Small-molecule degraders for oncogenic KRASG12C and pan-KRAS mutations.

EDITORIAL article Front. Oncol., 05 December 2012 | https://doi.org/10.3389/fonc.2012.00187

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, 'fit-for-purpose' PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable.Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.

The Ubiquitin Proteasome System (UPS) is responsible for the degradation of misfolded or aggregated proteins via a multistep ATP-dependent proteolytic mechanism. This process involves a cascade of ubiquitin (Ub) transfer steps from E1 to E2 to E3 ligase. The E3 ligase transfers Ub to a targeted protein that is brought to the proteasome for degradation. The inability of the UPS to remove misfolded or aggregated proteins due to UPS dysfunction is commonly observed in neurodegenerative diseases, such as Alzheimer’s disease (AD). UPS dysfunction in AD drives disease pathology and is associated with the common hallmarks such as amyloid-β (Aβ) accumulation and tau hyperphosphorylation, among others. E3 ligases are key members of the UPS machinery and dysfunction or changes in their expression can propagate other aberrant processes that accelerate AD pathology. The upregulation or downregulation of expression or activity of E3 ligases responsible for these processes results in changes in protein levels of E3 ligase substrates, many of which represent key proteins that propagate AD. A powerful way to better characterize UPS dysfunction in AD and the role of individual E3 ligases is via the use of high-quality chemical tools that bind and modulate specific E3 ligases. Furthermore, through combining gene editing with recent advances in 3D cell culture, in vitro modeling of AD in a dish has become more relevant and possible. These cell-based models of AD allow for study of specific pathways and mechanisms as well as characterization of the role E3 ligases play in driving AD. In this review, we outline the key mechanisms of UPS dysregulation linked to E3 ligases in AD and highlight the currently available chemical modulators. We present several key approaches for E3 ligase ligand discovery being employed with respect to distinct classes of E3 ligases. Where possible, specific examples of the use of cultured neurons to delineate E3 ligase biology have been captured. Finally, utilizing the available ligands for E3 ligases in the design of proteolysis targeting chimeras (PROTACs) to degrade aberrant proteins is a novel strategy for AD, and we explore the prospects of PROTACs as AD therapeutics.

Repurposing old drugs as new inhibitors of the ubiquitin-proteasome pathway for cancer treatment.

The role of ubiquitin ligases in cardiac disease

Protein degradation has become an area of focus for modern research due to the technological advancement of targeted protein degradation as a highly specific and selective therapeutic approach [1]. However, as can be said of most scientific discoveries, the cell itself has long utilized and fine-tuned this regulatory process to create intricate layers of regulation surrounding protein degradation to attune the proteome to intracellular and extracellular cues. Thus far, the answer to this question has indicated that post-translational modifications (PTMs) on target proteins are responsible for tuning protein degradation to the cell's needs [2]. As our understanding of protein degradation delves further into the mechanistic and biological implications of the relationship between the cellular environment, PTMs, and protein degradation, further questions are uncovered regarding why PTMs have evolved to play these central roles in protein degradation. This hypothesis by Hwang et al., [3] takes a refreshing perspective on this next step: Can the ubiquitination machinery itself be directly subject to environmental cues to add an additional regulatory layer to protein degradation? Protein degradation largely occurs through the ubiquitin-proteasome system (UPS). The UPS utilizes a three-step enzymatic cascade to processively attach ubiquitin (Ub) onto surface lysine residues of proteins targeted for degradation. The ubiquitination process is highly coordinated, thereby allowing for regulation at almost every step before, during, and after the ubiquitination cascade. The most common regulatory step occurs at ubiquitin attachment, wherein E3 ligase complexes function to coordinate transfer of the ubiquitin from an E2 ubiquitin-conjugating enzyme onto the target protein. These complexes are often made up of multiple adaptor and scaffold proteins, again indicating the level of regulation required for ubiquitination. Cullin-RING ligases (CRLs), the majority of known E3 ligases, consist of a Cullin scaffold protein and a RING adaptor protein which functions to bind the E2-Ub complex. Families of CRLs are characterized based on the other proteins that make up the complex. Skp1-cullin1-Fbox (SCF) are a subset of CRLs that utilize adaptor protein Skp1 to bind an F-box protein (FBP) bound to the target protein [4]. Intuitively, the recruitment of the target protein to the SCF complex via FBPs is crucial for protein ubiquitination via SCF complexes. This article posits the hypothesis that FBPs function as environmental sensors to control stress response mechanisms, particularly in response to heavy metals [3]. FBPs are well-evidenced to respond to various PTMs (including but not limited to phosphorylation, methylation, and acetylation), which are arguably all ways in which the cell can transmit environmental signals to protein degradation. The authors emphasis of hydroxylation [5] within this context is, therefore, an expansion on well-known regulatory processes dictating how PTMs allow FBP-target protein interactions to remain sensitive to environmental perturbations. The author's expansion into heavy metal sensing, however, is an exciting new hypothesis that is so novel that it is potentially under-evidenced. Canonically, the FBP is made up of two domains: (1) an F-box domain highly conserved for the interaction to adaptor protein Skp1 and (2) a protein interaction domain that binds the protein targeted for degradation. The authors hypothesize that the F-box domain may also sense heavy metals with functional implications to protein degradation, in addition to being an anchor to Skp1 and the rest of the SCF complex. The foundation for this hypothesis is found in the examples of yeast FBPs outlined within the article [6, 7], leading to the suggestion that human FBPs may also contain these functions. However, initial phenotypic verification of this hypothesis is not currently available. Correlative studies demonstrating statistically significant links between SCF complex activity and/or stability in the presence of various metals via cell-wide techniques (i.e., proteomics) could address this issue. In summary, the authors present an intriguing next step for the field of protein homeostasis: the integration of findings from other species to delve into domains and mechanisms long believed to contain just one simple function. This hypothesis builds on a strong foundation of FBPs as conditionally sensitive via PTM-mediated or -hindered protein-protein interactions with proteins targeted for degradation and looks ahead to new possibilities for the regulation of protein degradation. This article comments on the hypothesis paper by Jiwon Hwang, Linda Lauinger, and Peter Kaiser https://doi.org/10.1002/bies.202400249. The authors declare no conflicts of interest.

Proteolysis targeting chimera (PROTAC) degraders are typically bifunctional with one E3 ligase ligand connected to one target protein ligand via a linker. While augmented valency has been shown with trivalent PROTACs targeting two binding sites within a given target protein, or used to recruit two different targets, the possibility of recruiting two different E3 ligases within the same compound has not been demonstrated. Here we present dual-ligase recruitment as a strategy to enhance targeted protein degradation. We designed heterotrivalent PROTACs composed of CRBN, VHL and BET targeting ligands, separately tethered via a branched trifunctional linker. Structure-activity relationships of 12 analogues qualifies AB3067 as the most potent and fastest degrader of BET proteins, with minimal E3 ligase cross-degradation. Comparative kinetic analyses in wild-type and ligase single and double knockout cell lines revealed that protein ubiquitination and degradation induced by AB3067 was contributed to by both CRBN and VHL in an additive fashion. We further expand the scope of the dual-ligase approach by developing a heterotrivalent CRBN/VHL-based BromoTag degrader and a tetravalent PROTAC comprising of two BET ligand moieties. In summary, we provide proof-of-concept for dual-E3 ligase recruitment as a strategy to boost degradation fitness by recruiting two E3 ligases with a single degrader molecule. This approach could potentially delay the outset of resistance mechanisms involving loss of E3 ligase functionality.

Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.

Targeted protein degradation by the ubiquitin-proteasome system (UPS) has an essential role in carcinogenesis and cancer cell survival. Thus, the search for proteasome inhibitors is of great interest as a potential anticancer therapy.1 Recent studies by the Ferbeyre group suggest that certain UPS-targeted proteins play a role not just in carcinogenesis, but also in cellular senescence. The limited replicative potential of cells or tissues, called senescence, serves as a ‘mitotic clock’ for normal cell proliferation. During cancer development, it is often bypassed through inactivation of DNA damage checkpoints, and thus senescence is often considered a tumor suppressive mechanism.2 Senescence is induced by many causes such as telomere shortening, DNA damage, chromatin perturbations, oncogenes and reactive oxygen species (ROS).2 How these diverse triggers lead to cell cycle arrest and what common features define the senescent state is a topic under intensive investigation. The ubiquitin proteasome system (UPS) is a major protein degradation network in eukaryotic cells. One of the signaling networks that regulate the UPS is the Ras family of kinases. Ras is mutated in various cancer types and Ras signaling regulates cellular differentiation, survival, and proliferation. Extracellular signal regulated kinases (ERK) or MAP kinases (MAPK) are also commonly disrupted in cancer, and ubiquitinylation of Ras is essential to activate ERK/MAPK.3 Previously, Deschenes-Simard and colleagues uncovered a common feature of protein degradation in both Ras-induced and telomere dysfunction-induced senescence that they termed senescence-associated protein degradation (SAPD).4 In particular, they found that inhibition of ERK signaling attenuated Ras-induced senescence and permitted cells to adopt a transformed state.4 In order to model the specific proteome changes characteristic of SAPD, in the present study the authors compared the proteome of cells undergoing Ras-induced senescence in the absence or presence of the proteosome inhibitor MG132.5 The authors showed that Ras-induced senescence led to increased turnover of proteins whose functions are implicated in diverse cellular processes such as mitochondrion dysfunction (ATP5B, STAT3, TOM complex), proteotoxic stress (HSP27, HSP60, HSP90AB1), the DNA damage response, (CCDC6, SOD1, TOP2, Rap1), nucleolar and ribosome biogenesis dysfunction (NOLC1, NOP56/58, DDX1, NOL6, NOC2L, NCL, RPLP1, RSL1D1, NPM1), cell cycle arrest (YAP1, MCM2, ORCA, MYC, JUN, KAP1, TBX2), and impaired mRNA metabolism and translation (YBX1, SRRM1, SRRM2).5 Furthermore, some of these proteins, including RAP1, YAP1, MYC and STAT3, are also localized to PML nuclear bodies (PML-NBs) and are targets for sumoylation and phosphorylation followed by ubiquitylation, leading the authors to suggest that PML-NBs could be a site for nuclear SAPD. Figure 1. A schematic of the cellular processes affected by targeted protein degradation during Ras-induced senescence. Hyperactive ERK/MAPK (in red) leads to targeting of proteins to proteasomes for degradation (in blue). The authors suggest that PML nuclear bodies ... Other recently published findings support a role for the UPS as a trigger for the changes that accompany cellular aging; for example, proteosomal dysfunction can lead to mitochondrial ROS production, which is a common feature of the senescent state,6 and Mdm2-induced proteolysis of phosphoglycerate mutase is a hallmark of DNA damage-induced senescence.7 The precise mechanisms that link Ras, ERK and SAPD will be interesting to determine. For example, what are the key E3 ubiquitin ligases that trigger SAPD? The authors suggest CUL4A-DDB1 may be involved in Ras-induced SAPD5; if so, would this E3 ligase also be instrumental in DNA damage-induced or telomere dysfunction-induced senescence? An understanding of how these cellular networks become deranged will no doubt lead to further mechanistic insights not only in cellular senescence, but will hopefully also aid our understanding of aging and cancer.

The anaphase-promoting complex or cyclosome (APC/C) is a large ubiquitin ligase essential for the mitotic and meiotic cell cycle. It ligates chains of ubiquitin to its target proteins marking them for proteolysis by the 26S proteasome. APC/C activity and thereby stage-specific proteolysis are precisely regulated during the cell cycle to maintain the chronology of key events. This work characterized some of the mechanisms involved in APC/C regulation.In the first part, the requirement of post-translational modification with the small ubiquitin-like modifier SUMO on efficient APC/C-mediated protein degradation was tested. The anaphase inhibitor protein, Pds1, was found to be stabilized in cells defective for SUMOylation and due to this altered degradation, cells displayed defects in chromosome segregation. Furthermore also proteolysis of other APC/C target proteins, such as the mitotic cyclins Clb2, Clb3 and the polo-like kinase Cdc5, was delayed whereas degradation of unstable proteins that are not APC/C targets was unaffected. SUMOylation does not seem to be required for proteolysis in general but specifically for APC/C-mediated degradation.In the second part, cyclin Clb5 degradation and its importance for the exit from mitosis was analyzed. Clb5, which was previously identified as a target of the APC/C, was found to be degraded by a second, alternative mechanism independently of APC/C. It could be shown that the SCF ubiquitin-ligase is not involved in this process. Indeed, Clb5 is unstable even in mutant cells defective in both ubiquitin ligases, APC/C and SCF. The APC/C-independent mechanism remains to be identified, but under physiological conditions, this mechanism is obviously sufficient for Clb5 degradation in the cell cycle. Only under abnormal conditions, such as CLB5 overexpression, APC/C becomes essential for Clb5 degradation. In the third part of this work the regulation of the APC/C in meiosis by the meiosis-specific kinase Ime2 was investigated. Ime2, a negative APC/C regulator, is a highly unstable protein, and thus proteolysis could bear a regulatory mechanism. To investigate this, a truncated Ime2 protein was analyzed with regard to its functionality to induce a mitotic cell cycle arrest and to phosphorylate Cdh1. We showed that this truncated Ime2DC, lacking 241 amino acids at its C-terminus, is despite the truncation highly active and stable throughout meiosis. Furthermore, we found that homozygous strains for the truncated IME2 allele display a severe defect in meiosis II. These results indicate that either high Ime2 levels or loss of an unknown function causes the defect in meiosis II.

The KRASG12C mutation is a critical therapeutic target in the management of solid tumors, owing to its role in oncogenic signaling. Recent advances in covalent inhibitors that target mutant KRAS cysteine-12 have demonstrated the potential to halt aberrant signaling associated with this historically "undruggable" target. Here, we report the identification of 6-cyanoquinazoline covalent irreversible KRASG12C inhibitors. Lead optimization used structure-based design to identify novel switch-II pocket-binding motifs and in silico models to forecast in vitro metabolic stability and permeability. Human dose was improved by maximizing the rate of covalent modification (kobs/[I]) of KRASG12C-GDP, along with optimizing ADME parameters, to identify potent, orally bioavailable lead molecule 13de which demonstrated significant antitumor efficacy in the NCI-H1373 human lung adenocarcinoma xenograft model. Studies evaluating KRASG12C-GDP covalent target engagement, pharmacokinetics, and tumor growth inhibition estimated the efficacious human dose of 13de to be 192 mg administered once daily (QD), using allometric scaling.

Protein degraders are currently under rapid development as a promising modality for drug discovery. They are compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting intracellular protein degradation through proteasomal pathway. More protein degraders identification will greatly promote the development of this field. BAG3 is widely recognized as an excellent therapeutic target in cancer treatments. Exploring protein degraders that target BAG3 degradation has profound implications. Herein, molecular docking was applied to assess binding energy between 81 clinical phase I kinase inhibitors and BAG3. BAG3 protein and mRNA level were detected by western blot and quantitative real-time PCR. CCK8 assay and colony formation assay were applied to detect the cell viability and proliferation rate. Cell death was accessed using flow cytometry combined with PI and Annexin V double staining. AZD7762, a Chk1 kinase inhibitor, was identified to induce BAG3 degradation in a ubiquitin-proteasome pathway. AZD7762-induced BAG3 degradation was not dependent on Chk1 expression or activity. CRBN, an E3 ligase, was identified to bind to BAG3 and mediated BAG3 ubiquitination in the presence of AZD7762. By targeting Chk1 and BAG3, two ideal therapeutic targets in cancer treatment, AZD7762 would be a powerful chemotherapy agent in the future.

The majority of critically ill intensive care unit (ICU) patients with severe sepsis develop ICU-acquired weakness (ICUAW) characterized by loss of muscle mass, reduction in myofiber size and decreased muscle strength leading to persisting physical impairment. This phenotype results from a dysregulated protein homeostasis with increased protein degradation and decreased protein synthesis, eventually causing a decrease in muscle structural proteins. The ubiquitin proteasome system (UPS) is the predominant protein-degrading system in muscle that is activated during diverse muscle atrophy conditions, e.g., inflammation. The specificity of UPS-mediated protein degradation is assured by E3 ubiquitin ligases, such as atrogin-1 and MuRF1, which target structural and contractile proteins, proteins involved in energy metabolism and transcription factors for UPS-dependent degradation. Although the regulation of activity and function of E3 ubiquitin ligases in inflammation-induced muscle atrophy is well perceived, the contribution of the proteasome to muscle atrophy during inflammation is still elusive. During inflammation, a shift from standard- to immunoproteasome was described; however, to which extent this contributes to muscle wasting and whether this changes targeting of specific muscular proteins is not well described. This review summarizes the function of the main proinflammatory cytokines and acute phase response proteins and their signaling pathways in inflammation-induced muscle atrophy with a focus on UPS-mediated protein degradation in muscle during sepsis. The regulation and target-specificity of the main E3 ubiquitin ligases in muscle atrophy and their mode of action on myofibrillar proteins will be reported. The function of the standard- and immunoproteasome in inflammation-induced muscle atrophy will be described and the effects of proteasome-inhibitors as treatment strategies will be discussed.

The current interest in protein homeostasis as a therapeutic strategy has highlighted the importance of protein ubiquitination as a post-translational modification. Protein degradation has been enabled by several approaches (e.g. small molecule degraders, molecular glues, and PROTACsTM). Deubiquitinating enzymes (DUBs) are proteases that catalyse the de-ubiquitination of protein substrates and as such offer an alternative way to regulate protein homeostasis. Furthermore, since ubiquitination also controls a plethora of regulatory functions beyond direct degradation, inhibition of DUBs provides additional opportunities to manipulate critical cellular processes. As a result of this dual role in protein degradation and signalling, as well as their increasing linkage to the etiology of numerous pathological conditions including cancer and neurodegeneration, DUBs have emerged as an attractive target class for the development of first-in-class medicines with high therapeutic impact. However until recently, DUBs have proven largely refractory to drug discovery efforts despite significant industry efforts. We have developed a purpose-built drug discovery platform (UbiPlex™) for the identification and development of DUB inhibitors. Herein, we will highlight significant recent developments to this platform including the addition of multiple DUB crystal structures (including fragments and small molecule co-crystals), biophysics, chemo/bio-informatics capabilities and focussed screening libraries. These developments have allowed the identification of novel, highly potent (e.g. IC50 < 100 nM), reversible inhibitors with drug like properties against multiple therapeutically relevant DUB targets. Selectivity profiles of representative inhibitors against panels of DUBs and other non-related enzymes (e.g. kinases, proteases) will be described, demonstrating that excellent selectivity is achievable. Finally, we will describe our progress towards the development of lead molecules with drug-like properties with the aim of rapidly establishing proof-of-concept studies in vivo for previously unexplored DUB targets. This work exemplifies the broad tractability and druggability of the DUBome and provides an alternative strategy for manipulation of the UPS. Furthermore, these potent, selective, cell penetrant molecules represent important chemical probes for pharmacological validation of the biological pathways associated with deubiquitinase inhibition as well as starting points for the development of new therapeutics for cancer and associated disorders. Citation Format: Tim Harrison, Xavier Jacq, Colin O'Dowd, Gerald Gavory, Oliver Barker, Christina Bell, Frank Burkamp, Stephanie Burton, Eamon Cassidy, Matthew Helm, Peter Hewitt, Natalie Page, Shane Rountree, Ewelina Rozycka, Steven Shepherd, Steven Whitehead, Mark Wappett. Targeting the ubiquitin-proteasome system by small molecule inhibition of the DUBome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-049.

Insulin resistance in the brain is a pathological mechanism that is shared between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Although aberrant expression and phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to insulin resistance, the underlying mechanism remains elusive. In this study, we used several approaches, including adeno-associated virus-based protein overexpression, immunoblotting, immunoprecipitation, immunohistochemistry, and in situ proximal ligation assays, to investigate the function of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) in IRS-1 regulation and the downstream insulin signaling in neurons. We found that DYRK1A overexpression up-regulated IRS-1 expression by slowing turnover of the IRS-1 protein. We further observed that DYRK1A directly interacted with IRS-1 and phosphorylated IRS-1's multiple serine residues. Of note, DYRK1A and IRS-1 were coordinately up-regulated in the prefrontal cortex of db/db mice brain. Furthermore, DYRK1A overexpression ameliorated chronic high insulin-induced insulin resistance in SH-SY5Y cells as well as in primary rat neurons. These findings suggest that DYRK1A protects against insulin resistance in the brain by elevating IRS-1 expression.