Potent Degrader Research Articles

Targeted degradation of METTL3 against acute myeloid leukemia and gastric cancer

Accumulating evidence reveals the oncogenic role of methyltransferase-like 3 (METTL3) in a variety of cancers, either dependent or independent of its m6A methyl transferase activity. We have explored PROTACs targeting METTL3 and identified KH12 as a potent METTL3 degrader. Treatment of KH12 on MOLM-13 cells causes degradation of METTL3 with a DC50 value of 220 nM in a dose-, time- and ubiquitin-dependent fashion. In addition, KH12 is capable of reversing differentiation and possesses anti-proliferative effects surpassing the small molecule inhibitors on MOLM-13 cells. Notably, we first present that METTL3 degrader significantly suppresses the growth of various gastric cancer (GC) cells, where the m6A-independent activity of METTL3 plays a crucial role in tumorigenesis. The anti-GC effects of KH12 were further confirmed in patient-derived organoids (PDOs). This study offers therapeutic potentials of targeted degradation of METTL3 against GC implicated with non-catalytic function of METTL3 as well as against AML.

Programming DNA Nanoassemblies into Polyvalent Lysosomal Degraders for Potent Degradation of Pathogenic Membrane Proteins.

Lysosome-targeting chimera (LYTAC) shows great promise for protein-based therapeutics by targeted degradation of disease-associated membrane or extracellular proteins, yet its efficiency is constrained by the limited binding affinity between LYTAC reagents and designated proteins. Here, we established a programmable and multivalent LYTAC system by tandem assembly of DNA into a high-affinity protein degrader, a heterodimer aptamer nanostructure targeting both pathogenic membrane protein and lysosome-targeting receptor (insulin-like growth factor 2 receptor, IGF2R) with adjustable spatial distribution or organization pattern. The DNA-based multivalent LYTACs showed enhanced efficacy in removing immune-checkpoint protein programmable death-ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR2) in tumor cell membrane that respectively motivated a significant increase in T cell activity and a potent effect on cancer cell growth inhibition. With high programmability and versatility, this multivalent LYTAC system holds considerable promise for realizing protein therapeutics with enhanced activity.

Novel potent molecular glue degraders against broad range of hematological cancer cell lines via multiple neosubstrates degradation

BackgroundTargeted protein degradation of neosubstrates plays a crucial role in hematological cancer treatment involving immunomodulatory imide drugs (IMiDs) therapy. Nevertheless, the persistence of inevitable drug resistance and hematological toxicities represents a significant obstacle to their clinical effectiveness.MethodsPhenotypic profiling of a small molecule compounds library in multiple hematological cancer cell lines was conducted to screen for hit degraders. Molecular dynamic-based rational design and cell-based functional assays were conducted to develop more potent degraders. Multiple myeloma (MM) tumor xenograft models were employed to investigate the antitumor efficacy of the degraders as single or combined agents with standard of care agents. Unbiased proteomics was employed to identify multiple therapeutically relevant neosubstrates targeted by the degraders. MM patient-derived cell lines (PDCs) and a panel of solid cancer cell lines were utilized to investigate the effects of candidate degrader on different stage of MM cells and solid malignancies. Unbiased proteomics of IMiDs-resistant MM cells, cell-based functional assays and RT-PCR analysis of clinical MM specimens were utilized to explore the role of BRD9 associated with IMiDs resistance and MM progression.ResultsWe identified a novel cereblon (CRBN)-dependent lead degrader with phthalazinone scaffold, MGD-4, which induced the degradation of Ikaros proteins. We further developed a novel potent candidate, MGD-28, significantly inhibited the growth of hematological cancer cells and induced the degradation of IKZF1/2/3 and CK1α with nanomolar potency via a Cullin-CRBN dependent pathway. Oral administration of MGD-4 and MGD-28 effectively inhibited MM tumor growth and exhibited significant synergistic effects with standard of care agents. MGD-28 exhibited preferentially profound cytotoxicity towards MM PDCs at different disease stages and broad antiproliferative activity in multiple solid malignancies. BRD9 modulated IMiDs resistance, and the expression of BRD9 was significant positively correlated with IKZF1/2/3 and CK1α in MM specimens at different stages. We also observed pronounced synergetic efficacy between the BRD9 inhibitor and MGD-28 for MM treatment.ConclusionsOur findings present a strategy for the multi-targeted degradation of Ikaros proteins and CK1α against hematological cancers, which may be expanded to additional targets and indications. This strategy may enhance efficacy treatment against multiple hematological cancers and solid tumors.

53901 Potent and selective oral STAT6 degraders inhibit IL-4 and IL-13 functions in human cells and block TH2 inflammation in a mouse model of atopic dermatitis

53901 Potent and selective oral STAT6 degraders inhibit IL-4 and IL-13 functions in human cells and block TH2 inflammation in a mouse model of atopic dermatitis

54020 Potent and selective TYK2 degraders, devoid of JAK activity, potently and completely suppress IL12/23 and IFN-a signaling pathways.

54020 Potent and selective TYK2 degraders, devoid of JAK activity, potently and completely suppress IL12/23 and IFN-a signaling pathways.

Design, synthesis and biological evaluation of a novel PAK1 degrader for the treatment of triple negative breast cancer

Triple-negative breast cancer is one of the most malignant subtypes in clinical practice, and it is urgent to find new therapies. The p21-activated kinase I (PAK1) has been considered to be an attractive therapeutic target for TNBC. In this study, we designed and synthesized a series of novel PROTAC PAK1 degraders by conjugating VHL or CRBN ligase ligands to PAK1 inhibitors which are connected by alkyl chains or PEG chains. The most promising compound, 19s, can significantly degrade PAK1 protein at concentrations as low as 0.1 μM, and achieves potent anti-proliferative activity with an IC50 value of 1.27 μM in MDA-MB-231 cells. Additionally, 19s exhibits potent anti-migration activity in vitro and induces rapid tumor regression in vivo. Collectively, these findings document that 19s is a potent and novel PAK1 degrader with promising potential for TNBC treatment.

Discovery of Novel, Potent and Orally Bioavailable SMARCA2 PROTACs with Synergistic Anti-tumor Activity in Combination with KRAS G12C Inhibitors.

Cancer genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 in non-small cell lung cancer with a frequency of up to 33% in advanced stage disease, making it the most frequently mutated complex in lung cancer. We and others have identified SMARCA2 to be synthetic lethal to SMARCA4, indicating SMARCA2 is a high value therapeutic target. Here, we disclose the discovery and characterization of potent, selective and orally bioavailable Cereblon-based SMARCA2 PROTACs. Biochemically, YDR1 and YD54 are potent SMARCA2 degraders with an average DC 50 of 7.7nM and 3.5nM respectively in SMARCA4 mutant lung cancer cells. Phenotypically, both YDR1 and YD54 selectively inhibited growth of SMARCA4 mutant cancer cells. Further, we showed anti-tumor growth inhibitory activity of YDR1 and YD54 in SMARCA4 mutant xenograft models of lung cancer. Finally, we show that YDR1 and YD54 synergize with the KRAS G12C inhibitor sotorasib to inhibit growth of SMARCA4 and KRAS G12C co-mutant lung cancer cells. These findings provide additional evidence for the utility of single agent or combination regimens containing SMARCA2 PROTACs as synthetic lethal therapeutics against SMARCA4 mutant cancers.

Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells.

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.

Discovery of CW-3308 as a Potent, Selective, and Orally Efficacious PROTAC Degrader of BRD9.

The bromodomain-containing protein BRD9 has emerged as an attractive therapeutic target. In the present study, we successfully identified a number of highly potent BRD9 degraders by using two different cereblon ligands developed in our laboratory. Further optimization led to the discovery of CW-3308 as a potent, selective, and orally bioavailable BRD9 degrader. It displayed degradation potency (DC50) < 10 nM and efficiency (Dmax) > 90% against BRD9 in the G401 rhabdoid tumor and HS-SY-II synovial sarcoma cell lines and had a high degradation selectivity over BRD7 and BRD4 proteins. CW-3308 achieved 91% of oral bioavailability in mice. A single oral dose efficiently reduced the BRD9 protein by >90% in the synovial sarcoma HS-SY-II xenograft tumor tissue. Oral administration effectively inhibited HS-SY-II xenograft tumor growth in mice. CW-3308 is a promising lead compound for further optimization and extensive evaluation for the treatment of synovial sarcoma, rhabdoid tumor, and other BRD9-dependent human diseases.

Design, synthesis, and biological evaluation of RSL3-based GPX4 degraders with hydrophobic tags

Ferroptosis is a new type of programmed cell death characterized by iron-dependent lipid peroxidation, during which glutathione peroxidase 4 (GPX4) plays an essential role and is well-recognized as a promising therapeutic target for cancer treatment. Although some GPX4 degradation molecules have been developed to induce ferroptosis, the discovery of GPX4 degraders with hydrophobic tagging (HyT) as an innovative approach is more challenging. Herein, we designed and synthesized a series of HyT degraders by linking the GPX4 inhibitor RSL3 with a hydrophobic and bulky group of adamantane. Among them, compound R8 is a potent degrader (DC50, 24h = 0.019 μM) which can effectively degrade GPX4 in a dose- and time-dependent manner. Furthermore, compound R8 exhibited superior in vitro antitumor potency against HT1080 and MDA-MB-231 cell lines with IC50 values of 24 nM and 32 nM respectively, which are 4 times more potent than parental compound RSL3. Mechanistic investigation evidenced that R8 consumes GPX4 protein mainly through the ubiquitin proteasome (UPS) and enables to induce the accumulation of LPO, thereby triggering ferroptosis. Our work presented the novel GPX4 degrader of R8 by HyT strategy, and provided a promising pathway of degradation agents for the treatment of ferroptosis relevant diseases.

Discovery of a Series of Orally Bioavailable Androgen Receptor Degraders for the Treatment of Prostate Cancer.

Androgen receptor (AR) signaling plays a key role in the progression of prostate cancer. This study describes the discovery and optimization of a novel series of AR PROTAC degraders that recruit the Cereblon (CRBN) E3 ligase. Having identified a series of AR ligands based on 4-(4-phenyl-1-piperidyl)-2-(trifluoromethyl)benzonitrile, our PROTAC optimization strategy focused on linker connectivity and CRBN ligand SAR to deliver potent degradation of AR in LNCaP cells. This work culminated in compounds 11 and 16 which demonstrated good rodent oral bioavailability. Subsequent SAR around the AR binding region brought in an additional desirable feature, degradation of the important treatment resistance mutation L702H. Compound 22 (AZ'3137) possessed an attractive profile showing degradation of AR and L702H mutant AR with good oral bioavailability across species. The compound also inhibited AR signaling in vitro and tumor growth in vivo in a mouse prostate cancer xenograft model.

Discovery of Potent and Selective G9a Degraders for the Treatment of Pancreatic Cancer.

G9a, which was initially identified as a histone H3 Lys9 (H3K9) methyltransferase, is potentially an attractive therapeutic target for human cancers. Despite its importance, there is no available selective G9a chemical probe because its homologous protein GLP shares approximately 80% of its sequence with G9a. The development of G9a chemical probes with high selectivity for G9a over GLP is a big challenge but is extremely valuable for understanding G9a-related biology. Herein, we developed a first-in-class selective G9a degrader G9D-4, which induced a dose- and time-dependent G9a degradation without degradation of GLP. G9D-4 exhibited effective antiproliferative activities in a panel of pancreatic cancer cell lines and was able to sensitize KRASG12D mutant pancreatic cancer cells to KRASG12D inhibitor MRTX1133. These data clearly demonstrated the practicality and importance of a selective G9a degrader as a preliminary chemical probe suitable for understanding G9a-related biology and a promising strategy for the treatment of pancreatic cancer.

De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Peptide Prioritization via CLIP (PepPrCLIP) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro. Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.

Discovery of a Highly Potent, Selective and Efficacious USP7 Degrader for the Treatment of Acute Lymphoblastic Leukemia.

USP7 is an attractive therapeutic target for cancers, especially for acute lymphoblastic leukemia (ALL) with wild-type p53. Herein, we report the discovery of XM-U-14 as a highly potent, selective and efficacious USP7 proteolysis-targeting chimera degrader. XM-U-14 achieves DC50 values of 0.74 nM and Dmax of 93% in inducing USP7 degradation in RS4;11 cell lines, and also significantly inhibits ALL cell growth. XM-U-14 even at 5 mg/kg dosed daily effectively inhibits RS4;11 tumor growth with 64.7% tumor regressions and causes no signs of toxicity in mice. XM-U-14 is a promising USP7 degrader for further optimization for ALL treatment.

Recruitment of FBXO22 for targeted degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Discovery of a Highly Potent and Selective HDAC8 Degrader: Advancing the Functional Understanding and Therapeutic Potential of HDAC8.

HDAC8 plays crucial roles in biological processes, from gene regulation to cell motility, making it a highly desirable target for therapeutic intervention. HDAC8 also has deacetylase-independent activity which cannot be blocked by a conventional inhibitor. In this study, we report the discovery of YX862, a highly potent and selective hydrazide-based HDAC8-proteolysis targeting chimera (PROTAC) degrader. The selectivity is achieved through rational design of the warhead to spare HDAC3 activity from the previous HDAC3/8 dual degrader YX968. We demonstrate that the degradation of HDAC8 by YX862 increases acetylation levels of its nonhistone substrates such as SMC3 without significantly triggering histone PTM, supporting HDAC8's major role in nonhistone PTM regulation. YX862 exhibits promising on-target antiproliferative activity against DLBCL cells with higher potency than the HDAC8 selective inhibitor PCI-34051. As a selective HDAC8 degrader that avoids pan-HDAC inhibition, YX862 represents a valuable tool for exploring the biological and therapeutic potential of HDAC8.

Discovery of a peptide proteolysis-targeting chimera (PROTAC) drug of p300 for prostate cancer therapy

The E1A-associated protein p300 (p300) has emerged as a promising target for cancer therapy due to its crucial role in promoting oncogenic signaling pathways in various cancers, including prostate cancer. This need is particularly significant in prostate cancer. While androgen deprivation therapy (ADT) has demonstrated promising efficacy in prostate cancer, its long-term use can eventually lead to the development of castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC). Notably, p300 has been identified as an important co-activator of the androgen receptor (AR), highlighting its significance in prostate cancer progression. Moreover, recent studies have revealed the involvement of p300 in AR-independent oncogenes associated with NEPC. Therefore, the blockade of p300 may emerge as an effective therapeutic strategy to address the challenges posed by both CRPC and NEPC. We employed AI-assisted design to develop a peptide-based PROTAC (proteolysis-targeting chimera) drug that targets p300, effectively degrading p300 invitro and invivo utilizing nano-selenium as a peptide drug delivery system. Our p300-targeting peptide PROTAC drug demonstrated effective p300 degradation and cancer cell-killing capabilities in both CRPC, AR-negative, and NEPC cells. This study demonstrated the efficacy of a p300-targeting drug in NEPC cells. In both AR-positive and AR-negative mouse models, the p300 PROTAC drug showed potent p300 degradation and tumor suppression. The design of peptide PROTAC drug targeting p300 is feasible and represents an efficient therapeutic strategy for CRPC, AR-negative prostate cancer, and NEPC. The funding details can be found in the Acknowledgements section.

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 bivalent small molecule degraders of cyclin-dependent kinase 7 (CDK7)

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

Discovery of a Highly Potent and Selective BRD9 PROTAC Degrader Based on E3 Binder Investigation for the Treatment of Hematological Tumors.

BRD9 is a pivotal epigenetic factor involved in cancers and inflammatory diseases. Still, the limited selectivity and poor phenotypic activity of targeted agents make it an atypically undruggable target. PROTAC offers an alternative strategy for overcoming the issue. In this study, we explored diverse E3 ligase ligands for the contribution of BRD9 PROTAC degradation. Through molecular docking, binding affinity analysis, and structure-activity relationship study, we identified a highly potent PROTAC E5, with excellent BRD9 degradation (DC50 = 16 pM) and antiproliferation in MV4-11 cells (IC50 = 0.27 nM) and OCI-LY10 cells (IC50 = 1.04 nM). E5 can selectively degrade BRD9 and induce cell cycle arrest and apoptosis. Moreover, the therapeutic efficacy of E5 was confirmed in xenograft tumor models, accompanied by further RNA-seq analysis. Therefore, these results may pave the way and provide the reference for the discovery and investigation of highly effective PROTAC degraders.