Heterobifunctional Molecules Research Articles

Abstract 5307: Critical platforms of PROTAC validation

Abstract In recent years, proteolysis targeting chimeric (PROTAC) technology has become an effective endogenous protein degradation tool in drug discovery. As a class of heterobifunctional small molecules, PROTACs recruit an E3 ubiquitin ligase to a given substrate protein, resulting in its targeted degradation through the endogenous ubiquitin-proteasome system (UPS). PROTAC consists of three chemical elements: a ligand that binds to a target protein, a ligand that binds to an E3 ubiquitin ligase, and a linker that connects the two ligands. Compared with traditional drug development, PROTAC technology possesses unique advantages including overcoming drug resistance and targeting the “undruggable”. Here we generated a kind of PROTAC target, like KRAS, BRD4, ESR1, associated cell lines to accelerate the discovery of new protein degradation drugs and the target protein degradation therapeutics by an optimized insertion system. In the beginning, we develop a system of different tag targeted cell lines, which can be easy to introduce HiBiT, Flag, GFP and also Halo Tag, et.al to target protein by CRISPaint or CRISPR Cas9 mediated HDR system. We also developed an arbitrary point mutation system based on the optimized PE3 mutation system, which can randomly modify any point mutation to simulate in vivo disease models in vitro. During this process, we have also optimized a series of validation system, like Targeted Protein Degradation (TPD), High-throughput flow cytometry (HTFC) and In-Cell Western (ICW) to shorten the detection time and improve detection stability. It is worth mentioning that we also develop a HiBiT and GFP dual-signal system, which can easily validate the target protein degradation and cell viability. We hope that our detection system can speed up the development of new drugs and contribute to human anti-tumor.Keywords: PROTAC, UPS, drug development, therapeutics Citation Format: Ruifeng Wang. Critical platforms of PROTAC validation [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 5307.

Abstract 3729: Discovery of orally bioavailable SMARCA2/4 dual degraders for treatment of acute myeloid leukemia

Abstract Background: The BAF (SWI/SNF) chromatin remodeling complex comprises of two mutually exclusive ATPases, SMARCA2 (BRM) and SMARCA4 (BRG1), that affect the mobilization and positioning of nucleosomes on DNA and thereby regulates important cellular functions including transcription, DNA recombination, DNA repair and chromosome decatenation during mitosis. SMARCA4 is frequently overexpressed in several types of cancers. Overexpression has been linked to increased proliferation and survival, as well as aggressive tumors and poor prognosis. SMARCA4 knockdown in these tumors lead to inhibition of proliferation and increased sensitivity to known chemotherapeutic agents, supporting the validity of targeting SMARCA4. Genetic silencing studies have established that the oncogenic activity of tumors lacking SMARCA4 is primarily driven by SMARCA2-containing residual SWI/SNF complex, suggesting the importance of dual inhibition of SMARCA2 and SMARCA4. While SMARCA4 is known to play a vital role in maintaining the oncogenic transcription program and driving proliferation in leukemia, the impact of dual SMARCA2 and SMARCA4 inhibition/degradation in acute myeloid leukemia (AML) is largely unexplored. Methods and Results: As part of the initial design plan, selective SMARCA2/4 Bromodomain inhibitors and specific ligands of several E3 ligases were chosen to arrive at different degrader designs. A choice of linkers and different exit vectors were considered to construct a variety of hetero bifunctional molecules. Our proprietary ternary complex modeling algorithm, ALMOND (ALgorithm for MOdeling Neosubstrate Degraders) helped in prioritizing the designs. Short listed compounds were synthesized and profiled in multiple cellular assays to understand their degradation potential. Several compounds that degrade SMARCA2, SMARCA4 & PBRM1 with pico molar DC50 were identified. These compounds have shown very potent anti-proliferative activity in both SMARCA2/4 proficient (MV-4-11, VCaP etc) and SMARCA4 mutant cell lines (SK-MEL-5 & RERF-LC-A1 etc). Further, potent compounds were optimized for their pharmacokinetic properties. Multiple lead compounds with low IV clearance and good oral bioavailability in rodents were identified. Advanced lead compounds are currently being evaluated in rodent tolerability and PK-PD experiments to select doses for the efficacy study. Conclusions: Highly potent degraders of SMARCA2, SMARCA4 & PBRM1 were identified by conjugating selective SMARCA2/4 Bromodomain inhibitors and several E3 ligase specific ligands. Further optimization of the linkers resulted in compounds with improved pharmacokinetic profile and very good oral bioavailability in rodents. Highly potent and orally available degraders of SMARCA2, SMARCA4 are efficacious in AML xenograft models and advanced profiling of candidate molecule is in progress. Citation Format: Chandrasekhar Abbineni, Leena Khare, Bilash Kuila, Abdul Rawoof Khaji, Dhaytadak Bhagwan Mahadeo, Sandeep Vitthal Dukare, Bhagya M S Kumar, Suraj T Gore, Vijay Kamal Ahuja, Amit A Dhudashiya, Raghavendra N R, Nagesh Gowda, Charamanna K B, Kiran Aithal B, Samiulla D S, Subhendu Mukherjee, Thomas Antony, Sanjeev Giri, Shekar Chelur, Kavitha Nellore, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Discovery of orally bioavailable SMARCA2/4 dual degraders for treatment of acute myeloid leukemia [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 3729.

Beyond Proteolysis-Targeting Chimeric Molecules: Designing Heterobifunctional Molecules Based on Functional Effectors.

In recent years, with the successful development of proteolysis-targeting chimeric molecules (PROTACs), the potential of heterobifunctional molecules to contribute to reenvisioning drug design, especially small-molecule drugs, has been increasingly recognized. Inspired by PROTACs, diverse heterobifunctional molecules have been reported to simultaneously bind two or more molecules and bring them into proximity to interaction, such as ribonuclease-recruiting, autophagy-recruiting, lysosome-recruiting, kinase-recruiting, phosphatase-recruiting, glycosyltransferase-recruiting, and acetyltransferase-recruiting chimeras. On the basis of the heterobifunctional principle, more opportunities for advancing drug design by linking potential effectors to a protein of interest (POI) have emerged. Herein, we introduce heterobifunctional molecules other than PROTACs, summarize the limitations of existing molecules, list the main challenges, and propose perspectives for future research directions, providing insight into alternative design strategies based on substrate-proximity-based targeting.

Comparative analysis of NX-2127 and ibrutinib in treating B-cell malignancies

Oncology is an ever-changing field of medicine with the constant development of innovative treatments. Small-molecule drugs have entered the spotlight in the past few decades for their efficacy, selectivity, and ability to target intracellular proteins. Small-molecule inhibitors (SMIs) are small-molecule drugs that inhibit proteins involved in tumour growth [1,2]. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that degrade proteins involved in tumour growth [3,4]. Both treatments have revolutionized the field of oncology as they have proven to be more beneficial than traditional treatments.

Selective IRAK4 degradation, not kinase inhibition, blocks TLR-activated NF-Kb and p38 Signaling leading to broad cytokine inhibition

Abstract IL-1R/TLR activation plays a central role in the pathophysiology of multiple autoimmune and inflammatory diseases via the Myddosome that is dependent on both the kinase and scaffolding functions of Interleukin-1 receptor associated kinase 4 (IRAK4). Therefore, hetero-bifunctional molecules that selectively target IRAK4 for degradation and elimination by the ubiquitin proteasome pathway have the greatest potential to block IL-1R/TLR signaling, including NF-kb activation and cytokine production. To interrogate downstream signaling, phosphorylation events were monitored in monocytes and B cells following TLR7/8 or TLR9 activation, respectively. IRAK4 degradation, but not kinase inhibition, inhibited NF-kb p65 activation and phosphorylation of p38 pathway members by TLR agonists in both cell types. PBMCs pretreated with an IRAK4 degrader and then stimulated with the TLR7/8 agonist, R848, exhibited significantly broader inhibition of cytokines (IL-6, TNFa, IL-8 and IL-1b) compared to those pretreated with a selective IRAK4 kinase inhibitor. Compound washout experiments demonstrated a sustained effect of IRAK4 degrader on both target pharmacodynamics and cytokine inhibition that was differentiated from IRAK4 kinase inhibition. Overall, through removal of both scaffolding and kinase functions, IRAK4 degradation demonstrated greater activity compared to kinase inhibition across multiple TLR stimuli and cell types. These data highlight the potential for IRAK4 degraders to block multiple TLR signaling pathways across different immune cell types and thereby impact TLR/IL-1R-driven inflammatory and autoimmune diseases.

STAT3 degraders inhibit cellular activation, cytokine production, and Th17 development, resulting in inhibition of autoimmunity in the MOG-EAE model of CNS inflammation.

Abstract Signal transducer and activator of transcription 3 (STAT3), an “undruggable” transcription factor activated by a variety of receptor- and non-receptor tyrosine kinases, plays a critical role in activation pathways triggered by cytokines, hormones, and growth factors, which makes it an attractive target for the treatment of autoimmune and autoinflammatory disorders. Kymera has developed heterobifunctional molecules that selectively target STAT3 for degradation by the ubiquitin-proteasome pathway. These degraders have broad and potent activity in-vitro against TLR receptor and cytokine-induced activation of immune and stromal cells and attendant mediators release such as MCP-1 (CCL2) and collagen1a1. STAT3 degradation in CD4+ T cells potently inhibited Th17 development, decreasing IL-17, IL-22, IL-8/CXCL8, and TNFa production, with concomitant increase in Treg numbers, that was superior to JAK1/2 kinase inhibition. STAT3 degradation was subsequently evaluated in-vivo in murine models of autoimmune disease. In the Th17-driven inflammatory model of MOG-induced Experimental Autoimmune Encephalomyelitis (EAE), dose-dependent decrease of incidence, disease onset, clinical scores, and histopathology were observed in comparison to a S1P1 inverse agonist or steroid treatment. Ex-vivo MOG-stimulated cytokine release by leukocytes isolated from draining lymph node was also robustly inhibited. These data demonstrate the broad activity of STAT3 degradation in alleviating autoimmune inflammation in systems relevant to human disease.

Linkers as Game-changers in PROTAC Technology: Emphasizing General Trends in PROTAC Pharmacokinetics for their Rational Design

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules that act as degraders. They selectively remove disease-associated proteins by hijacking the Ubiquitin-Proteasome System (UPS). Chemically, they consist of three parts: an E3 ligase ligand, a target of interest (TOI) ligand, and a linker, which connects the two moieties. The rapid expansion of PROTAC Technology as an innovative therapeutic modality in cancer fostered the drug discovery effort to optimize their physicochemical properties. Due to their large size, their features are far from the traditional ‘drug-like’ properties. This short review highlights some of the structural modifications in the linker component to optimize the PROTAC Drug Metabolism and Pharmacokinetics (DMPK) profile. In particular, we discussed aspects related to solubility, cell permeability, active transporters efflux and, metabolic stability.

Advances in the Design and Development of PROTAC-mediated HDAC Degradation.

Due to developments in modern chemistry, previously uundruggable substrates are now targetable thanks to selective degradation using the ubiquitin-proteasomal degradation system. PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules designed specifically to degrade target proteins. They are of significant interest to industry and academia as they are highly specific and can target previously undruggable target proteins from transcription factors to enzymes. More than 15 degraders are expected to be evaluated in clinical trials by the end of 2021. Herein, we describe recent advances in the design and development of PROTAC-mediated degradation of histone deacetylases (HDACs). PROTAC-mediated degradation of HDACs can offer some significant advantages over direct inhibition, such as the use of substoichiometric doses and the potential to disrupt enzyme-independent HDAC function. We discuss the potential implication of the degradation of HDACs in comparison with HDAC knockout studies. Along with the selection of HDAC inhibitors and E3 ligase ligands for the design of PROTACs. The potential utility of HDAC PROTACs in various disease pathologies from cancer to inflammation to neurodegeneration is driving the interest in this field.

Heterobifunctional molecules tackle targeted protein dephosphorylation

Recently, Chen and colleagues reported the development of phosphatase-targeting chimeric molecules (PhosTACs), heterobifunctional small molecules that promote targeted, proximity-induced protein dephosphorylation. This strategy represents an innovative approach to selectively manipulate phosphoprotein function and provides proof-of-concept for a new class of bifunctional small molecules in the chemical biologist's toolbox.

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

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

Expanding the landscape of E3 ligases for targeted protein degradation

Targeted protein degradation (TPD) is a rapidly developing field in chemical biology and drug discovery. Various TPD modalities have emerged, with proteolysis-targeting chimeras (PROTACs) being the most well-developed at present. In PROTACs, a heterobifunctional molecule is used to recruit an E3 ligase to degrade a protein of therapeutic interest. Most of the PROTAC candidates that have been developed thus far use either CRBN or VHL as the hijacked E3 ligase, which poses several limitations. In order to overcome these limitations and furthermore realize the full potential of TPD as a therapeutic modality, the field will need to unlock additional E3 ligases. This review will therefore present 11 alternative E3 ligases for TPD. It will also describe some of the ongoing platform development that is facilitating the discovery of additional E3 ligases for TPD.

PROTACs: past, present and future.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules consisting of one ligand that binds to a protein of interest (POI) and another that can recruit an E3 ubiquitin ligase. The chemically-induced proximity between the POI and E3 ligase results in ubiquitination and subsequent degradation of the POI by the ubiquitin-proteasome system (UPS). The event-driven mechanism of action (MOA) of PROTACs offers several advantages compared to traditional occupancy-driven small molecule inhibitors, such as a catalytic nature, reduced dosing and dosing frequency, a more potent and longer-lasting effect, an added layer of selectivity to reduce potential toxicity, efficacy in the face of drug-resistance mechanisms, targeting nonenzymatic functions, and expanded target space. Here, we highlight important milestones and briefly discuss lessons learned about targeted protein degradation (TPD) in recent years and conjecture on the efforts still needed to expand the toolbox for PROTAC discovery to ultimately provide promising therapeutics.

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity.

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.

Selective STAT3 Degraders Dissect Peripheral T-Cell Lymphomas Vulnerabilities Empowering Personalized Regimens

Introduction: Peripheral T-cell lymphomas (PTCLs) include heterogeneous entities of rare and aggressive neoplasms. The improved understanding of the biological/molecular mechanisms driving T-cell transformation and tumor maintenance has powerfully propelled new therapeutic programs. However, despite this progress, PTCLs remain an unmet medical need. Recurrent aberrations and the deregulated activation of distinct signaling pathways have been mapped and linked to selective subtypes. The JAK/STAT signaling pathway's deregulated activation plays a pathogenetic role in PTCL, including ALCL subtypes. STATs regulate the differentiation/phenotype, survival and cell-growth, metabolism, and drug resistance of T-cell lymphomas as well as host immunosuppressive microenvironments. Although many drugs' discovery programs were launched, a plethora of compounds has failed.Methods: We have discovered heterobifunctional molecules by an iterative medicinal chemistry SAR campaign that potently and selectively degrade STAT3 in a proteasome-dependent manner. Conventional PTCL cell lines and Patient Derived Tumor Xenograft (PDTX) and/or derived cell lines (PDTX-CL), carrying either WT- or mutated-STAT3, were exposed to increasing amounts (50nM⇒5µM) of STAT3-degraders. Proteins and mRNA transcripts (2⇒144hrs) were assessed by deep-proteomics and paired-end RNA sequencing, combined with WB/flow cytometry and qRT-PCR. Cell-titer-glo, cell titer blue, Annexin-V and S-cell cycle analyses were used as readouts. Chromatin accessibility, STAT3 DNA binding, 3D chromosomal architecture reorganization and 5-hmC profiling were assessed by ATACseq, CHIPseq and Hi-C and H3K27ac Hi-CHIP and mass-spectrometry. Drug testing/discovery combinations (96-well-plate) were performed using a semi-automated flow-cytometry. A battery of PTCL PDTX models were tested in pre-clinical trials.Results: Treatment of ALK+ ALCL (SU-DHL1) led to the rapid (~6hrs.) and profound down-regulation of STAT3 followed by the loss of canonical STAT3-regulated proteins (SOCS3, MYC, Granzyme B, GAS1, and IL2RA), without appreciable changes for other STAT family members (STAT1, STAT5b). In vitro, cytoplasmic, nuclear, and mitochondrial STAT3 downregulation was maintained up to 144 hrs. Loss of STAT3 in ALK+/- ALCL and BIA-ALCL cells was associated with major transcriptional changes (7116-10615 and 15114 DEGs in ALK- and ALK+ ALCL, respectively), underscoring public/shared as well as private time-dependent signatures. Main down-regulated pathways included JAK-STAT, MAPK, NF-kB, PI3K, TGFb, and TNFa. Comparison of STAT3 shRNA (ALK+ ALCL) and STAT3 degrader (ALK-/ALK+ ALCL) signatures demonstrated a substantial and concordant gene modulation (24hrs) among all models with the highest overlaps between ALK+ ALCL (Figure 3). To identify direct STAT3 gene targets, we analyzed CHIPseq peaks and predicted bindings sites, demonstrating that canonical genes, i.e., SOCS3, Granzyme B, GAS1, IL2RA, STAT3, and CD30, were significantly downregulated. Conversely, CD58, CD274, and MCH-I/II were upregulated at late time points. By mapping the STAT3 binding sites in ALK+ and ALK- ALCL, we have identified 1077 and 2763 STAT3 peaks within promoter/5‘-/3‘- and distant intergenic regions, corresponding to both coding and non-coding genes. Therapeutically, in vitro treatments led to cell cycle arrest and profound growth inhibition, and over time increased cell death of PTCL cells, including ALCL. Accordingly, growth inhibition of ALCL xenograft and PDTX tumors was also achieved (Figure 2). To identify drugs that could synergize withSTAT3-degrader activity, we tested a compound library (40) targeting pro-tumorigenic PTCL pathways as well as FDA-approved compounds. Ongoing studies are in progress.Conclusion: We have discovered selective STAT3 degraders which control PTCL growth. STAT3 degraders are powerful tools to define the STAT3 pathogenetic mechanisms and dissect genes/pathways to be targeted for T-cell lymphoma eradication. These data provide additional rationale for testing STAT3 degraders in the clinic for the treatment of aggressive malignancies including PTCL/ALCL. [Display omitted] DisclosuresYang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: Owkin: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Johnson and Johnson: Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; One Three Biotech: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Horwitz: Affimed: Research Funding; Aileron: Research Funding; ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Inhibitors, PROTACs and Molecular Glues as Diverse Therapeutic Modalities to Target Cyclin-Dependent Kinase.

Simple SummaryCyclin-dependent kinases (CDKs) are rich and viable therapeutic targets for various cancers. The emergence of event-driven pharmacology as an alternative to occupancy-driven pharmacology has begun to address the challenges associated with selectively targeting CDKs. In this review article, we summarize the CDK inhibitors that are currently in clinical trials. In addition, we provide an overview of PROTAC- and molecular glue-based strategies to modulate CDK function.The cyclin-dependent kinase (CDK) family of proteins play prominent roles in transcription, mRNA processing, and cell cycle regulation, making them attractive cancer targets. Palbociclib was the first FDA-approved CDK inhibitor that non-selectively targets the ATP binding sites of CDK4 and CDK6. In this review, we will briefly inventory CDK inhibitors that are either part of over 30 active clinical trials or recruiting patients. The lack of selectivity among CDKs and dose-limiting toxicities are major challenges associated with the development of CDK inhibitors. Proteolysis Targeting Chimeras (PROTACs) and Molecular Glues have emerged as alternative therapeutic modalities to target proteins. PROTACs and Molecular glues utilize the cellular protein degradation machinery to destroy the target protein. PROTACs are heterobifunctional molecules that form a ternary complex with the target protein and E3-ligase by making two distinct small molecule–protein interactions. On the other hand, Molecular glues function by converting the target protein into a “neo-substrate” for an E3 ligase. Unlike small molecule inhibitors, preclinical studies with CDK targeted PROTACs have exhibited improved CDK selectivity. Moreover, the efficacy of PROTACs and molecular glues are not tied to the dose of these molecular entities but to the formation of the ternary complex. Here, we provide an overview of PROTACs and molecular glues that modulate CDK function as emerging therapeutic modalities.

Targeted Protein Acetylation in Cells Using Heterobifunctional Molecules.

Protein acetylation is a central event in orchestrating diverse cellular processes. However, current strategies to investigate protein acetylation in cells are often nonspecific or lack temporal and magnitude control. Here, we developed an acetylation tagging system, AceTAG, to induce acetylation of targeted proteins. The AceTAG system utilizes bifunctional molecules to direct the lysine acetyltransferase p300/CBP to proteins fused with the small protein tag FKBP12F36V, resulting in their induced acetylation. Using AceTAG, we induced targeted acetylation of a diverse array of proteins in cells, specifically histone H3.3, the NF-κB subunit p65/RelA, and the tumor suppressor p53. We demonstrate that targeted acetylation with the AceTAG system is rapid, selective, reversible and can be controlled in a dose-dependent fashion. AceTAG represents a useful strategy to modulate protein acetylation and should enable the exploration of targeted acetylation in basic biological and therapeutic contexts.

Structural Characterization of Degrader-Induced Ternary Complexes Using Hydrogen-Deuterium Exchange Mass Spectrometry and Computational Modeling: Implications for Structure-Based Design.

The field of targeted protein degradation (TPD) has grown exponentially over the past decade with the goal of developing therapies that mark proteins for destruction leveraging the ubiquitin-proteasome system. One common approach to achieve TPD is to employ a heterobifunctional molecule, termed as a degrader, to recruit the protein target of interest to the E3 ligase machinery. The resultant generation of an intermediary ternary complex (target-degrader-ligase) is pivotal in the degradation process. Understanding the ternary complex geometry offers valuable insight into selectivity, catalytic efficiency, linker chemistry, and rational degrader design. In this study, we utilize hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify degrader-induced protein-protein interfaces. We then use these data in conjunction with constrained protein docking to build three-dimensional models of the ternary complex. The approach was used to characterize complex formation between the E3 ligase CRBN and the first bromodomain of BRD4, a prominent oncology target. We show marked differences in the ternary complexes formed in solution based on distinct patterns of deuterium uptake for two degraders, CFT-1297 and dBET6. CFT-1297, which exhibited positive cooperativity, altered the deuterium uptake profile revealing the degrader-induced protein-protein interface of the ternary complex. For CFT-1297, the ternary complexes generated by the highest scoring HDX-constrained docking models differ markedly from those observed in the published crystal structures. These results highlight the potential utility of HDX-MS to provide rapidly accessible structural insights into degrader-induced protein-protein interfaces in solution. They further suggest that degrader ternary complexes exhibit significant conformation flexibility and that biologically relevant complexes may well not exhibit the largest interaction surfaces between proteins. Taken together, the results indicate that methods capable of incorporating linker conformation uncertainty may prove an important component in degrader design moving forward. In addition, the development of scoring functions modified to handle interfaces with no evolved complementarity, for example, through consideration of high levels of water infiltration, may prove valuable. Furthermore, the use of crystal structures as validation tools for novel degrader methods needs to be considered with caution.

Discovery of Potent and Selective CDK9 Degraders for Targeting Transcription Regulation in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is highly aggressive with very limited treatment options due to the lack of efficient targeted therapies and thus still remains clinically challenging. Targeting transcription-associated cyclin-dependent kinases to remodel transcriptional regulation shows great promise in cancer therapy. Herein, we report the synthesis, optimization, and evaluation of new series of heterobifunctional molecules as highly selective and efficacious CDK9 degraders, enabling potent inhibition of TNBC cell growth and rapidly targeted degradation of CDK9. Moreover, the most potent CDK9 degrader (compound 45) induces cell apoptosis in vitro and inhibits tumor growth in the MDA-MB-231 TNBC model. Furthermore, the RNA-seq, immunohistochemistry assays demonstrate that the CDK9 degrader downregulates the downstream targets, such as MYC, at the transcriptional level, resulting apoptosis in TNBC cells. Our work establishes that 45 is a highly potent and efficacious CDK9 degrader for targeting transcription regulation, which represents an effective strategy and great potential as a new targeted therapy for TNBC.

Abstract 1348: Targeted degradation of PARP14 Using a heterobifunctional small molecule

Abstract PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types and has been shown to promote the pro-tumor M2 polarization of macrophages and support Th2/Th17 signaling in models of allergic airway disease. PARP14 is a large 203 kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates, three macrodomains that bind mono-ADP-ribose, a WWE domain that serves as a binding module for poly-ADP-ribose, and an RNA recognition motif. We have previously shown that the potent and reversible enzymatic inhibitor, RBN012759 (IC50 < 0.003 μM, 300-fold selective over monoPARPs, > 1,000-fold selective over polyPARPs), links PARP14 catalytic inhibition with suppression of the antitumor immune response in human primary macrophages and human kidney cancer explants. While this catalytic inhibitor of PARP14 was able to suppress IL-4-driven pro-tumor gene expression in macrophages, it is unknown what roles the non-enzymatic biomolecular recognition motifs play in the biological function of PARP14. To further understand this, we describe a heterobifunctional small molecule, RBN012811, based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+-binding site and recruits the E3 ligase cereblon to ubiquitinate PARP14 and selectively target it for degradation. RBN012811 has a IC50 of 0.01 μM against PARP14 in a biophysical assay and is at least 200-fold selective over all other PARPs. In KYSE-270 cancer cells, RBN012811 has a half-maximal degradation concentration (DC50) of 0.005 μM and it does not cause degradation of other PARP enzymes. In human primary macrophages PARP14 degradation by RBN012811 led to a dose-dependent decrease of IL-10 release induced by IL-4 stimulation. Our data demonstrates that RBN012811 is a useful tool to enable further exploration of the role of PARP14 in inflammation and cancer. Citation Format: Tim Wigle, Yue Ren, Jennifer Molina, Danielle Blackwell, Laurie Schenkel, Kerren Swinger, Anne Cheug, Ryan Abo, Elena Minissale, Alvin Lu, Christina Majer, William Church, Bryan Dorsey, Mario Niepel, Nicholas Perl, Kristy Kuplast-Barr, Kristen McEachern, Melissa Vasbinder, Heike Keilhack, Kevin Kuntz. Targeted degradation of PARP14 Using a heterobifunctional small molecule [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1348.

Abstract 44: The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer

Abstract ARV-471, an estrogen receptor (ER) alpha PROTAC® protein degrader, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ~ 1 nM. PROTAC® mediated ER degradation decreases the expression of classically regulated ER-target genes and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant anti-tumor activity of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (131% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also significantly reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC® protein degrader. These preclinical data supported the clinical development of ARV-471 for the treatment of patients with breast cancer. The discovery, chemical structure and initial clinical data of ARV-471 will be presented. Citation Format: Lawrence B. Snyder, John J. Flanagan, Yimin Qian, Sheryl M. Gough, Monica Andreoli, Mark Bookbinder, Gregory Cadelina, John Bradley, Emma Rousseau, Julian Chandler, Ryan Willard, Jennifer Pizzano, Craig M. Crews, Andrew P. Crew, John Houston, Marcia Dougan Moore, Ron Peck, Ian Taylor. The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 44.