Potent Proteolysis Targeting Chimeras Research Articles

Discovery of a potent PROTAC degrader for RNA demethylase FTO as antileukemic therapy

The fat mass and obesity-associated protein (FTO) is an RNA demethylase required for catalytic demethylation of N6-methyladenosine (m6A); it is highly expressed and functions as an oncogene in acute myeloid leukemia (AML). Currently, the overarching objective of targeting FTO is to precisely inhibit the catalytic activity. Meanwhile, whether FTO degradation also exerts antileukemic effects remains unknown. Herein, we designed the first FTO-targeting proteolysis targeting chimera (PROTAC) degrader QP73 using our FTO inhibitor Dac85—which potently inhibits FTO demethylation in AML cell lines—as a warhead. Notably, QP73 significantly induced FTO degradation in a time-, dose-, and ubiquitin–proteasome system-dependent manner and had superior antiproliferative activities to the FTO inhibitor Dac85 in various AML cell lines. Moreover, QP73 treatment significantly increased m6A modification on mRNA, promoted myeloid differentiation, and induced apoptosis of AML cells. Quantitative proteomics analysis showed that QP73 induced complete FTO degradation, upregulating RARA and ASB2 abundance and downregulating CEBPA, MYC, PFKP, and LDHB levels in AML cells. Lastly, QP73 exhibited antileukemic activity by increasing m6A modification and decreasing FTO levels in xenograft AML tumors. This proof-of-concept study shows that FTO-targeting PROTAC degraders can regulate the FTO signaling pathway and have potential antileukemia applications.

Discovery of Selective Proteolysis-Targeting Chimera Degraders Targeting PTP1B as Long-Term Hypoglycemic Agents.

PTP1B, a promising target for insulin sensitizers in type 2 diabetes treatment, can be effectively degraded using proteolysis-targeting chimera (PROTAC). This approach offers potential for long-acting antidiabetic agents. We report potent bifunctional PROTACs targeting PTP1B through the E3 ubiquitin ligase cereblon. Western blot analysis showed significant PTP1B degradation by PROTACs at concentrations from 5 nM to 5 μM after 48 h. Evaluation of five highly potent PROTACs revealed compound 75 with a longer PEG linker (23 atoms), displaying remarkable degradation activity after 48 and 72 h, with DC50 values of 250 nM and 50 nM, respectively. Compound 75 induced selective degradation of PTP1B, requiring engagement with both the target protein and CRBN E3 ligase, in a ubiquitination and proteasome-dependent manner. It significantly reduced blood glucose AUC0-2h to 29% in an oral glucose tolerance test and activated the IRS-1/PI3K/Akt signaling pathway in HepG2 cells, showing promise for long-term antidiabetic therapy.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

Abstract 4514: Discovery of potent and orally bioavailable degraders of HPK1 based on a novel HPK1 binder

Abstract Background: Proteolysis targeting chimeras (PROTACs) are novel structures designed to target a protein of interest (POI) for ubiquitination and degradation, leading to the selective reduction in the expression levels of the POI. Hematopoietic progenitor kinase 1 (HPK1) is considered a promising target for tumor immunotherapy. PROTAC-involved disruption of HPK1 protein activity enhanced the treatment efficacy of CAR-T-cell-based immunotherapies in different solid tumors and mouse models. Thus, targeting HPK1 by PROTAC to restore T-cell activity is an attractive approach to induce a greater immune responses in cancer. Results: We designed and synthesized a series of HPK1 degraders by tethering our previously discovered novel HPK1 inhibitor with CRBN binder through various linkage. The most potent PROTAC effectively degrades HPK1 proteins in the Ramous cell in a dose-dependent manner and achieves DC50 value <50 nM. The maximum degradation (D max ) values achieved are >90%. Our PROTAC showed EC50 value <100 nM for IL-2 and IFNγ production. PK studies revealed a low clearance, high plasma exposure and oral bioavailability in mice and rats. Furthermore, our PROTAC is shown to induce robust and statistically significant tumor growth inhibition in the MC38 and CT26 syngeneic model with increased T cell signatures observed within the tumor. The PROTAC in combination with anti-PD1 also resulted in robust anti-tumor activity with TGI > 90%. The 14 days DRF experiment shows that the compound has a great safety window(>200 folds). Conclusion: We show here that a potent oral HPK1 degrader demonstrates strong immune cell activation and robust anti-tumor activity in mouse syngeneic tumor models, as a single agent and in combination with anti-PD1. Further evaluation of these potent degraders in additional in vivo studies will continue to build upon our mechanistic understanding of HPK1 degradation as a novel immunomodulatory approach for anti-tumor immunity. Citation Format: Zhimin Zhang, Mengqiang Wu, Ling Wang, Xi Yang, Zhiping Zhang, Liubin Guo, Hao Pan, Mengting Zhao, Linli Wang, Sirui Liu, Zhao Dong, Chunhua Jiang, Haowen Zheng, Dongzhou Liu. Discovery of potent and orally bioavailable degraders of HPK1 based on a novel HPK1 binder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4514.

High-Throughput Miniaturized Synthesis of PROTAC-Like Molecules.

The development of miniaturized high-throughput in situ screening platforms capable of handling the entire process of drug synthesis to final screening is essential for advancing drug discovery in the future. In this study, an approach based on combinatorial solid-phase synthesis, enabling the efficient synthesis of libraries of proteolysis targeting chimeras (PROTACs) in an array format is presented. This on-chip platform allows direct biological screening without the need for transfer steps. UV-induced release of target molecules into individual droplets facilitates further on-chip experimentation. Utilizing a mitogen-activated protein kinase kinases (MEK1/2) degrader as a template, a series of 132 novel PROTAC-like molecules is synthesized using solid-phase Ugi reaction. These compounds are further characterized using various methods, including matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging, while consuming only a few milligrams of starting materials in total. Furthermore, the feasibility of culturing cancer cells on the modified spots and quantifying the effect of MEK suppression is demonstrated. The miniaturized synthesis platform lays a foundation for high-throughput in situ biological screening of potent PROTACs for potential anticancer activity and offers the potential for accelerating the drug discovery process by integrating miniaturized synthesis and biological steps on the same array.

A close-up shot of protein-protein docking, from experiment to theory and reverse with the PROTAC performers

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. Communicated by Ramaswamy H. Sarma

Entdeckung eines potenten PROTAC ermöglicht die gezielte Ausschaltung der Gerüstfunktionen von FKBP51

AbstractDas FK506‐bindende Protein 51 (FKBP51) stellt ein vielversprechendes Wirkstoffziel zur Behandlung von verschiedenen Krankheiten dar, darunter Depression, chronischer Schmerz und Fettleibigkeit. Bisherige FKBP51‐gerichtete Wirkstoffe waren auf die Blockade der FK506‐Bindestelle beschränkt, wodurch Kernfunktionen von FKBP51 jedoch nicht beeinträchtigt werden. Hier präsentieren wir die Entwicklung des ersten FKBP51 Proteolysis Targeting Chimeras (PROTAC), der den Abbau von FKBP51 ermöglicht und damit auch die Gerüstfunktion von FKBP51 ausschalten kann. Die initiale Synthese von 220 FKBP‐gerichteten PROTACs ergab eine Vielzahl aktiver PROTACs für FKBP12, sechs für FKBP51 und keinen für FKBP52. Die Strukturanalyse eines binären FKBP12:PROTAC‐Komplexes offenbarte die molekulare Grundlage für negative Kooperativität. Die Linker‐Optimierung eines FKBP51 PROTACs der ersten Generation führte zur Entwicklung von SelDeg51 mit verbesserter zellulärer Aktivität, Selektivität und hoher Kooperativität. Die Struktur des ternären FKBP51:SelDeg51:VCB Komplexes zeigte, wie SelDeg51 durch Dimerisierung von FKBP51 und VHL Kooperativität herstellt, die dem Bindungsmodus von molekularen Klebern ähnelt. SelDeg51 baut FKBP51 auf effiziente Weise in Zellen ab und reaktiviert den GR‐Signalweg, was die erhöhte Wirksamkeit des Proteinabbaus im Vergleich zur klassischen FKBP51‐Besetzung demonstriert.

PROTAC derivatization of natural products for target identification and drug discovery: Design of evodiamine-based PROTACs as novel REXO4 degraders

IntroductionNatural products (NPs) play a crucial role in the development of therapeutic drugs. However, it is still highly challenging to identify the targets of NPs. Besides, NPs usually exert their pharmacological activities via acting on multiple targets or pathways, which also poses great difficulties for the target identification of NPs. ObjectivesInspired by our continuous efforts in designing drug-like protein degraders, this study introduced a successful example for the target identification and drug discovery of natural products evodiamine by employing PROTAC technology. MethodsTaking advantages of proteolysis targeting chimera (PROTAC), herein an integrated strategy combining PROTAC derivatization, quantitative proteomic analysis and binding affinity validation was developed for target identification and drug discovery of antitumor NP evodiamine. ResultsIn this study, both highly potent PROTACs and negative controls were designed for quantitative proteomic analysis. Furthermore, REXO4 was confirmed as a direct target of 3-fluoro-10-hydroxylevodiamine, which induced cell death through ROS. In addition, the PROTAC 13c effectively degraded REXO4 both in vitro and in vivo, leading to potent antitumor activities and reduced toxic side effects. ConclusionIn summary, we developed an integrated strategy for the target identification and drug discovery of NPs, which was successfully applied to the PROTAC derivatization and target characterization of evodiamine. This proof-of-concept study highlighted the superiority of PROTAC technology in target identification of NPs and accelerated the process of NPs-based drug discovery, exhibiting broad application in NP-based drug development.

Abstract 5018: PROTAC degraders of CDK8/CDK19 Mediator kinases potently suppress multiple myeloma proliferation

Abstract CDK8 and CDK19 are kinase components of the CDK module that comprises CDK8 or CDK19 together with their binding partner cyclin C (CCNC) and two other proteins; the CDK module is associated with transcriptional Mediator complex. CDK8/19 Mediator kinases potentiate transcription induced by different signals, regulate protein levels of Mediator complex components and protect CCNC from proteolytic degradation; the latter activity is exerted in a kinase-independent manner. CDK8/19 kinase inhibitors have entered clinical trials for solid tumors and leukemias. To extend the effects of CDK8/19 inhibition and to suppress their kinase-independent activities, we have developed three series of PROteolysis TArgeting Chimeras (PROTACs) based on different selective inhibitors of CDK8/19 kinases, connected via different linkers to a moiety binding cereblon (CRBN) E3 ligase. The most potent PROTACs degraded both CDK8 and CDK19 in different cell types with DC50s of 10-20 nM. RNA-Seq analysis of 293 cells treated with a CDK8/19 kinase inhibitor or PROTAC showed that the bulk of the PROTAC’s transcriptomic effects matched the effects of the kinase inhibitor. In contrast to the kinase inhibitors, CDK8/19-degrading PROTACs also induced CCNC degradation. Cancer Dependency Map (DepMap) analysis revealed that multiple myelomas (MM), where CRBN is an established therapeutic target, show greater dependency on CCNC than most of the tumor cell lines. We have tested two strongly CCNC-dependent MM cell lines and one CCNC-independent MM line for sensitivity to different CDK8/19 kinase inhibitors and PROTACs. Kinase inhibitors showed low to moderate anti-proliferative activity in all three MM lines, whereas CDK8/19 PROTACs were an order of magnitude more potent than the kinase inhibitors in the two CCNC-dependent MM lines (IC50s of 20-30 nM) but not in the CCNC-independent line. The effects of CDK8/19-degrading PROTACs were stronger than those of the CRBN-targeting drug pomalidomide (approved for MM), a CRBN-binding PROTAC acting on androgen receptor or a CDK8 degradation-inactive PROTAC analog containing CDK8/19- and CRBN-binding moieties. These results suggest the potential of CDK8/CDK19-degrading PROTACs for the treatment of MM. Citation Format: Li Zhang, Jing Li, Charles E. Dowling, Eugenia V. Broude, Igor B. Roninson, Campbell McInnes, Mengqian Chen. PROTAC degraders of CDK8/CDK19 Mediator kinases potently suppress multiple myeloma proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5018.

Abstract 1685: Overcoming acquired resistance to PROTAC degraders

Abstract Background: Proteolysis-targeting chimera (PROTAC) technology has been widely investigated for cancer treatment and there have been several PROTAC degrader-based drugs used for clinical trials in the past few years. PROTAC degraders are able to target crucial proteins traditionally thought to be “undruggable”, such as transcriptional factors. Hence, there is increasing interest in these drugs due to their highly efficacy and low off-target toxicity. However, upon further investigation, it has been found that after a period of treatment with PROTAC degraders, cancer cells can acquire resistance to these compounds. Thus, it is of increasing importance to figure out the mechanisms of the resistance and to overcome the acquired resistance to these PROTAC degraders. In our study, we classified resistance mechanisms of prostate cancer cells to PROTAC degraders into two classes and identified factors that can induce them to preferentially develop specific resistance mechanisms. Methods and Results: AU-15330 is a PROTAC that specifically degrades the subunits (BRG1, PBRM1, BRM) of SWI/SNF complex. We treated 22rv1, a prostate cancer cell line, with different AU-15330 concentrations over a month to establish AU-15330-resistant cell lines. Whole exome sequencing (WES) analysis of these resistant cell lines found that the resistant cell lines that were developed through high-dose (1uM) AU-15330 treatment acquired multiple point mutations within or adjacent to the AU-15330 targeting bromodomain of BRG1. However, BRG1 point mutations were absent in the resistant cell lines that developed through lower concentration of AU-15330 treatment (100nM). Instead, RNA-seq analysis revealed that ABCB1 (also known as Multidrug Resistance Protein 1) expression was upregulated in cells that developed resistance to AU-15330 at 100nM. This was further confirmed by Western blot and qPCR. Next, we overexpressed ABCB1 in LNCaP and 22RV1, and found that overexpression of ABCB1 indeed made prostate cancer cells resistant to AU15330. In addition, we tested with several different potent PROTAC degraders (ARD616, ZBC260, etc) in ABCB1-expressing AU-15530 resistant cells, and found the ABCB1 expression drove the cells to become resistant to all tested PROATC degraders compared to the parental cells. Lastly, we demonstrated that Zosuquidar, an ABCB1 inhibitor, can overcome the ABCB1-mediated resistance to AU-15330 and other PROTAC degraders, suggesting that inhibiting ABCB1 might be a direct and effective strategy to restore PROTAC degrader’s potency when ABCB1-mediated drug resistance is developed. Conclusion: Our study found that the mechanisms of resistance of PROTAC degraders can vary and may be dependent on drug concentrations. Such concepts may inform future clinical decision-making regarding drug dosing. Citation Format: Tongchen He, Caleb Cheng, Abhijit Parolia, Alex Hopkins, Yuanyuan Qiao, Lanbo Xiao, Arul Chinnaiyan. Overcoming acquired resistance to PROTAC degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1685.

Identification of novel and potent PROTACs targeting FAK for non-small cell lung cancer: Design, synthesis, and biological study

The intracellular non-receptor tyrosine protein kinase Focal adhesion kinase (FAK) is a key signalling regulator, which mediates tumor survival, invasion, metastasis, and angiogenesis through its kinase catalytic functions and non-kinase scaffolding functions. Previous efforts have clarified that it is crucial to address both FAK kinase and scaffolding functions instead of just inhibiting FAK kinase activity because it may be insufficient to completely block FAK signaling. Proteolysis targeting chimera (PROTAC) technology is a method of targeting a specific protein and inducing its degradation in the cell, which can simultaneously eliminate both kinase-dependent enzymatic functions and scaffolding functions. In current study, we designed and synthesized a series of novel FAK PROTACs and the optimal PROTAC B5 exhibited potent FAK affinity with an IC50 value of 14.9 nM. Furthermore, in A549 cells, PROTAC B5 presented strong FAK degradation activity (86.4% degradation @ 10 nM), powerful antiproliferative activity (IC50 = 0.14 ± 0.01 μM) and inhibited cell migration and invasion in a concentration-dependent manner. Additionally, the in vitro preliminary drug-like properties evaluation of PROTAC B5 showed outstanding plasma stability and moderate membrane permeability. Together, current results provided a promising FAK PROTAC B5 as lead compound for cancer-related drug discovery and FAK-degradation functions exploration in biological systems.

Discovery of Potent PROTACs Targeting EGFR Mutants through the Optimization of Covalent EGFR Ligands.

Drug resistance caused by epidermal growth factor receptor (EGFR) mutation has largely limited the clinical use of EGFR tyrosine kinase inhibitors (EGFR-TKIs) for the treatment of non-small-cell lung cancer (NSCLC). Herein, to overcome the intractable problem of drug resistance, proteolysis targeting chimeras (PROTACs) targeting EGFR mutants were developed by optimizing covalent EGFR ligands. Covalent or reversible covalent pyrimidine- or purine-containing PROTACs were designed, synthesized, and evaluated. As a consequence, covalent PROTAC CP17, with a novel purine-containing EGFR ligand, was discovered as a highly potent degrader against EGFRL858R/T790M and EGFRdel19, reaching the lowest DC50 values among all reported EGFR-targeting PROTACs. Furthermore, CP17 exhibited excellent cellular activity against the H1975 and HCC827 cell lines with high selectivity. Mechanism investigation indicated that the lysosome was involved in the degradation process. Importantly, the covalent binding strategy was proven to be an effective approach for the design of PROTACs targeting EGFRL858R/T790M, which laid the practical foundation for further development of potent EGFR-targeting PROTACs.

28P Discovery of potent PROTAC degraders of KRASG12C based on a reversible non-covalent KRAS binder

Targeted protein degradation (TPD) using proteolysis targeting chimeras (PROTACs) has arisen as a powerful therapeutic modality for eliminating disease-causing proteins from cells. PROTACs employ heterobifunctional small molecules to chemically induce the proximity of target proteins with E3 ubiquitin ligases to ubiquitinate and degrade specific proteins via the proteasome. This study is to design and synthesize a series of novel PROTAC compounds targeting the KRAS G12C mutationwhich make up over 50% of all KRAS mutant LUAD (Lung Adenocarcinoma). In our study, targets mutant KRAS G12C has been designed and developed which is composed of reversible non-covalent KRAS binder linked to E3. We conducted the design, synthesis, and evaluation of PROTAC KRAS degraders using the VHL or cereblon ligands, and different classes of non-covalent KRAS binder. I We fully determined the optimal linker lengths and types needed in our PROTAC molecules for potent and effective KRAS degradation. Through extensive optimization of the linker and modifications of the KRAS binder portion of the compounds, we have discovered a set of exceptionally potent KRAS degraders with moderate membrane permeability and good plasma stability. More than 50 compounds were designed and synthesized using various linkers, E3 ligands and KRASG12C binders. Almost all compounds maintained good selectivity to KRASG12C.Our degrader series based on a non-covalent inhibitor and a ligand that recruits VHL successfully engaged VHL in cells, bound KRASG12C in vitro, induced VHL/ KRASG12C dimerization, and degraded KRASG12C in cells in a VHL-dependent manner. The representative compound induced KRASG12C ubiquitination and degradation with the DC50 value of 0.1 μM and Dmax value of 90%. Its discovered that the PROTAC degrader, the structure of the linker plays a key role in inducing degradation of the target protein. This current study demonstrated that conformational restriction of the linker in PROTAC KRASG12C degraders, coupled with modifications of KRASG12 binder portion are critical in the finding of potent KRASG12C degraders.

Profiling of diverse tumor types establishes the broad utility of VHL-based ProTaCs and triages candidate ubiquitin ligases

SummaryThe success of small molecule therapeutics that promotes degradation of critical cancer targets has fueled an intense effort to mimic this activity with bispecific molecules called PROTACs (proteolysis targeting chimeras). The simultaneous binding of PROTACs to a ligase and target can induce proximity-driven ubiquitination and degradation. VHL and CRBN are the two best characterized PROTAC ligases, but the rules governing their cellular activities remain unclear. To establish these requirements and extend them to new ligases, we screened a panel of 56 cell lines with two potent PROTACs that utilized VHL, MZ1, or CRBN, dBET1 to induce degradation of BRD4. With notable exceptions, MZ1 was broadly active in the panel whereas dBET1 was frequently inactive. A search for predictive biomarkers of PROTAC activity found that expression and mutation of VHL and CRBN were themselves predictors of PROTAC activity in the cell line panel.

Novel, highly potent PROTACs targeting AURORA-A kinase

The family of AURORA kinases is essential for cell cycle progression and dysregulation of AURORA-A in cancer led to a large number of clinical and pre-clinical inhibitors. However, ATP competitive AURORA-A inhibitors usually do not target non-catalytic functions that have also been identified as mechanisms promoting tumorigenesis. To target non-catalytic as well as catalytic functions, we developed a series of PROTACs (PROteolysis TArgeting Chimeras) based on the selective AURORA-A kinase inhibitor MK-5108 (VX-689) and the CEREBLON E3-ligase ligand thalidomide. The most potent PROTAC, JB301, had good physicochemical properties and cell penetration resulting in degradation of AURORA-A in leukemic cells at single digit nM concentration.

Noncovalent CDK12/13 dual inhibitors-based PROTACs degrade CDK12-Cyclin K complex and induce synthetic lethality with PARP inhibitor

Cyclin-dependent kinase 12 (CDK12) plays a crucial role in DNA-damage response gene transcription and has recently been validated as a promising target in cancer therapy. However, existing CDK12 inhibitors potently inhibit its closest isoform CDK13, which could cause potential toxicity. Therefore, the development of CDK12 inhibitors with isoform-selectivity against CDK13 continues to be a challenge. By taking advantage of the emerging PROteolysis-TArgeting Chimeras (PROTACs) approach, we have synthesized a potent PROTAC degrader PP-C8 based on the noncovalent dual inhibitors of CDK12/13 and demonstrated its specificity for CDK12 over CDK13. Notably, PP-C8 induces profound degradation of cyclin K simultaneously and downregulates the mRNA level of DNA-damage response genes. Global proteomics profiling revealed PP-C8 is highly selective toward CDK12-cyclin K complex. Importantly, PP-C8 demonstrates profound synergistic antiproliferative effects with PARP inhibitor in triple-negative breast cancer (TNBC). The potent and selective CDK12 PROTAC degrader developed in this study could potentially be used to treat CDK12-dependent cancers as combination therapy.

Integrative Modeling of PROTAC-Mediated Ternary Complexes.

Proteolysis-targeting chimeras (PROTACs), which selectively induce targeted protein degradation, represent an emerging drug discovery technology. Although numerous PROTACs have been reported, designing potent PROTACs still remains a great challenge, to some extent, due to insufficient structural data of Target-PROTAC-E3 ternary complexes. In this work, PROTAC-Model, an integrative computational method by combining the FRODOCK-based protocol and RosettaDock-based refinement, was developed to predict PROTAC-mediated ternary complex structures and tested on 14 cases. The quality of the models was evaluated using the criteria of the critical assessment of predicted interactions (CAPRI). Using the unbound structures, the FRODOCK-based protocol can generate the ternary complex structures with medium or high quality for 8 cases out of 14. With the refinement by RosettaDock, the cases with medium or high quality increase to 12. Compared with PRosettaC and the method developed by Drummond et al., PROTAC-Model shows better performance. In summary, PROTAC-Model should be useful for the rational design of PROTACs.

Strategies toward Discovery of Potent and Orally Bioavailable Proteolysis Targeting Chimera Degraders of Androgen Receptor for the Treatment of Prostate Cancer

Proteolysis targeting chimera (PROTAC) small-molecule degraders have emerged as a promising new type of therapeutic agents, but the design of PROTAC degraders with excellent oral pharmacokinetics is a major challenge. In this study, we present our strategies toward the discovery of highly potent PROTAC degraders of androgen receptor (AR) with excellent oral pharmacokinetics. Employing thalidomide to recruit cereblon/cullin 4A E3 ligase and through the rigidification of the linker, we discovered highly potent AR degraders with good oral pharmacokinetic properties in mice with ARD-2128 being the best compound. ARD-2128 achieves 67% oral bioavailability in mice, effectively reduces AR protein and suppresses AR-regulated genes in tumor tissues with oral administration, leading to the effective inhibition of tumor growth in mice without signs of toxicity. This study supports the development of an orally active PROTAC AR degrader for the treatment of prostate cancer and provides insights and guidance into the design of orally active PROTAC degraders.

Discovery of selective CDK9 degraders with enhancing antiproliferative activity through PROTAC conversion.

Cyclin-dependent kinase 9 (CDK9) is an increasingly important potential cancer treatment target. Nowadays, developing selective CDK9 inhibitors has been extremely challenging as its ATP-binding sites are similar with other CDKs. Here, we report that the CDK9 inhibitor BAY-1143572 is converted into a series of proteolysis targeting chimeras (PROTACs) which leads to several compounds inducing the degradation of CDK9 in acute myeloid leukemia cells at a low nanomolar concentration. In addition, the most potent PROTAC molecule B03 could inhibit cell growth more effectively than warhead alone, with little inhibition of other kinases. This enhanced antiproliferative activity is mediated by a slight increase in kinase inhibitory activity and an increase in the level of apoptosis induction. Moreover, B03 could induce the degradation of CDK9 invivo. Our work provides evidence that B03 represents a lead for further development and that CDK9 degradation is a potential valuable therapeutic strategy in acute myeloid leukemia.

AML-399: A Pan-BET Degrader Shows Broad Pre-Clinical Activity in a Large Acute Myeloid Leukemia Cohort

Bromodomain and Extra Terminal (BET) proteins are ubiquitously expressed epigenetic “readers” that play a major role in regulating expression of oncogenic genes such as c-Myc. BET protein family are crucial for acute myeloid leukemia (AML) maintenance, and expression of BRD4 is a poor prognostic indicator in AML. We have developed novel and a highly potent proteolysis-targeting, chimera (PROTAC)-based pan-BET degrader, QCA570, that depletes BET. This BET-degrader is derived from a new class of inhibitor, HYD276. We tested QCA570 in a panel of 104 primary AML samples in ex-vivo cultures. Leukemia stem cells (LSCs) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors and drugs for 72 hrs, and cell death and apoptosis induction was measured by flow cytometry for DAPI-Annexin V binding. Primary AML cells were obtained from newly diagnosed AML (N=89) or relapse/refractory AML (N=15). The median IC50 for BET-degrader was 120 pM, and only 13 samples had an IC50 >1 nM. In contrast, the median IC50 for the inhibitor HYD276 was 30,000 fold higher at 3.5 μM. An IC50 of greater than 10 μM HYD276 was considered as resistant to the BET inhibitor. BET inhibitor-resistant LSCs were found to be sensitive to the degrader QCA570, irrespective of mutation status and chromosomal aberrations, suggesting that there are additional mechanisms of action of the degrader as compared to the inhibitor. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBP-alpha (2%), DNMT3A (2%), and other mutations (10%). Based on classical karyotype, 30% of them had a normal karyotype, and 55% had abnormal karyotype. The degradation of BRD4 and repression of c-MYC was confirmed by immunoblotting after a 4-hr exposure of LSCs to the degrader. Moreover, treatment of normal CD34-positive hematopoietic cells in an apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. Evaluation of this new class of drugs in patient-derived xenografts is ongoing. In conclusion, QCA570 represents a highly potent and efficacious pan-BET-BRD degrader undergoing extensive pre-clinical evaluation for the treatment of acute leukemias. Bromodomain and Extra Terminal (BET) proteins are ubiquitously expressed epigenetic “readers” that play a major role in regulating expression of oncogenic genes such as c-Myc. BET protein family are crucial for acute myeloid leukemia (AML) maintenance, and expression of BRD4 is a poor prognostic indicator in AML. We have developed novel and a highly potent proteolysis-targeting, chimera (PROTAC)-based pan-BET degrader, QCA570, that depletes BET. This BET-degrader is derived from a new class of inhibitor, HYD276. We tested QCA570 in a panel of 104 primary AML samples in ex-vivo cultures. Leukemia stem cells (LSCs) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors and drugs for 72 hrs, and cell death and apoptosis induction was measured by flow cytometry for DAPI-Annexin V binding. Primary AML cells were obtained from newly diagnosed AML (N=89) or relapse/refractory AML (N=15). The median IC50 for BET-degrader was 120 pM, and only 13 samples had an IC50 >1 nM. In contrast, the median IC50 for the inhibitor HYD276 was 30,000 fold higher at 3.5 μM. An IC50 of greater than 10 μM HYD276 was considered as resistant to the BET inhibitor. BET inhibitor-resistant LSCs were found to be sensitive to the degrader QCA570, irrespective of mutation status and chromosomal aberrations, suggesting that there are additional mechanisms of action of the degrader as compared to the inhibitor. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBP-alpha (2%), DNMT3A (2%), and other mutations (10%). Based on classical karyotype, 30% of them had a normal karyotype, and 55% had abnormal karyotype. The degradation of BRD4 and repression of c-MYC was confirmed by immunoblotting after a 4-hr exposure of LSCs to the degrader. Moreover, treatment of normal CD34-positive hematopoietic cells in an apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. Evaluation of this new class of drugs in patient-derived xenografts is ongoing. In conclusion, QCA570 represents a highly potent and efficacious pan-BET-BRD degrader undergoing extensive pre-clinical evaluation for the treatment of acute leukemias.