Chapter Two - Covalency in PROTACs: Mechanisms and applications

Special Focus Issue - Targeted protein degradation: a new paradigm in medicinal chemistry.

Advances and opportunities in targeted protein degradation

Therapeutic targeting of RNA-binding protein by RNA-PROTAC

Recent years have seen great advancement of targeted protein degradation technology, in particular, proteolysis targeting chimeras (PROTACs) are now widely used in developing therapeutics for treating cancer. A PROTAC is a heterobifunctional small molecule with three distinct moieties: a ligand to bind a targeted protein of interest (POI), a second ligand to recruit E3 ubiquitin ligase to form a ternary complex, and a linker for bridging the two ligands. Through the E3 ubiquitin ligase pathway, properly designed PROTACs can effectively degrade POIs with high specificity, which can be pathogenic proteins, thus regulate related pathways and inhibit tumor growth. Currently, there is no high-throughput biochemical assay to measure the POI degradation in vitro, hindering the application of the PROTAC technology. Here we report the development of a high-throughput cell based assay to quantitatively measure the endogenous drug target degradation induced by PROTACs. This assay encompasses over 800 cancer cell lines, many of which are CRISPR-engineered ones on common drug targets. A unique feature of our assay is the incorporation of HiBiT short sequence tags on either N-terminal or C-terminal of endogenous protein in E3 ligase matched cell line through site-specific gene homologous recombination technology. By highly specific and sensitive detection of HiBiT tag content in cell lysates with biochemical assay, we can determine target protein degradation by PROTAC treatment. We demonstrate the utility of the assay on a wide array of drug targets including RAS, LDHA, BTK and HPK1, and show that it can accelerate PROTAC drug discovery. Citation Format: Yunpeng Zhai, Ming Tan, Defu Liu, Guoqian Wang, Jinying Ning, Feng Hao. Cell line panel with HIBIT tagged endogenous proteins to accelerate PROTAC drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6181.

ERα-positive breast cancers comprise approximately 80% of all newly diagnosed cases. Current treatment approaches targeting ER signaling include antagonizing and/or downregulating ER or reducing estrogen levels. Faslodex (fulvestrant) is the only clinically-approved agent that is both a potent ER antagonist and downregulator but has limitations given its pharmacokinetics and route of administration. Over the past several years, targeted ER therapies have focused on developing selective estrogen receptor downregulators (SERDs, i.e, GDC-0810, GDC-0927, AZD9496, RAD1901). The mechanisms involved in ER downregulation by SERD binding are not completely understood, but evidence suggests that conformational changes in the receptor upon ligand binding combined with specific co-regulator interactions destabilize the receptor making it a target for passive proteasomal degradation. We hypothesized that the complex ER pharmacology required for SERD-based passive degradation might be different across various ER-positive cell lines and that targeted degradation of the receptor by actively recruiting the ubiquitin-proteasome machinery would provide a better approach for reducing ER levels. To test this hypothesis, we developed potent molecules directed against ER using our pioneering technology—proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional molecules that actively recruit specific E3 ligases resulting in ubiquitylation and degradation of target proteins. When testing for ER degradation using several SERDs and ER PROTACs, we discovered that both fulvestrant and ER PROTACs provided robust degradation in all ER-positive lines (<1 nM 50% degradation; >90% reduction) whereas other SERDs did not degrade or only modestly degraded the receptor. Importantly, MCF-7 cells were uniquely sensitive to SERD-based degradation of ER compared to other cell lines. Subcutaneous administration of fulvestrant (1mpk) or ER PROTACs (10 mpk) reduced uterine ER alpha levels in immature rats (>65% reduction). PROTAC-mediated degradation of ER was also achieved in breast cancer xenografts. To further validate the PROTAC mechanism, incubation of ER-positive cells with ER PROTACs resulted in increased levels of poly-ubiquitylated ERα when compared to SERDs. Lastly, to demonstrate the specificity of PROTAC-mediated ERα degradation, we utilized a cellular expression proteomics-based approach to examine over 7,000 proteins. In this experiment, only ERα and several known proteins whose genes are regulated by ERα, were significantly reduced by PROTACs. It remains to be seen how the current class of investigational downregulators will perform in the clinic. More importantly, a better understanding of the therapeutic potential and benefit of degrading the receptor instead of inhibiting the receptor needs to be explored. To that end, we continue to develop and characterize novel ER PROTACs with the anticipation that targeted ERα degradation will provide a greater clinical benefit than receptor antagonism. Citation Format: Flanagan JJ, Rossi AK, Anderoli M, Willard RR, Gordon D, Harling J, Churcher I, Smith I, Zinn N, Bantscheff M, Crews CM, Crew A, Coleman KG, Winkler JD, Qian Y. Targeted and selective degradation of estrogen receptor (ER) alpha by PROTACs [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S4-03.

Expanding the landscape of E3 ligases for targeted protein degradation

A heterobifunctional molecule recruits cereblon to an RNA scaffold and activates its PROTAC function

PROTACs (Proteolysis Targeting Chimeras), heterobifunctional molecules, exhibit selectivity in degrading target proteins through E3 ubiquitin ligases. Designing effective PROTACs requires a deep understanding of the intricate binding interactions in the ternary complex (POI/PROTAC/E3 ligase), crucial for efficient target protein degradation. To address this challenge, we introduce a novel computational virtual screening method that considers essential amino acid interactions between the protein of interest and the chosen E3 ligase. This approach enhances accuracy and reliability, facilitating the strategic development of potent PROTACs. Utilizing a crystallized model of the VHL:PROTAC:SMARCA2BD ternary complex (PDB: 7Z6L), we assessed the effectiveness of our method. Our study reveals that increasing the number of essential restraints between the two proteins reduces the generated docking poses, leading to closer alignment with the experimental ternary complex. Specifically, utilizing three restraints showed the closest resemblance to the published complex, highlighting crucial interactions such as an H-bond between A:Gln 89 and B:Asn 67, along with two hydrophobic interactions: A:Gly 22 with B:Arg 69 and A:Glu 37 with B:Pro 99. This resulted in a significant decrease in the mean RMSD value from 31.8 and 31.0 Å to 24.4 Å, respectively. This underscores the importance of incorporating multiple essential restraints to enhance docking accuracy. Building on this progress, we introduce a systematic approach to design potential PROTACs between the Estrogen receptor and the E3 ligase, utilizing bridging intermediates with 4, 6, or 7 carbon atoms. By providing a more accurate and efficient means of identifying optimal PROTAC candidates, this approach has the potential to accelerate the development of targeted therapies and reduce the time and costs associated with drug discovery.

Pancreatic cancer remains one of the most lethal malignancies, with limited therapeutic options and a 5-year survival rate below 12%. MYC, a key driver of pancreatic cancer progression, remains a difficult target due to its lack of defined binding pockets. BRD4, an upstream regulator of MYC, has emerged as a viable target for indirect MYC suppression. However, despite the promise of BRD4 inhibitors, their rapid clearance, low potency, and dose-limiting toxicities have hampered clinical translation. Targeted protein degradation (TPD) using proteolysis-targeting chimeras (PROTACs) offers an alternative approach, leveraging endogenous degradation pathways to achieve sustained target depletion. PROTACs are heterobifunctional molecules that bind a protein of interest and an E3 ubiquitin ligase, promoting proximity-induced ubiquitination and subsequent proteasomal degradation. This strategy can overcome limitations of traditional small molecules, such as resistance mechanisms, lack of sustained inhibition, and difficulty in targeting undruggable proteins. While BRD4-targeting PROTACs show efficacy in cancer types other than pancreatic, their safety profiles remain a concern due to potential on-target effects in normal tissues. The selectivity of PROTACs is governed in part by the recruited E3 ligase, presenting an opportunity to enhance tumor specificity by leveraging E3 ligases differentially expressed in cancer versus normal tissues. Commonly targeted E3 ligases include VHL and CRBN; the ubiquitous, uniform expression profile of VHL limits cancer-cell selectivity, while CRBN expression is decreased in cancerous tissues compared to non-cancerous tissues and cell lines. To promote cancer-cell selectivity, we hypothesize that tuning the E3 ligase-recruiting moiety of a PROTAC to one that is enriched in pancreatic tumors may yield improved tumor selectivity and therapeutic index. To explore this, we have synthesized a panel of novel BRD4-directed PROTACs based on the ARV-771 scaffold, each recruiting a different E3 ligase (CRBN, DCAF16, RNF114). These degraders have been evaluated in pancreatic cancer cell lines and non-cancerous HEK-293 cells, where we observe differential cytotoxic responses and varying potential safety indices, which could be suggestive of E3-dependent effects. Notably, SJM-012, which targets CRBN, had decreased efficacy and safety as compared to the other PROTACs, in agreement with the lower expression of that E3 ligase system in cancers. Work is ongoing examining changes in BRD4 and MYC protein expression over time across pancreatic cancer cell lines. In future studies, we plan to assess target binding and ternary complex formation, as well as evaluate the physicochemical properties. We also intend to explore live-cell degradation and target engagement using NanoBRET and HiBiT-based approaches. Together, these efforts aim to define how E3 ligase selection influences the potency, selectivity, and therapeutic index of degraders in pancreatic cancer, ultimately guiding the rational design of next-generation tumor-selective degraders. Citation Format: Steven J. McKay, Sarah I. Korn, Tracy A. Brooks, Tony D. Davis. Breaking BRD4: Tailoring E3 ligase recruitment for tumor-selective MYC suppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A112.

Proteolysis targeting chimeras (PROTACs) technology has emerged as a novel therapeutic paradigm in recent years. PROTACs are heterobifunctional molecules that degrade target proteins by hijacking the ubiquitin–proteasome system. Currently, about 20–25% of all protein targets are being studied, and most works focus on their enzymatic functions. Unlike small molecules, PROTACs inhibit the whole biological function of the target protein by binding to the target protein and inducing subsequent proteasomal degradation. PROTACs compensate for limitations that transcription factors, nuclear proteins, and other scaffolding proteins are difficult to handle with traditional small-molecule inhibitors. Currently, PROTACs have successfully degraded diverse proteins, such as BTK, BRD4, AR, ER, STAT3, IRAK4, tau, etc. And ARV-110 and ARV-471 exhibited excellent efficacy in clinical II trials. However, what targets are appropriate for PROTAC technology to achieve better benefits than small-molecule inhibitors are not fully understood. And how to rationally design an efficient PROTACs and optimize it to be orally effective poses big challenges for researchers. In this review, we summarize the features of PROTAC technology, analyze the detail of general principles for designing efficient PROTACs, and discuss the typical application of PROTACs targeting different protein categories. In addition, we also introduce the progress of relevant clinical trial results of representative PROTACs and assess the challenges and limitations that PROTACs may face. Collectively, our studies provide references for further application of PROTACs.

Expansion of targeted degradation by Gilteritinib-Warheaded PROTACs to ALK fusion proteins

A new series of therapeutic modalities resulting in degradation of target proteins, termed proteolysis targeting chimeras (PROTACs), hold significant therapeutic potential with possible prolonged pharmacodynamics, improved potency, and ability to target proteins previously thought of as "undruggable". PROTACs are heterobifunctional small molecules consisting of a target binding handle bridged via a chemical linker to an E3 ligase handle which recruit the E3 ligase and ubiquitin machinery to target proteins, resulting in subsequent ubiquitination and degradation of the target. With the generation of small molecule PROTAC compound libraries for drug discovery, it becomes essential to have sensitive screening technologies to rapidly profile activity and have assays which can clearly inform on performance at the various cellular steps required for PROTAC-mediated degradation. For PROTAC compounds, this has been particularly challenging using either biochemical or cellular assay approaches. Biochemical assays are highly informative for the first part of the degradation process, including optimization of compound binding to targets and interrogation of target:PROTAC:E3 ligase ternary complex formation, but struggle with the remaining steps; recruitment of ternary complex into larger active E3 ligase complexes, ubiquitination, and proteasomal degradation. On the other hand, cellular assays are excellent at determining if the PROTAC successfully degrades the target in its relevant setting but struggle as early development PROTAC compounds are often poorly cell-permeable given their high molecular weight. Additionally, if degradation is not observed in a cellular assay, it is difficult to deconvolute the reason why or at which step there was failure. In this review we will highlight the current approaches along with recent advances to overcome the challenges faced for cellular PROTAC screening, which will enable and advance drug discovery of therapeutic degradation compounds.

Development and Characterization of Third Generation MDM2 PROTACs for the Treatment of Acute Myeloid Leukemia Utilizing an Achiral Ligand for E3 Ligase Cereblon

Background: First-generation targeted protein degraders (TPDs) known as Proteolysis-Targeting Chimeras (PROTACs) are hetero-bifunctional molecules that simultaneously bind both a target protein and an E3 ubiquitin ligase, marking the target for proteasomal degradation. PROTACS have shown promise in Phase I studies but are limited by their large size, long development timelines, and emergence of resistance. We sought to overcome these limitations with next-generation TPDs known as CURE-PROs. These small molecule monomers self-assemble and dimerize inside the cancer cell in the presence of the target oncoprotein and E3 ligase to form a quaternary complex that facilitates ubiquitination of the target protein. Due to their small size, the individual CURE-PRO monomer ligands have superior oral and tumor cell absorption and can be rapidly developed or modified utilizing a large library of monomers and linker components. We selected BRD4 as our initial oncoprotein target to validate in vitro and in vivo activity of CURE-PROs. Methods: Twenty-seven CURE-PRO monomers consisting of derivatives of the BRD4 protein ligands, JQ1 and OXT-015, and ligand monomers that bind E3 ligases (Cereblon, VHL, and MDM2) were combined to create 170 unique CURE-PRO dimers. These combinations were tested in cancer cell lines, in the presence/absence of proteasome inhibitors, and BRD4 protein degradation was assessed by Western Blot. CURE-PRO-mediated degradation of BRD4 was also evaluated in nude mice with bilateral human cancer xenografts using three doses over eight hours. A parallel arm using a BRD4 PROTAC (ARV-825) served as a positive control. The tumors were excised 1, 2, 4, 16 and 40 hours after the last dose and protein expression was analyzed by Western Blot. Results: Cell screening identified 49 CURE-PRO combinations, consisting of different E3 ligase partners and BRD4 ligands, that induced greater than 50% BRD4 degradation. BRD4 degradation occurred only in the presence of both CURE-PRO monomers and was dependent on the pairs forming a reversible dimer. Five CURE-PRO pairs reduced protein levels by more than 95%, with a half-maximal degradation concentrations (DC50) of 30-100 nM. Consistent with a PROTAC mechanism-of-action, CURE-PRO-induced degradation of BRD4 was inhibited by proteasome inhibitors. In the xenograft models, BRD4 protein levels were reduced to ~40% at 2-4 hours after exposure to a CURE-PRO pair, with full recovery by 40 hours. Conclusions: Several CURE-PRO pairs, using different E3 ligase partners, achieved BRD4 protein degradation in vitro comparable to PROTACs, with mechanism-of-action through the proteasome system. Our lead CURE-PRO pair significantly degraded BRD4 in a cancer xenograft mouse model. This work constitutes the first proof-of-mechanism of reversible self-assembling drug dimers for targeted protein degradation in vitro and in vivo. Future studies are planned to optimize degradation efficacy and identify if resistance emerges. Citation Format: Sarah F Giardina, Elena Valdambrini, Micheal Peel, Manny D Bacolod, Mace L Rothenberg, Richard B Lanman, J David Warren, Francis Barany. CURE-PROs: Proof-of-mechanism for the first reversible self-assembling targeted protein degraders [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B072.

Journey of Von Hippel-Lindau (VHL) E3 ligase in PROTACs design: From VHL ligands to VHL-based degraders