Confounding Factors in Targeted Degradation of Short-Lived Proteins.

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.

Stereochemical inversion at a 1,4-cyclohexyl PROTAC linker fine-tunes conformation and binding affinity.

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.

Ternary Complex-Templated Dynamic Combinatorial Chemistry for the Selection and Identification of Homo-PROTACs.

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.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2.

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.

Expanding the Structural Diversity at the Phenylene Core of Ligands for the von Hippel-Lindau E3 Ubiquitin Ligase: Development of Highly Potent Hypoxia-Inducible Factor-1α Stabilizers.

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.

A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo.

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.

Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders.

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.

Functional genomic analysis of epithelioid sarcoma reveals distinct proximal and distal subtype biology.

BACKGROUND: Metastatic epithelioid sarcoma (EPS) remains a largely unmet clinical need in children, adolescents and young adults despite the advent of EZH2 inhibitor tazemetostat. METHODS: In order to realise consistently effective drug therapies, a functional genomics approach was used to identify key signalling pathway vulnerabilities in a spectrum of EPS patient samples. EPS biopsies/surgical resections and cell lines were studied by next-generation DNA exome and RNA deep sequencing, then EPS cell cultures were tested against a panel of chemical probes to discover signalling pathway targets with the most significant contributions to EPS tumour cell maintenance. RESULTS: Other biologically inspired functional interrogations of EPS cultures using gene knockdown or chemical probes demonstrated only limited to modest efficacy in vitro. However, our molecular studies uncovered distinguishing features (including retained dysfunctional SMARCB1 expression and elevated GLI3, FYN and CXCL12 expression) of distal, paediatric/young adult-associated EPS versus proximal, adult-associated EPS. CONCLUSIONS: Overall results highlight the complexity of the disease and a limited chemical space for therapeutic advancement. However, subtle differences between the two EPS subtypes highlight the biological disparities between younger and older EPS patients and emphasise the need to approach the two subtypes as molecularly and clinically distinct diseases.

Targeting epigenetic modulators using PROTAC degraders: Current status and future perspective.

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.

SMARCA4 biology in alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.

Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity.

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.

Von Hippel-Lindau (VHL) small-molecule inhibitor binding increases stability and intracellular levels of VHL protein.

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.

Targeting non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex improves the efficacy of HEDGEHOG pathway inhibition in melanoma.

Despite the development of new targeted and immune therapies, the prognosis of metastatic melanoma remains bleak. Therefore, it is critical to better understand the mechanisms controlling advanced melanoma to develop more effective treatment regimens. Hedgehog/GLI (HH/GLI) signaling inhibitors targeting the central pathway transducer Smoothened (SMO) have shown to be clinical efficacious in skin cancer; however, several mechanisms of non-canonical HH/GLI pathway activation limit their efficacy. Here, we identify a novel SOX2-BRD4 transcriptional complex driving the expression of GLI1, the final effector of the HH/GLI pathway, providing a novel mechanism of non-canonical SMO-independent activation of HH/GLI signaling in melanoma. Consistently, we find a positive correlation between the expression of GLI1 and SOX2 in human melanoma samples and cell lines. Further, we show that combined targeting of canonical HH/GLI pathway with the SMO inhibitor MRT-92 and of the SOX2-BRD4 complex using a potent Proteolysis Targeted Chimeras (PROTACs)-derived BRD4 degrader (MZ1), yields a synergistic anti-proliferative effect in melanoma cells independently of their BRAF, NRAS, and NF1 mutational status, with complete abrogation of GLI1 expression. Combination of MRT-92 and MZ1 strongly potentiates the antitumor effect of either drug as single agents in an orthotopic melanoma model. Together, our data provide evidence of a novel mechanism of non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex, and describe the efficacy of a new combinatorial treatment for a subset of melanomas with an active SOX2-BRD4-GLI1 axis.

Transforming targeted cancer therapy with PROTACs: A forward-looking perspective.

Small-molecule targeted protein degraders have in recent years made a great impact on the strategies of many industry and academic cancer research endeavours. We seek here to provide a concise perspective on the opportunities and challenges that lie ahead for bifunctional degrader molecules, so-called 'Proteolysis Targeting Chimeras (PROTACs),' in the context of cancer therapy. We highlight high-profile studies that support the potential for PROTAC approaches to broaden drug target scope, address drug resistance, enhance target selectivity and provide tissue specificity, but also assess where the modality is yet to fully deliver in these contexts. Future opportunities presented by the unique bifunctional nature of these molecules are also discussed.

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 (IC50) 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.

E3 Ligase Ligands for PROTACs: How They Were Found and How to Discover New Ones.

Bifunctional degrader molecules, also called proteolysis-targeting chimeras (PROTACs), are a new modality of chemical tools and potential therapeutics to understand and treat human disease. A required PROTAC component is a ligand binding to an E3 ubiquitin ligase, which is then joined to another ligand binding to a protein to be degraded via the ubiquitin-proteasome system. The advent of nonpeptidic small-molecule E3 ligase ligands, notably for von Hippel-Lindau (VHL) and cereblon (CRBN), revolutionized the field and ushered in the design of drug-like PROTACs with potent and selective degradation activity. A first wave of PROTAC drugs are now undergoing clinical development in cancer, and the field is seeking to extend the repertoire of chemistries that allow hijacking new E3 ligases to improve the scope of targeted protein degradation.Here, we briefly review how traditional E3 ligase ligands were discovered, and then outline approaches and ligands that have been recently used to discover new E3 ligases for PROTACs. We will then take an outlook at current and future strategies undertaken that invoke either target-based screening or phenotypic-based approaches, including the use of DNA-encoded libraries (DELs), display technologies and cyclic peptides, smaller molecular glue degraders, and covalent warhead ligands. These approaches are ripe for expanding the chemical space of PROTACs and usher in the advent of other emerging bifunctional modalities of proximity-based pharmacology.

Targeting epigenetic reader domains by chemical biology.

Over the past years, growing interest toward post-translational modifications (PTMs) of histones and nonhistone proteins has prompted academia and industrial research groups to develop different approaches to better understand the link between PTMs and pathological states. Selective recognition of PTMs is carried out by reader modules, which mediate the biological readout of epigenetic mechanisms. Progress in medicinal chemistry and chemical biology has contributed to corroborate the role of reader domains in chromatin-binding proteins as potential therapeutic targets. Here, we review the state-of-the-art of the most important small molecules developed to date, with a particular attention on contemporary chemical biology approaches, including photoaffinity probes, cyclic peptides, bifunctional inhibitors, and PROTAC degraders.

Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System.

The affinity-directed protein missile (AdPROM) system utilizes specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we use Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-AdPROM) system. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to the Halo-tag, we achieve robust degradation of GFP-tagged POIs only upon treatment of cells with the HaloPROTAC. For GFP-tagged POIs, ULK1, FAM83D, and SGK3 were knocked in with a GFP-tag using CRISPR/Cas9. By substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.

Design and Characterization of SGK3-PROTAC1, an Isoform Specific SGK3 Kinase PROTAC Degrader.

SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to class 1 PI3K or Akt inhibitors, by substituting for the loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3 and obstruct its role in inhibitor resistance. Here, we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation.  SGK3-PROTAC1 (0.3 µM) induced 50% degradation of endogenous SGK3 within 2 h, with maximal 80% degradation observed within 8 h, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1-0.3 µM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.