Potent Degrader Research Articles

Targeted degradation of Pin1 by protein-destabilizing compounds

The concept of targeted protein degradation is at the forefront of modern drug discovery, which aims to eliminate disease-causing proteins using specific molecules. In this paper, we explored the idea to design protein degraders based on the section of ligands that cause protein destabilization, hence that facilitate the cellular breakdown of the target. Our studies present covalent agents targeting Pin1, a cis-trans prolyl isomerase that plays a crucial role in tumorigenesis. Our design strategy entailed iterative optimizations of agents for potency and Pin1 destabilization in vitro. Biophysical and cellular studies suggest that the agents may act like molecular crowbars, displacing protein-stabilizing interactions that open the structure for recognition by the proteasome degradation machinery. This approach resulted in a series of potent and effective Pin1 degraders with potential applications in target validation and in therapeutic development. We propose that our design strategy can identify molecular degraders without engineering bifunctional agents that artificially create interactions between a disease-causing protein and a ubiquitin ligase.

Development of Oral, Potent, and Selective CK1α Degraders for AML Therapy

Development of Oral, Potent, and Selective CK1α Degraders for AML Therapy

Discovery of SD-436: A Potent, Highly Selective and Efficacious STAT3 PROTAC Degrader Capable of Achieving Complete and Long-Lasting Tumor Regression.

STAT3 is an attractive therapeutic target for cancer and other human diseases. We have previously reported the discovery of potent, selective, and efficacious PROTAC STAT3 degraders SD-36 and SD-91. In this study, we have designed and synthesized a novel series of STAT3 degraders using a new, high-affinity STAT3 ligand with excellent chemical stability and cereblon ligands. Our efforts led to the discovery of SD-436, a highly potent and selective STAT3 degrader. A single intravenous administration of SD-436 at 5 mg/kg effectively induces rapid, complete, and durable depletion of STAT3 in mouse native and xenograft tumor tissues. SD-436 achieves complete and long-lasting tumor regression even with a weekly dosing schedule in leukemia and lymphoma xenograft models in mice. SD-436 represents a promising STAT3 degrader for advanced preclinical development as a new therapy for the treatment of human cancers and other human diseases.

Discovery of the first selective and potent PROTAC degrader for the pseudokinase TRIB2

Pseudokinase TRIB2, a member of the CAMK Ser/Thr protein kinase family, regulates various cellular processes through phosphorylation-independent mechanisms. Dysregulation of TRIB2 has been implicated in promoting tumor growth, metastasis, and therapy resistance, making it a promising target for cancer treatment. In this study, we designed and synthesized a series of TRIB2 PROTAC degraders by conjugating a TRIB2 binder 1 with VHL or CRBN ligands via linkers of varying lengths and compositions. Among these compounds, 5k demonstrated potent TRIB2 degradation with a DC50 value of 16.84 nM (95 % CI: 13.66–20.64 nM) in prostate cancer PC3 cells. Mechanistic studies revealed that 5k directly interacted with TRIB2, selectively inducing its degradation through a CRBN-dependent ubiquitin-proteasomal pathway. Moreover, 5k outperformed the TRIB2 binder alone in inhibiting cell proliferation and inducing apoptosis, confirming that TRIB2 protein degradation could be a promising therapeutic strategy for TRIB2-associated cancers. Additionally, compound 5k also serves as an effective tool for probing TRIB2 biology.

Targeting METTL3 protein by proteolysis-targeting chimeras: A novel therapeutic approach for acute myeloid leukemia

Despite numerous studies suggesting that RNA m6A transferase core complex including METTL3 and METTL14 play essential roles in both the initiation and maintenance of acute myeloid leukemia (AML), effective pharmacological targeting of these two proteins remains elusive. Here, we report the development and evaluation of a novel METTL3 degrader, ZW27941, designed to induce METTL3 degradation via the VHL-mediated proteasomal degradation pathway. ZW27941 exhibited potent and selective degradation of METTL3 and its binding partner METTL14, leading to significant anti-leukemic activity in AML cell lines. Furthermore, ZW27941 demonstrated synergistic or additive effects when combined with standard AML therapeutics, such as cytarabine and venetoclax. Our findings suggest that selective METTL3 degraders, exemplified by ZW27941, hold promise as a novel therapeutic approach for AML, particularly when used in combination with existing treatments to enhance efficacy and overcome resistance mechanisms.

Design, Synthesis, and Activity Evaluation of BRD4 PROTAC Based on Alkenyl Oxindole-DCAF11 Pair.

Proteolytic targeting chimera (PROTAC) represent an advanced strategy for targeting undruggable proteins, and the molecular warheads targeting E3 ligases play a crucial role. Recently, we explored an alkenyl oxindole warhead targeting the E3 ligase DCAF11 and sought to validate its potential. In this study, we synthesized a range of BRD4 PROTACs (8a-8o, 14a-14f, 22a-22m) with modified alkenyl oxindole warheads and developed a high-throughput screening system based on high-content imaging. We identified L134 (22a) as a potent BRD4 degrader, achieving BRD4 degradation (Dmax > 98%, DC50 = 7.36 nM) and demonstrating antitumor activity. Mechanically, BRD4 degradation by L134 was mediated through the ubiquitin-proteasome system in a DCAF11-dependent manner. Therefore, this study provides a rapid screening method for effective PROTACs and highlights the PROTAC L134 based on alkenyl oxindole-DCAF11 pair as a promising candidate for treating BRD4-driven cancers.

Development of 2-Aminoadenine-Based Proteolysis-Targeting Chimeras (PROTACs) as Novel Potent Degraders of Monopolar Spindle 1 and Aurora Kinases.

Monopolar spindle 1 (Mps1, also known as TTK) and Aurora kinase (AURK) A and B are critical regulators of mitosis and have been linked to the progression of various cancers. Here, we report the design, synthesis, and biological evaluation of a series of PROTACs (proteolysis-targeting chimeras) targeting TTK and AURKs. We synthesized various degrader molecules based on four different 2-aminoadenine-based ligands, recruiting either cereblon or VHL as the E3-ligase. Our research showed that the nature of the linker and modification of the ligand significantly influence the target specificity and degradation efficacy. Notably, compound 19, among the most potent degraders, demonstrated robust proteasome-mediated degradation of TTK with D max of 66.5% and DC50 value (6 h) of 17.7 nM as compared to its structurally akin inhibitor control, 23. The cytotoxicity of most of the synthesized chimeras against acute myeloid leukemia cell line MV4-11 was lower than that of the corresponding parent inhibitors. However, we could also identify degraders such as 15 and 26 that induce potent AURKA degradation and display comparable antiproliferative activities to their parent compound SF1. Compound 15 degrades AURKA with low DC50 value of 2.05 nM, which is 77-fold and 21-fold more selective toward AURKB and TTK and has an EC50 value of 39 nM against cancer MV4-11 cells. Overall, the observations we made with the degrader molecules we developed can further aid in the design and development of optimized TTK or AURK degraders for cancer therapy.

Structure-Activity Relationship of Potent, Selective, and Orally Bioavailable Molecular Glue Degraders of CK1α.

The original molecular glue degraders (thalidomide, lenalidomide, and pomalidomide) are known to bind to cereblon (CRBN) and alter its surface to induce recruitment, ubiquitination, and degradation of therapeutically valuable neosubstrates (IKZF1, IKZF3, and CK1α). With the aim of understanding and modulating neosubstrate specificity, we recently reported the discovery of SJ3149 (4), a selective and potent molecular glue degrader of CK1α, that is active in multiple cancer cell lines. Herein, we describe the medicinal chemistry efforts that resulted in the discovery of SJ3149 as well as other potent and selective CK1α degraders. We report kinetic profiling and parameters of CK1α degradation, ternary complex, antiproliferative effects, in vitro ADME data, and in vivo pharmacokinetic studies with demonstrated oral bioavailability.

Discovery of a Potent, selective and orally bioavailable CDK9 degrader for targeting transcription regulation in Triple-Negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous and invasive subtype of breast cancer with very limited effective modalities of treatment. Degrading the critical transcription regulator cyclin-dependent kinase 9 (CDK9) by proteolysis targeting chimeras (PROTACs) has shown promising potential for treating TNBC. However, to date, CDK9-targeting PROTACs for oral administration in treatment of cancers have not been reported. We herein present the design, synthesis, and extensive biological evaluation of a series of novel PROTACs as orally bioavailable, potent and selective degraders of CDK9 for targeting transcription regulation in triple-negative breast cancer. The developed compound 29 exhibited a desired potency (DC50 = 3.94 nM) with high efficacy (Dmax = 96 %) on CDK9 degradation, and effectively inhibited the proliferation of TNBC MDA-MB-231 cells. Mechanistic investigations revealed that compound 29 is a bona fide CDK9 degrader and can substantially downregulate the downstream targets c-Myc and MCL-1. Furthermore, compound 29 displayed favorable oral bioavailability in mice, and oral administration of degrader 29 significantly depleted CDK9 protein in TNBC tumor tissues and exhibited tumor growth inhibition in TNBC xenograft mice models. Collectively, our work established that degrader 29 is a highly potent and selective degraders of CDK9 with satisfactory oral bioavailability, which holds promising potential for the treatment of TNBC.

Regulate catalytic performance by engineering non-regular structure of extradiol dioxygenase: An entrance to bottom strategy

Extradiol dioxygenase Tcu3516 is a home-sourced enzyme demonstrating potent aromatic phenol degradation capacity. To add to the advantageous modifications inside active cavity, this work reported a novel strategy to engineer rarely concerned non-regular structures around the entrance towards the active site at the bottom of cavity. Three structures, Loop region 1 (Loop1: Met173-Arg185), Loop region 2 (Loop2: Ala201-Val212) and C-terminal (C-tail: His290-Lys306) were therefore identified through structural flexibility analysis. Highly rigid prolines within the structures were mutated into smaller alanine, glycine, or serine to improve structural flexibilities; while only P183S on Loop1 showed 3-fold activity enhancement vs the WT when subjected to cleavage of mono-cyclic catechol analogues. The analysis of Root Mean Square Fluctuation showed that P183S presents certain enhancement on Loop1 flexibility without dramatic changes of other domains. Furthermore, the synergetic effects from mutation P183S and cavity-based mutations V186L, V212N and D285A were evaluated by characterizing combinatorial mutants. Temperature dependence and thermostability of the combined mutants showed a more flexible catalytic domain without sacrificing structural integrity and stability. kcat value of P183S/V186L (SL) towards monocyclic catechols significantly surpasses any other combinatorial mutants around Tcu3516 active sites. Moreover, the synergetic effects on conformational plasticity were analyzed by molecular dynamic simulations to shed light into the interplay between structural changes and catalytic performance.

Probing class I histone deacetylases (HDAC) with proteolysis targeting chimera (PROTAC) for the development of highly potent and selective degraders

Class I HDACs are considered promising targets for cancer due to their role in epigenetic modifications. The main challenges in developing a new, potent and non-toxic class I HDAC inhibitor are selectivity and appropriate pharmacokinetics. The PROTAC technique (Proteolysis Targeting Chimera) is a new method in drug development for the production of active substances that can degrade a protein of interest (POI) instead of inhibiting it. This technique will open the era to produce selective and potent drugs with a high margin of safety. Previously, we reported different inhibitors targeting class I HDACs functionalized with aminobenzamide or hydroxamate groups. In the current research work, we will employ PROTAC technique to develop class I HDAC degraders based on our previously reported inhibitors. We synthesized two series of aminobenzamide-based PROTACs and hydroxamate-based PROTACs and tested them in vitro against class I HDACs. To ensure their degradation, all of them were screened against HDAC2 as representative example of class I. The best candidates were evaluated at different concentrations at various HDAC subtypes. This resulted in the PROTAC (32a) (HI31.1) that degrades HDAC8 with a DC50 of 8.9 nM with a proper margin of selectivity against other isozymes. Moreover, PROTAC 32a is able to degrade HDAC6 with DC50 = 14.3 nM. Apoptotic study on leukemic cells (MV-4–11) displayed more than 50 % apoptosis took place at 100 nM. PROTAC 32a (HI31.1) showed a good margin of safety against normal cell line and proper chemical stability.

94 (PB082): AUTX-703, a novel and potent KAT2A and KAT2B protein degrader, induces profound cell state changes and inhibits growth in Small Cell Lung Cancer (SCLC) model systems

94 (PB082): AUTX-703, a novel and potent KAT2A and KAT2B protein degrader, induces profound cell state changes and inhibits growth in Small Cell Lung Cancer (SCLC) model systems

183 (PB171): A novel kinase degrader-based payload for the development of potent Degrader Antibody Conjugates (DACs)

183 (PB171): A novel kinase degrader-based payload for the development of potent Degrader Antibody Conjugates (DACs)

Discovery of a Highly Potent PROTAC Degrader of p300/CBP Proteins for the Treatment of Enzalutamide-Resistant Prostate Cancer.

Prostate cancer therapies against androgen receptor (AR) eventually develop lethal resistance; thus, exploring new therapeutic approaches is urgent for prostate cancer treatment. Acetyltransferase p300/CBP are key coactivators for AR-mediated transcription and represent promising therapeutic targets to inhibit AR activity in prostate cancer. We describe the design synthesis and evaluation of a new class of p300/CBP PROTAC degraders. We identified an excellent p300/CBP degrader MJP6412, which effectively induced degradation of p300/CBP proteins, downregulated AR target genes, and inhibited cell growth of human prostate cancer cell lines and enzalutamide-resistant cells with IC50 even at nanomolar concentrations. Furthermore, MJP6412 demonstrated significant inhibition of tumor growth in a VCaP xenograft model. Collectively, MJP6412 is a promising lead compound for the treatment of prostate cancer, especially enzalutamide-resistant prostate cancer.

Discovery of electrophilic degraders that exploit SNAr chemistry.

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly 'undruggable' targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

Discovery of Novel PROTAC SIRT6 Degraders with Potent Efficacy against Hepatocellular Carcinoma.

Sirtuin 6 (SIRT6), a member of the SIRT family, plays essential roles in the regulation of metabolism, inflammation, aging, DNA repair, and cancer development, making it a promising anticancer drug target. Herein, we present our use of proteolysis-targeting chimera (PROTAC) technology to formulate a series of highly potent and selective SIRT6 degraders. One of the degraders, SZU-B6, induced the near-complete degradation of SIRT6 in both SK-HEP-1 and Huh-7 cell lines and more potently inhibited hepatocellular carcinoma (HCC) cell proliferation than the parental inhibitors. In preliminary mechanistic studies, SZU-B6 hampered DNA damage repair, promoting the cellular radiosensitization of cancer cells. Our SIRT6 degrader SZU-B6 displayed promising antitumor activity, particularly when combined with the well-known kinase inhibitor sorafenib or irradiation in an SK-HEP-1 xenograft mouse model. Our results suggest that these PROTACs might constitute a potent therapeutic strategy for HCC.

Non-Markovian Dynamic Models Identify Non-Canonical KRAS-VHL Encounter Complex Conformations for Novel PROTAC Design.

Targeted protein degradation (TPD) is emerging as a promising therapeutic approach for cancer and other diseases, with an increasing number of programs demonstrating its efficacy in human clinical trials. One notable method for TPD is Proteolysis Targeting Chimeras (PROTACs) that selectively degrade a protein of interest (POI) through E3-ligase induced ubiquitination followed by proteasomal degradation. PROTACs utilize a warhead-linker-ligand architecture to bring the POI (bound to the warhead) and the E3 ligase (bound to the ligand) into proximity. The resulting non-native protein-protein interactions (PPIs) formed between the POI and E3 ligase lead to the formation of a stable ternary complex, enhancing cooperativity for TPD. A significant challenge in PROTAC design is the screening of the linkers to induce favorable non-native PPIs between POI and E3 ligase. Here, we present a physics-based computational protocol to predict noncanonical and metastable PPI interfaces between an E3 ligase and a given POI, aiding in the design of linkers to stabilize the ternary complex and enhance degradation. Specifically, we build the non-Markovian dynamic model using the Integrative Generalized Master equation (IGME) method from ∼1.5 ms all-atom molecular dynamics simulations of linker-less encounter complex, to systematically explore the inherent PPIs between the oncogene homologue protein and the von Hippel-Lindau E3 ligase. Our protocol revealed six metastable states each containing a different PPI interface. We selected three of these metastable states containing promising PPIs for linker design. Our selection criterion included thermodynamic and kinetic stabilities of PPIs and the accessibility between the solvent-exposed sites on the warheads and E3 ligand. One selected PPIs closely matches a recent cocrystal PPI interface structure induced by an experimentally designed PROTAC with potent degradation efficacy. We anticipate that our protocol has significant potential for widespread application in predicting metastable POI-ligase interfaces that can enable rational design of PROTACs.

Translational modelling to predict human pharmacokinetics and pharmacodynamics of a Bruton's tyrosine kinase-targeted protein degrader BGB-16673.

Bifunctional small molecule degraders, which link the target protein with E3 ubiquitin ligase, could lead to the efficient degradation of the target protein. BGB-16673 is a Bruton's tyrosine kinase (BTK) degrader. A translational PK/PD modelling approach was used to predict the human BTK degradation of BGB-16673 from preclinical in vitro and in vivo data. A simplified mechanistic PK/PD model was used to establish the correlation between the in vitro and in vivo BTK degradation by BGB-16673 in a mouse model. Human and mouse species differences were compared using the parameters generated from in vitro human or mouse blood, and human or mouse serum spiked TMD-8 cells. Human PD was then predicted using the simplified mechanistic PK/PD model. BGB-16673 showed potent BTK degradation in mouse whole blood, human whole blood, and TMD-8 tumour cells in vitro. Furthermore, BGB-16673 showed BTK degradation in a murine TMD-8 xenograft model in vivo. The PK/PD model predicted human PD and the observed BTK degradation in clinical studies both showed robust BTK degradation in blood and tumour at clinical dose range. The presented simplified mechanistic model with reduced number of model parameters is practically easier to be applied to research projects compared with the full mechanistic model. It can be used as a tool to better understand the PK/PD behaviour for targeted protein degraders and increase the confidence when moving to the clinical stage.

Discovery of Covalent MLKL PROTAC Degraders via Optimization of a Theophylline Derivative Ligand for Treating Necroptosis.

Mixed lineage kinase domain-like pseudokinase (MLKL) initiates necroptosis and could serve as a therapeutic target related to a series of human diseases. Proteolysis-targeting chimeras (PROTACs) are useful tools for degrading pathological proteins and blocking disease processes. Using computer-aided modeling and molecular dynamics simulations, we developed a series of covalent MLKL PROTACs by linking and optimizing a theophylline derivative that covalently targets MLKL. Via structure-activity relationship studies, MP-11 was identified as a potent MLKL PROTAC degrader. Furthermore, MP-11 showed lower toxicity than the original MLKL ligand, exhibiting nanomolar-scale antinecroptotic activity on human cell lines. Xenograft model studies showed that MP-11 effectively degraded MLKL in vivo. Importantly, our study demonstrates that the covalent binding strategy is an effective approach for designing MLKL-targeting PROTACs, serving as a model for developing PROTACs to treat future necroptosis-related human diseases.

Structure-Activity Relationship Studies of Substituted 2-Phenyl-1,2,4-triazine-3,5(2H,4H)-dione Analogues: Development of Potent eEF2K Degraders against Triple-Negative Breast Cancer.

eEF2K, an atypical alpha-kinase, is responsible for regulating protein synthesis and energy homeostasis. Aberrant eEF2K function has been linked to various human cancers, including triple-negative breast cancer (TNBC). However, limited cellular activity of current eEF2K modulators impedes their clinical application. Based on the 2-phenyl-1,2,4-triazine-3,5(2H,4H)-dione scaffold of our hits I4 and C1, structure-activity relationship analysis led to the discovery of several more active derivatives (e.g., 19, 34, and 36) in inhibiting the viability of TNBC cell line MDA-MB-231. Moreover, the most potent compound 36 significantly suppresses the viability, proliferation, and migration of both MDA-MB-231 and HCC1806 cell lines. Mechanistically, compound 36 has a high binding affinity for the eEF2K protein and effectively induces its degradation. Additionally, 36 exerts a comparable tumor-suppressive effect to paclitaxel in an MDA-MB-231 cell xenograft mouse model with no obvious toxicity, demonstrating that compound 36 could be developed as a potential novel therapeutic for TNBC treatment.