Epigenetic balance ensures mechanistic control of MLL amplification and rearrangement.

MLL/KMT2A amplifications and translocations are prevalent in infant, adult, and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here, we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the crosstalk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, in turn promoting amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications.

Structurally Specific Z-DNA Proteolysis Targeting Chimera Enables Targeted Degradation of Adenosine Deaminase Acting on RNA 1.

Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus broadening the scope and repertoire of DNA-based PROTACs. Unlike conventional A- or B-form DNA, Z-form DNA is a configuration that exclusively manifests itself under specific stress conditions and with specific target sequences, which can be recognized by specific reader proteins, such as ADAR1 or ZBP1, to exert downstream biological functions. The core of our innovation lies in the strategic engagement of Z-form DNA with ADAR1 and its degradation is achieved by leveraging a VHL ligand conjugated to Z-form DNA to recruit the E3 ligase. This ingenious construct engendered a series of Z-PROTACs, which we utilized to selectively degrade the Z-DNA-binding protein ADAR1, a molecule that is frequently overexpressed in cancer cells. This meticulously orchestrated approach triggers a cascade of PANoptotic events, notably encompassing apoptosis and necroptosis, by mitigating the blocking effect of ADAR1 on ZBP1, particularly in cancer cells compared with normal cells. Moreover, the Z-PROTAC design exhibits a pronounced predilection for ADAR1, as opposed to other Z-DNA readers, such as ZBP1. As such, Z-PROTAC likely elicits a positive immunological response, subsequently leading to a synergistic augmentation of cancer cell death. In summary, the Z-DNA-based PROTAC (Z-PROTAC) approach introduces a modality generated by the conformational change from B- to Z-form DNA, which harnesses the structural specificity intrinsic to potentiate a selective degradation strategy. This methodology is an inspiring conduit for the advancement of PROTAC-based therapeutic modalities, underscoring its potential for selectivity within the therapeutic landscape of PROTACs to target undruggable proteins.

Chemically induced degradation of epigenetic targets.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that induce the ternary complex formation between a protein-of-interest (POI) and an E3 ligase, leading to targeted polyubiquitination and degradation of the POI. Particularly, PROTACs have the distinct advantage of targeting both canonical and noncanonical functions of epigenetic targets over traditional inhibitors, which typically target canonical functions only, resulting in greater therapeutic efficacy. In this review, we methodically analyze published PROTAC degraders of epigenetic writer, reader, and eraser proteins and their in vitro and in vivo effects. We highlight the mechanism of action of these degraders and their advantages in targeting both canonical and noncanonical functions of epigenetic targets in the context of cancer treatment. Furthermore, we present a future outlook for this exciting field. Overall, pharmacological degradation of epigenetic targets has emerged as an effective and attractive strategy to thwart cancer progression and growth.

Acetylation Targeting Chimera Enables Acetylation of the Tumor Suppressor p53.

With advances in chemically induced proximity technologies, heterobifunctional modalities such as proteolysis targeting chimeras (PROTACs) have been successfully advanced to clinics for treating cancer. However, pharmacologic activation of tumor-suppressor proteins for cancer treatment remains a major challenge. Here, we present a novel Acetylation Targeting Chimera (AceTAC) strategy to acetylate the p53 tumor suppressor protein. We discovered and characterized the first p53Y220C AceTAC, MS78, which recruits histone acetyltransferase p300/CBP to acetylate the p53Y220C mutant. MS78 effectively acetylated p53Y220C lysine 382 (K382) in a concentration-, time-, and p300-dependent manner and suppressed proliferation and clonogenicity of cancer cells harboring the p53Y220C mutation with little toxicity in cancer cells with wild-type p53. RNA-seq studies revealed novel p53Y220C-dependent upregulation of TRAIL apoptotic genes and downregulation of DNA damage response pathways upon acetylation induced by MS78. Altogether, the AceTAC strategy could provide a generalizable platform for targeting proteins, such as tumor suppressors, via acetylation.

Telomere Targeting Chimera Enables Targeted Destruction of Telomeric Repeat-Binding Factor Proteins.

Telomeres are naturally shortened after each round of cell division in noncancerous normal cells, while the activation of telomerase activity to extend telomere in the cancer cell is essential for cell transformation. Therefore, telomeres are regarded as a potential anticancer target. In this study, we report the development of a nucleotide-based proteolysis-targeting chimera (PROTAC) designed to degrade TRF1/2 (telomeric repeat-binding factor 1/2), which are the key components of the shelterin complex (telosome) that regulates the telomere length by directly interacting with telomere DNA repeats. The prototype telomere-targeting chimeras (TeloTACs) efficiently degrade TRF1/2 in a VHL- and proteosome-dependent manner, resulting in the shortening of telomeres and suppressed cancer cell proliferation. Compared to the traditional receptor-based off-target therapy, TeloTACs have potential application in a broad spectrum of cancer cell lines due to their ability to selectively kill cancer cells that overexpress TRF1/2. In summary, TeloTACs provide a nucleotide-based degradation approach for shortening the telomere and inhibiting tumor cell growth, representing a promising avenue for cancer treatment.

Methylated Nucleotide-Based Proteolysis-Targeting Chimera Enables Targeted Degradation of Methyl-CpG-Binding Protein 2.

Methyl-CpG-binding protein 2 (MeCP2), a reader of DNA methylation, has been extensively investigated for its function in neurological and neurodevelopmental disorders. Emerging evidence indicates that MeCP2 exerts an oncogenic function in cancer; however, the endeavor to develop a MeCP2-targeted therapy remains a challenge. This work attempts to address it by introducing a methylated nucleotide-based targeting chimera termed methyl-proteolysis-targeting chimera (methyl-PROTAC). The methyl-PROTAC incorporates a methylated cytosine into an oligodeoxynucleotide moiety to recruit MeCP2 for targeted degradation in a von Hippel-Lindau- and proteasome-dependent manner, thus displaying antiproliferative effects in cancer cells reliant on MeCP2 overexpression. This selective cytotoxicity endows methyl-PROTAC with the capacity to selectively eliminate cancer cells that are addicted to the overexpression of the MeCP2 oncoprotein. Furthermore, methyl-PROTAC-mediated MeCP2 degradation induces apoptosis in cancer cells. These findings underscore the therapeutic potential of methyl-PROTAC to degrade undruggable epigenetic regulatory proteins. In summary, the development of methyl-PROTAC introduces an innovative strategy by designing a modified nucleotide-based degradation approach for manipulating epigenetic factors, thereby representing a promising avenue for the advancement of PROTAC-based therapeutics.

TF-DUBTACs Stabilize Tumor Suppressor Transcription Factors.

Targeted protein degradation approaches have been widely used for degrading oncogenic proteins, providing a potentially promising therapeutic strategy for cancer treatment. However, approaches to targeting tumor suppressor proteins are very limited, and only a few agonists have been developed to date. Here, we report the development of a platform termed TF-DUBTAC, which links a DNA oligonucleotide to a covalent ligand of the deubiquitinase OTUB1 via a click reaction, to selectively stabilize tumor suppressor transcription factors. We developed three series of TF-DUBTACs, namely, FOXO-DUBTAC, p53-DUBTAC, and IRF-DUBTAC, which stabilize FOXO3A, p53, and IRF3 in cells, respectively, in an OTUB1-dependent manner. These results suggest that TF-DUBTAC is a generalizable platform to achieve selective stabilization of tumor suppressor transcription factors as a therapeutic means to suppress tumorigenesis.

Artificial intelligence and machine learning-aided drug discovery in central nervous system diseases: State-of-the-arts and future directions.

Neurological disorders significantly outnumber diseases in other therapeutic areas. However, developing drugs for central nervous system (CNS) disorders remains the most challenging area in drug discovery, accompanied with the long timelines and high attrition rates. With the rapid growth of biomedical data enabled by advanced experimental technologies, artificial intelligence (AI) and machine learning (ML) have emerged as an indispensable tool to draw meaningful insights and improve decision making in drug discovery. Thanks to the advancements in AI and ML algorithms, now the AI/ML-driven solutions have an unprecedented potential to accelerate the process of CNS drug discovery with better success rate. In this review, we comprehensively summarize AI/ML-powered pharmaceutical discovery efforts and their implementations in the CNS area. After introducing the AI/ML models as well as the conceptualization and data preparation, we outline the applications of AI/ML technologies to several key procedures in drug discovery, including target identification, compound screening, hit/lead generation and optimization, drug response and synergy prediction, de novo drug design, and drug repurposing. We review the current state-of-the-art of AI/ML-guided CNS drug discovery, focusing on blood-brain barrier permeability prediction and implementation into therapeutic discovery for neurological diseases. Finally, we discuss the major challenges and limitations of current approaches and possible future directions that may provide resolutions to these difficulties.

TF-PROTACs Enable Targeted Degradation of Transcription Factors.

Transcription factors (TFs) represent a major class of therapeutic targets for the treatment of human diseases including cancer. Although the biological functions and even crystal structures of many TFs have been clearly elucidated, there is still no viable approach to target the majority of TFs, thus rendering them undruggable for decades. PROTACs (proteolysis targeting chimeras) emerge as a powerful class of therapeutic modalities, which rely on induced protein-protein interactions between the proteins of interest (POIs) and E3 ubiquitin ligases to aid the degradation of POIs by the ubiquitin-proteasome system (UPS). Here, we report the development of a platform termed TF-PROTAC, which links an DNA oligonucleotide to an E3 ligase ligand via a click reaction, to selectively degrade the TF of interest. The selectivity of these TF-PROTACs depends on the DNA oligonucleotides utilized that can be specific to the TFs of interest. We have developed two series of VHL-based TF-PROTACs, NF-κB-PROTAC (dNF-κB) and E2F-PROTAC (dE2F), which effectively degrade endogenous p65 and E2F1 proteins in cells, respectively, and subsequently display superior antiproliferative effects in cells. Collectively, our results suggest that TF-PROTACs provide a generalizable platform to achieve selective degradation of TFs and a universal strategy for targeting most "undruggable" TFs.

Cancer Selective Target Degradation by Folate-Caged PROTACs.

PROTACs (proteolysis targeting chimeras) are an emerging class of promising therapeutic modalities that degrade intracellular protein targets by hijacking the cellular ubiquitin-proteasome system. However, potential toxicity of PROTACs in normal cells due to the off-tissue on-target degradation effect limits their clinical applications. Precise control of a PROTAC's on-target degradation activity in a tissue-selective manner could minimize potential toxicity/side-effects. To this end, we developed a cancer cell selective delivery strategy for PROTACs by conjugating a folate group to a ligand of the VHL E3 ubiquitin ligase, to achieve targeted degradation of proteins of interest (POIs) in cancer cells versus noncancerous normal cells. We show that our folate-PROTACs, including BRD PROTAC (folate-ARV-771), MEK PROTAC (folate-MS432), and ALK PROTAC (folate-MS99), are capable of degrading BRDs, MEKs, and ALK, respectively, in a folate receptor-dependent manner in cancer cells. This design provides a generalizable platform for PROTACs to achieve selective degradation of POIs in cancer cells.

Harnessing the E3 Ligase KEAP1 for Targeted Protein Degradation.

Proteolysis targeting chimeras (PROTACs) represent a new class of promising therapeutic modalities. PROTACs hijack E3 ligases and the ubiquitin-proteasome system (UPS), leading to selective degradation of the target proteins. However, only a very limited number of E3 ligases have been leveraged to generate effective PROTACs. Herein, we report that the KEAP1 E3 ligase can be harnessed for targeted protein degradation utilizing a highly selective, noncovalent small-molecule KEAP1 binder. We generated a proof-of-concept PROTAC, MS83, by linking the KEAP1 ligand to a BRD4/3/2 binder. MS83 effectively reduces protein levels of BRD4 and BRD3, but not BRD2, in cells in a concentration-, time-, KEAP1- and UPS-dependent manner. Interestingly, MS83 degrades BRD4/3 more durably than the CRBN-recruiting PROTAC dBET1 in MDA-MB-468 cells and selectively degrades BRD4 short isoform over long isoform in MDA-MB-231 cells. It also displays improved antiproliferative activity than dBET1. Overall, our study expands the limited toolbox for targeted protein degradation.

Inhibitors of Protein Methyltransferases and Demethylases.

Post-translational modifications of histones by protein methyltransferases (PMTs) and histone demethylases (KDMs) play an important role in the regulation of gene expression and transcription and are implicated in cancer and many other diseases. Many of these enzymes also target various nonhistone proteins impacting numerous crucial biological pathways. Given their key biological functions and implications in human diseases, there has been a growing interest in assessing these enzymes as potential therapeutic targets. Consequently, discovering and developing inhibitors of these enzymes has become a very active and fast-growing research area over the past decade. In this review, we cover the discovery, characterization, and biological application of inhibitors of PMTs and KDMs with emphasis on key advancements in the field. We also discuss challenges, opportunities, and future directions in this emerging, exciting research field.

A potent, selective and cell-active allosteric inhibitor of protein arginine methyltransferase†3 (PRMT3).

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell-active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 =31±2 nM, KD =53±2 nM) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well-characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease.

Discovery of a novel, highly potent EZH2 PROTAC degrader for targeting non-canonical oncogenic functions of EZH2.

Aberrant expression of EZH2, the main catalytic subunit of PRC2, has been implicated in numerous cancers, including leukemia, breast, and prostate. Recent studies have highlighted non-catalytic oncogenic functions of EZH2, which EZH2 catalytic inhibitors cannot attenuate. Therefore, proteolysis-targeting chimera (PROTAC) degraders have been explored as an alternative therapeutic approach to suppress both canonical and non-canonical oncogenic activity. Here we present MS8847, a novel, highly potent EZH2 PROTAC degrader that recruits the E3 ligase von Hippel-Lindau (VHL). MS8847 degrades EZH2 in a concentration-, time-, and ubiquitin-proteasome system (UPS)-dependent manner. Notably, MS8847 induces superior EZH2 degradation and anti-proliferative effects in MLL-rearranged (MLL-r) acute myeloid leukemia (AML) cells compared to previously published EZH2 PROTAC degraders. Moreover, MS8847 degrades EZH2 and inhibits cell growth in triple-negative breast cancer (TNBC) cell lines, displays efficacy in a 3D TNBC in vitro model, and has a pharmacokinetic (PK) profile suitable for in vivo efficacy studies. Overall, MS8847 is a valuable chemical tool for the biomedical community to investigate canonical and non-canonical oncogenic functions of EZH2.

Discovery of the First-in-class G9a/GLP PROTAC Degrader.

Aberrantly expressed lysine methyltransferases G9a and GLP, which catalyze mono- and di-methylation of histone H3 lysine 9 (H3K9), have been implicated in numerous cancers. Recent studies have uncovered both catalytic and non-catalytic oncogenic functions of G9a/GLP. As such, G9a/GLP catalytic inhibitors have displayed limited anticancer activity. Here, we report the discovery of the first-in-class G9a/GLP proteolysis targeting chimera (PROTAC) degrader, 10 (MS8709), as a potential anticancer therapeutic. 10 induces G9a/GLP degradation in a concentration-, time, and ubiquitin-proteasome system (UPS)-dependent manner, does not alter the mRNA expression of G9a/GLP and is selective for G9a/GLP over other methyltransferases. Moreover, 10 displays superior cell growth inhibition to the parent G9a/GLP inhibitor UNC0642 in prostate, leukemia, and lung cancer cells and has suitable mouse pharmacokinetic properties for in vivo efficacy studies. Overall, 10 is a valuable chemical biology tool to further investigate the functions of G9a/GLP and a potential therapeutic for treating G9a/GLP-dependent cancers.

Discovery of the First-in-Class G9a/GLP PROTAC Degrader.

Aberrantly expressed lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9), have been implicated in numerous cancers. Recent studies have uncovered both catalytic and noncatalytic oncogenic functions of G9a/GLP. As such, G9a/GLP catalytic inhibitors have displayed limited anticancer activity. Here, we report the discovery of the first-in-class G9a/GLP proteolysis targeting chimera (PROTAC) degrader 10 (MS8709), as a potential anticancer therapeutic. 10 induces G9a/GLP degradation in a concentration-, time-, and ubiquitin-proteasome system (UPS)-dependent manner. Futhermore, 10 does not alter the mRNA expression of G9a/GLP and is selective for G9a/GLP over other methyltransferases. Moreover, 10 displays superior cell growth inhibition to the parent G9a/GLP inhibitor UNC0642 in prostate, leukemia, and lung cancer cells and has suitable mouse pharmacokinetic properties for in vivo efficacy studies. Overall, 10 is a valuable chemical biology tool to further investigate the functions of G9a/GLP and a potential therapeutic for treating G9a/GLP-dependent cancers.

Recent advances in developing degraders & inhibitors of lysine methyltransferases.

Over the last several decades, there has been continued interest in developing novel therapeutic approaches targeting protein lysine methyltransferases (PKMTs). Along with PKMT inhibitors, targeted protein degradation (TPD) has emerged as a promising strategy to attenuate aberrant PKMT activity. Particularly, proteolysis targeting chimeras (PROTACs) effectively eliminate PKMTs of interest, suppressing all enzymatic and non-enzymatic functions. PROTACs and other TPD approaches add new depth to PKMT research and novel therapeutics discovery. This review focuses on recent advances in PKMT degrader and inhibitor development over the last several years.

Discovery of the First Lactate Dehydrogenase Proteolysis Targeting Chimera Degrader for the Treatment of Pancreatic Cancer.

Lactate dehydrogenase (LDH) is a key glycolytic enzyme and biomarker of aggressive cancers. LDHA and LDHB are two main LDH subunits, and both are frequently overexpressed in tumors and essential for tumor growth. A number of LDHA/B small-molecule inhibitors have been developed. Here, we report the discovery of the first LDH proteolysis targeting chimera (PROTAC) degrader, compound 22 (MS6105). 22 potently degraded LDHA in a time- and ubiquitin-proteasome system-dependent manner. Using an unbiased global proteomic study, we confirmed that 22 degraded both LDHA and LDHB significantly. 22 was significantly more potent than the parent LDH inhibitor in suppressing the growth of both quasi-mesenchymal state and epithelial state pancreatic cancer cell lines. Furthermore, 22 was bioavailable in mice through intraperitoneal injection. Overall, 22 could be a valuable chemical tool for the research community to explore pathophysiological functions of LDH in vitro and in vivo.

Inhibition of USP7 induces p53-independent tumor growth suppression in triple-negative breast cancers by destabilizing FOXM1.

Although numerous studies indicate that inhibition of USP7 suppresses tumor growth by activating p53, the precise mechanism by which USP7 contributes to tumor growth through the p53-independent manner is not well understood. p53 is frequently mutated in most triple-negative breast cancers (TNBC), characterized as the very aggressive form of breast cancers with limited treatment options and poor patient outcomes. Here, we found that the oncoprotein Forkhead Box M1 (FOXM1) acts as a potential driver for tumor growth in TNBC and, surprisingly, through a proteomic screen, we identified USP7 as a major regulator of FOXM1 in TNBC cells. USP7 interacts with FOXM1 both in vitro and in vivo. USP7 stabilizes FOXM1 through deubiquitination. Conversely, RNAi-mediated USP7 knockdown in TNBC cells, dramatically reduced the levels of FOXM1. Moreover, based upon the proteolysis targeting chimera (PROTAC) technology, we generated PU7-1 (protein degrader for USP7-1), as a USP7 specific degrader. PU7-1 induces rapid USP7 degradation at low nanomolar concentrations in cells but shows no obvious effect on other USP family proteins. Strikingly, the treatment of TNBC cells with PU7-1 significantly abrogates FOXM1 functions and effectively suppresses cell growth in vitro. By using xenograft mouse models, we found that PU7-1 markedly represses tumor growth in vivo. Notably, ectopic overexpression of FOXM1 can reverse the tumor growth suppressive effects induced by PU7-1, underscored the specific effect on FOXM1 induced by USP7 inactivation. Together, our findings indicate that FOXM1 is a major target of USP7 in modulating tumor growth in a p53-independent manner and reveals the USP7 degrader as a potential therapeutic tool for the treatment of triple-negative breast cancers.

De Novo Design of κ-Opioid Receptor Antagonists Using a Generative Deep Learning Framework.

Likely effective pharmacological interventions for the treatment of opioid addiction include attempts to attenuate brain reward deficits during periods of abstinence. Pharmacological blockade of the κ-opioid receptor (KOR) has been shown to abolish brain reward deficits in rodents during withdrawal, as well as to reduce the escalation of opioid use in rats with extended access to opioids. Although KOR antagonists represent promising candidates for the treatment of opioid addiction, very few potent selective KOR antagonists are known to date and most of them exhibit significant safety concerns. Here, we used a generative deep learning framework for the de novo design of chemotypes with putative KOR antagonistic activity. Molecules generated by models trained with this framework were prioritized for chemical synthesis based on their predicted optimal interactions with the receptor. Our models and proposed training protocol were experimentally validated by binding and functional assays.