Ap C-terminus (HECT) E3 Ub ligases, which includes the yeast protein Rsp5p and the mammalian homolog NEDD4, AIP4/Itch, and Smurf, has been shown to ubiquitylate membrane proteins and, in some instances, to induce their degradation. In this report, we have identified Syntaxin 8 as a binding protein to a novel HECT domain protein, HECT domain containing 3 (HECTd3), by yeast two-hybrid screen. Besides HECT domain, HECTd3 contains an anaphase-promoting complex, subunit 10 (APC10) domain. Our co-immunoprecipitation experiments show that Syntaxin 8 directly interacts with HECTd3 and that the overexpression of HECTd3 promotes the ubiquitination of Syntaxin 8. Immunofluorescence results show that Syntaxin 8 and HECTd3 have similar subcellular localization.     

The emerging role for Cullin 4 family of E3 ligases in tumorigenesis

As a member of the Cullin-RING ligase family, Cullin-RING ligase 4 (CRL4) has drawn much attention due to its broad regulatory roles under physiological and pathological conditions, especially in neoplastic events. Based on evidence from knockout and transgenic mouse models, human clinical data, and biochemical interactions, we summarize the distinct roles of the CRL4 E3 ligase complexes in tumorigenesis, which appears to be tissue- and context-dependent. Notably, targeting CRL4 has recently emerged as a noval anti-cancer strategy, including thalidomide and its derivatives that bind to the substrate recognition receptor cereblon (CRBN), and anticancer sulfonamides that target DCAF15 to suppress the neoplastic proliferation of multiple myeloma and colorectal cancers, respectively. To this end, PROTACs have been developed as a group of engineered bi-functional chemical glues that induce the ubiquitination-mediated degradation of substrates via recruiting E3 ligases, such as CRL4 (CRBN) and CRL2 (pVHL). We summarize the recent major advances in the CRL4 research field towards understanding its involvement in tumorigenesis and further discuss its clinical implications. The anti-tumor effects using the PROTAC approach to target the degradation of undruggable targets are also highlighted.     

CDK5-Mediated Phosphorylation-Dependent Ubiquitination and Degradation of E3 Ubiquitin Ligases GP78 Accelerates Neuronal Death in Parkinson’s Disease

The molecular mechanisms responsible for the loss of dopaminergic neurons in Parkinson’s disease (PD) remain obscure. Loss of function of E3 ubiquitin ligases is associated with mitochondria dysfunction, dysfunction of protein degradation, and α-synuclein aggregation, which are major contributors to neurodegeneration in PD. Recent research has thus focused on E3 ubiquitin ligase glycoprotein 78 (GP78); however, the role of GP78 in PD pathogenesis remains unclear. Notably, cyclin-dependent kinase 5 (CDK5) controls multiple cellular events in postmitotic neurons, and CDK5 activity has been implicated in the pathogenesis of PD. Thus, we addressed the relationship between CDK5 and GP78 in MPTP-based PD models. We found that GP78 expression is decreased in MPTP-based cellular and animal PD models, and CDK5 directly phosphorylated GP78 at Ser516, which promoted the ubiquitination and degradation of GP78. Importantly, overexpression of GP78 or interference of GP78 Ser516 phosphorylation protected neurons against MPP⁺-induced cell death. Thus, our research reveals that the CDK5-GP78 pathway is involved in the pathogenesis of PD and could be a novel candidate drug target for the treatment of PD.     

Recent advances in IAP-based PROTACs (SNIPERs) as potential therapeutic agents

Proteolytic targeting chimaeras (PROTACs) have been developed as an effective technology for targeted protein degradation. PROTACs are heterobifunctional molecules that can trigger the polyubiquitination of proteins of interest (POIs) by recruiting the ubiquitin-proteasome system, thereby inhibiting the intracellular level of POIs. To date, a variety of small-molecule PROTACs (CRBN, VHL, IAP, and MDM2-based PROTACs) have been developed. IAP-based PROTACs, also known as specific and nongenetic IAP-dependent protein erasers (SNIPERs), are used to degrade the target proteins closely related to diseases. Their structures consist of three parts, including target protein ligand, E3 ligase ligand, and the linker between them. So far, many SNIPERs have been extensively studied worldwide and have performed well in multiple diseases, especially cancer. In this review, we will present the most relevant advances in the field of SNIPERs and provide our perspective on the opportunities and challenges for SNIPERs to become therapeutic agents.     

Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family

Cullin-based E3 ligases target substrates for ubiquitin-dependent degradation by the 26S proteasome. The SCF (Skp1-Cul1-F-box) and ECS (ElonginC-Cul2-SOCS box) complexes are so far the best-characterized cullin-based ligases. Their atomic structure has been solved recently, and several substrates have been described in different organisms. In addition to Cul1 and Cul2, higher eucaryotic genomes encode for three other cullins: Cul3, Cul4, and Cul5. Recent results have shed light on the molecular composition and function of Cul3-based E3 ligases. In these complexes, BTB-domain-containing proteins may bridge the cullin to the substrate in a single polypeptide, while Skp1/F-box or ElonginC/SOCS heterodimers fulfill this function in the SCF and ECS complexes. BTB-containing proteins are evolutionary conserved and involved in diverse biological processes, but their function has not previously been linked to ubiquitin-dependent degradation. In this review, we present these new findings and compare the composition of Cul3-based ligases to the well-defined SCF and ECS ligases.     

Proteolysis-targeting chimaeras (PROTACs) as pharmacological tools and therapeutic agents: advances and future challenges

Proteolysis-targeting chimaeras (PROTACs) have been developed to be an emerging technology for targeted protein degradation and attracted the favour of academic institutions, large pharmaceutical enterprises, and biotechnology companies. The mechanism is based on the inhibition of protein function by hijacking a ubiquitin E3 ligase for protein degradation. The heterobifunctional PROTACs contain a ligand for recruiting an E3 ligase, a linker, and another ligand to bind with the protein targeted for degradation. To date, PROTACs targeting ∼70 proteins, many of which are clinically validated drug targets, have been successfully developed with several in clinical trials for diseases therapy. In this review, the recent advances in PROTACs against clinically validated drug targets are summarised and the chemical structure, cellular and in vivo activity, pharmacokinetics, and pharmacodynamics of these PROTACs are highlighted. In addition, the potential advantages, challenges, and prospects of PROTACs technology in disease treatment are discussed.     

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor–induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.     

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.     

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.     

Knockdown of CDK5 down-regulates PD-L1 via the ubiquitination-proteasome pathway and improves antitumor immunity in lung adenocarcinoma

Although immunotherapy (anti-PD-1/PD-L1 antibodies) has been approved for clinical treatment of lung cancer, only a small proportion of patients respond to monotherapy. Hence, understanding the regulatory mechanism of PD-L1 is particularly important to identify optimal combinations. In this study, we found that inhibition of CDK5 induced by shRNA or CDK5 inhibitor leads to reduced expression of PD-L1 protein in human lung adenocarcinoma cells, while the mRNA level is not substantially altered. The PD-L1 protein degradation is mediated by E3 ligase TRIM21 via ubiquitination-proteasome pathway. Subsequently, we studied the function of CDK5/PD-L1 axis in LUAD. In vitro, the absence of CDK5 in mouse Lewis lung cancer cell (LLC) has no effect on cell proliferation. However, the attenuation of CDK5 or combined with anti-PD-L1 greatly suppresses tumor growth in LLC implanted mouse models in vivo. Disruption of CDK5 elicits a higher level of CD3⁺, CD4⁺ and CD8⁺ T cells in spleens and lower PD-1 expression in CD4⁺ and CD8⁺ T cells. Our findings highlight a role for CDK5 in promoting antitumor immunity, which provide a potential therapeutic target for combined immunotherapy in LUAD.     

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.