Novel Acyl Thiourea-Based Hydrophobic Tagging Degraders Exert Potent Anti-Influenza Activity through Two Distinct Endonuclease Polymerase Acidic-Targeted Degradation Pathways.

The spread of the influenza virus has caused devastating pandemics and huge economic losses worldwide. Antiviral drugs with diverse action modes are urgently required to overcome the challenges of viral mutation and drug resistance, and targeted protein degradation strategies constitute excellent candidates for this purpose. Herein, the first degradation of the influenza virus polymerase acidic (PA) protein using small-molecule degraders developed by hydrophobic tagging (HyT) technology to effectively combat the influenza virus was reported. The SAR results revealed that compound 19b with Boc2-(L)-Lys demonstrated excellent inhibitory activity against A/WSN/33/H1N1 (EC50 = 0.015 µM) and amantadine-resistant strain (A/PR/8/H1N1), low cytotoxicity, high selectivity, substantial degradation ability, and good drug-like properties. Mechanistic studies demonstrated that the proteasome system and autophagic lysosome pathway were the potential drivers of these HyT degraders. Thus, this study provides a powerful tool for investigating the targeted degradation of influenza virus proteins and for antiviral drug development.

Fluorescence theranostic PROTACs for real-time visualization of ERα degradation.

Proteolysis targeting chimera (PROTAC) technology, a groundbreaking strategy for degradation of pathogenic proteins by hijacking of the ubiquitin-proteasome-system has become a promising strategy in drug design. However, the real-time monitoring and visualization of protein degradation processes have been long-standing challenges in the realm of drug development. In this research, we sought to amalgamate the highly efficient protein-degrading capabilities of PROTAC technology with the visualization attributes of fluorescent probes, with the potential to pave the path for the design and development of a novel class of visual PROTACs. These novel PROTACs uniquely possess both fluorescence imaging and therapeutic characteristics, all with the goal of enabling real-time observations of protein degradation processes. Our approach involved the utilization of a high ER-targeting fluorescent probe, previously reported in our laboratory, which served as a warhead that specifically binds to the protein of interest (POI). Additionally, a VHL ligand for recruiting E3 ligase and linkers of various lengths were incorporated to synthesize a series of novel ER-inherent fluorescence PROTACs. Among them, compound A3 demonstrated remarkable efficiency in degrading ERα proteins (DC50 = 0.12 µM) and displaying exceptional anti-proliferative activity against MCF-7 cells (IC50 = 0.051 µM). Furthermore, it exhibited impressive fluorescence imaging performance, boasting an emission wavelength of up to 582 nm, a Stokes shift of 116 nm, and consistent optical properties. These attributes make it especially suitable for the real-time, in situ tracking of ERα protein degradation processes, thus may serve as a privileged visual theranostic PROTAC for ERα+ breast cancer. This study not only broadens the application spectrum of PROTAC technology but also introduces a novel approach for real-time visualization of protein degradation processes, ultimately enhancing the diagnostic and treatment efficacy of PROTACs.

Novel estrogen receptor ß/histone deacetylase dual-targeted near-infrared fluorescent probes as theranostic agents for imaging and treatment of prostate cancer.

Estrogen receptor (ER) ß and histone deacetylases (HDACs), when overexpressed, are associated closely with the occurrence and development of prostate cancer and are, therefore, considered important targets and biomarkers used in the clinical treatment of prostate cancer. The present study involved the design and synthesis of the first ERß and HDAC dual-target near-infrared fluorescent probe with both imaging capacity and antitumor activity for prostate cancer. Both P1 and P2 probes exhibited excellent ERß selectivity, with P1 being almost exclusively selective for ERß compared to ERα. In addition, P1 exhibited good optical properties, such as strong near-infrared emission, large Stokes shift, and better anti-interference ability, along with excellent imaging ability for living cells. P1 also exhibited potent inhibitory activity against HDAC6 and DU-145 cells, with IC50 values of 52 nM and 0.96 µM, respectively. Further, P1 was applied successfully for the in vivo imaging of prostate cancer in a mouse model, and significant in vivo antitumor efficacy was achieved. The developed dual-target NIR fluorescent probe is expected to serve as an effective tool in the research on prostate cancer, leading to novel insights for the theranostic study of diseases related to ERß and HDACs.

Discovery of novel covalent selective estrogen receptor degraders against endocrine-resistant breast cancer.

Endocrine-resistance remains a major challenge in estrogen receptor α positive (ERα+) breast cancer (BC) treatment and constitutively active somatic mutations in ERα are a common mechanism. There is an urgent need to develop novel drugs with new mode of mechanism to fight endocrine-resistance. Given aberrant ERα activity, we herein report the identification of novel covalent selective estrogen receptor degraders (cSERDs) possessing the advantages of both covalent and degradation strategies. A highly potent cSERD 29c was identified with superior anti-proliferative activity than fulvestrant against a panel of ERα+ breast cancer cell lines including mutant ERα. Crystal structure of ERα‒29c complex alongside intact mass spectrometry revealed that 29c disrupted ERα protein homeostasis through covalent targeting C530 and strong hydrophobic interaction collied on H11, thus enforcing a unique antagonist conformation and driving the ERα degradation. These significant effects of the cSERD on ERα homeostasis, unlike typical ERα degraders that occur directly via long side chains perturbing the morphology of H12, demonstrating a distinct mechanism of action (MoA). In vivo, 29c showed potent antitumor activity in MCF-7 tumor xenograft models and low toxicity. This proof-of-principle study verifies that novel cSERDs offering new opportunities for the development of innovative therapies for endocrine-resistant BC.

Rational design and optimization of acylthioureas as novel potent influenza virus non-nucleoside polymerase inhibitors.

Evidence suggests that rapidly evolving virus subvariants risk rendering current vaccines and anti-influenza drugs ineffective. Hence, exploring novel scaffolds or new targets of anti-influenza drugs is of great urgency. Herein, we report the discovery of a series of acylthiourea derivatives produced via a scaffold-hopping strategy as potent antiviral agents against influenza A and B subtypes. The most effective compound 10m displayed subnanomolar activity against H1N1 proliferation (EC50 = 0.8 nM) and exhibited inhibitory activity toward other influenza strains, including influenza B virus and H1N1 variant (H1N1, H274Y). Additionally, druggability evaluation revealed that 10m exhibited favorable pharmacokinetic properties and was metabolically stable in liver microsome preparations from three different species as well as in human plasma. In vitro and in vivo toxicity studies confirmed that 10m demonstrated a high safety profile. Furthermore, 10m exhibited satisfactory antiviral activity in a lethal influenza virus mouse model. Moreover, mechanistic studies indicated that these acylthiourea derivatives inhibited influenza virus proliferation by targeting influenza virus RNA-dependent RNA polymerase. Thus, 10m is a potential lead compound for the further exploration of treatment options for influenza.

Identification of Novel Dual-Target Estrogen Receptor α Degraders with Tubulin Inhibitory Activity for the Treatment of Endocrine-Resistant Breast Cancer.

Endocrine resistance remains a significant problem in the clinical treatment of estrogen receptor α-positive (ERα+) breast cancer (BC). In this study, we developed a series of novel dual-functional ERα degraders based on a bridged bicyclic scaffold with selenocyano (SeCN) side chains. These compounds displayed potent ERα degradation and tubulin depolymerization activity. Among them, compounds 35s and 35t exhibited the most promising antiproliferative and ERα degradation activity in multiple ERα+ BC cell lines bearing either wild-type or mutant ERα. Meanwhile, compounds 35s and 35t disrupted the microtubule network by restraining tubulin polymerization, evidenced by 35t inducing cell cycle arrest in the G2/M phase. In MCF-7 and LCC2 xenograft models, compounds 35s and 35t remarkably suppressed tumor growth without noticeable poisonousness. Finally, this study provided guidance for developing new dual-target antitumor drug candidates for the ERα+ BC therapy, especially for the resistant variant.

Antiviral PROTACs: Opportunity borne with challenge.

Proteolysis targeting chimera (PROTAC) degradation of pathogenic proteins by hijacking of the ubiquitin-proteasome-system has become a promising strategy in drug design. The overwhelming advantages of PROTAC technology have ensured a rapid and wide usage, and multiple PROTACs have entered clinical trials. Several antiviral PROTACs have been developed with promising bioactivities against various pathogenic viruses. However, the number of reported antiviral PROTACs is far less than that of other diseases, e.g., cancers, immune disorders, and neurodegenerative diseases, possibly because of the common deficiencies of PROTAC technology (e.g., limited available ligands and poor membrane permeability) plus the complex mechanism involved and the high tendency of viral mutation during transmission and replication, which may challenge the successful development of effective antiviral PROTACs. This review highlights the important advances in this rapidly growing field and critical limitations encountered in developing antiviral PROTACs by analyzing the current status and representative examples of antiviral PROTACs and other PROTAC-like antiviral agents. We also summarize and analyze the general principles and strategies for antiviral PROTAC design and optimization with the intent of indicating the potential strategic directions for future progress.

Discovery of a Novel Class of PROTACs as Potent and Selective Estrogen Receptor α Degraders to Overcome Endocrine-Resistant Breast Cancer In Vitro and In Vivo.

The estrogen receptor (ER) is a well-established target for endocrine therapies of ER-positive breast cancer (ER+ BC), but endocrine resistance limits the efficacy of clinical drugs. Using proteolysis targeting chimera (PROTAC) technology to degrade ERα may be an effective alternative to endocrine therapies. Herein, we disclose a novel series of potent and selective ERα PROTACs based on an oxabicycloheptane sulfonamide (OBHSA) scaffold, with no associated ERß degradation. These PROTACs showed significant antiproliferation and ERα degradation activities against a broad spectrum of ER+ BC cells including tamoxifen-resistant and ERα mutant cell lines. Genomics analysis confirmed that these PROTACs inhibited the nascent RNA synthesis of ERα target genes and impaired genome-wide ERα binding. Compound ZD12 exhibited excellent antitumor potency and ERα degradation activity in both tamoxifen-sensitive and -resistant BC mice models, which are superior to fulvestrant. This study demonstrates the potential of these PROTACs as novel drug candidates for endocrine-resistant BC treatment.

CDK12 and Integrator-PP2A complex modulates LEO1 phosphorylation for processive transcription elongation.

Cyclin-dependent kinase 12 (CDK12) interacts with cyclin K to form a functional nuclear kinase that promotes processive transcription elongation through phosphorylation of the C-terminal domain of RNA polymerase II (Pol II). To gain a comprehensive understanding of CDK12's cellular function, we used chemical genetic and phosphoproteomic screening to identify a landscape of nuclear human CDK12 substrates, including regulators of transcription, chromatin organization, and RNA splicing. We further validated LEO1, a subunit of the polymerase-associated factor 1 complex (PAF1C), as a bona fide cellular substrate of CDK12. Acute depletion of LEO1, or substituting LEO1 phosphorylation sites with alanine, attenuated PAF1C association with elongating Pol II and impaired processive transcription elongation. Moreover, we discovered that LEO1 interacts with and is dephosphorylated by the Integrator-PP2A complex (INTAC) and that INTAC depletion promotes the association of PAF1C with Pol II. Together, this study reveals an uncharacterized role for CDK12 and INTAC in regulating LEO1 phosphorylation, providing important insights into gene transcription and its regulation.

Structure-guided identification of novel dual-targeting estrogen receptor α degraders with aromatase inhibitory activity for the treatment of endocrine-resistant breast cancer.

Drug resistance is a major challenge in conventional endocrine therapy for estrogen receptor (ER) positive breast cancer (BC). BC is a multifactorial disease, in which simultaneous aromatase (ARO) inhibition and ERα degradation may effectively inhibit the signal transduction of both proteins, thus potentially overcoming drug resistance caused by overexpression or mutation of target proteins. In this study, guided by the X-ray structure of a hit compound 30a in complex with ER-Y537S, a structure-based optimization was performed to get a series of multiacting inhibitors targeting both ERα and ARO, and finally a novel class of potent selective estrogen receptor degraders (SERDs) based on a three-dimensional oxabicycloheptene sulfonamide (OBHSA) scaffold equipped with aromatase inhibitor (AI) activity were identified. Of these dual-targeting SERD-AI hybrids, compound 31q incorporating a 1H-1,2,4-triazole moiety showed excellent ERα degradation activity, ARO inhibitory activity and remarkable antiproliferative activity against BC resistant cells. Furthermore, 31q manifested efficient tumor suppression in MCF-7 tumor xenograft models. Taken together, our study reported for the first time the highly efficient dual-targeting SERD-AI hybrid compounds, which may lay the foundation of translational research for improved treatment of endocrine-resistant BC.

A novel selective estrogen receptor degrader induces cell cycle arrest in breast cancer via ERα degradation and the autophagy-lysosome pathway.

Breast cancer (BC), a well-known estrogen-dependent cancer, is the most common cancer among women and the leading cause of cancer deaths. One of the most important therapeutic approaches for BC is endocrine therapy targeting estrogen receptor alpha (ERα) and thus blocking the estrogen receptor signaling pathway. Drugs, such as tamoxifen or fulvestrant, are developed based on this theory and have benefited numerous patients with BC for many years. However, many patients with advanced BC, such as tamoxifen-resistant BC, cannot benefit from these developed drugs anymore. Therefore, new drugs targeting ERα are urgently needed by patients with BC. Recently, elacestrant, a novel selective estrogen receptor degrader (SERD), was approved by the United States Food and Drug Administration (FDA), highlighting the importance of ERα degradation in endocrine therapy. Proteolysis targeting chimera (PROTAC) has been considered a powerful technique for targeting protein degradation (TPD). In this regard, we developed and studied a novel ERα degrader, which is a PROTAC-like SERD named 17e. We found that compound 17e can inhibit the growth of BC both in vitro and in vivo and induce the cell cycle arrest of BC. Importantly, 17e displayed no apparent toxicity toward healthy kidney and liver cells. Moreover, we observed that the presence of 17e led to a dramatic increase in the autophagy-lysosome pathway in an ERα-independent manner. Finally, we revealed that a decrease in MYC, a frequent deregulation oncogene in human cancers, was mediated by both ERα degradation and autophagy activation in the presence of 17e. Collectively, we discovered that compound 17e induced ERα degradation and exerts significant anti-cancer effects on BC mainly through promoting the autophagy-lysosome pathway and decreasing MYC level.

Identification of a novel acylthiourea-based potent broad-spectrum inhibitor for enterovirus 3D polymerase in vitro and in vivo.

Enterovirus infections have become a serious public health threat to young children, leading to hand-foot-and-mouth disease and more severe nervous system diseases. Due to the lack of licensed anti enterovirus drugs, we reported herein that a Tenovin-1 analog, acylthiourea-based 4-(tert-butyl)-N-((4-(4-(tert-butyl)benzamido)phenyl)carbamothioyl) benzamide (AcTU), displayed low nanomolar anti-EV-A71 activity with an EC50 of 1.0 nM in RD cells. Moreover, AcTU exhibited nanomolar to picomolar inhibitory activity against a series of enteroviruses including EV-D68, CV-A21, CV-A16 and CV-B1 (EC50 = 0.75-17.15 nM). Mechanistic studies indicated that AcTU inhibited enterovirus proliferation by targeting 3D polymerase. In addition, AcTU displayed moderate pharmacokinetic properties in rats (F = 7.4%, T1/2 = 3.26 h), and in vivo protection studies demonstrated that AcTU orally administered at 0.6 mg/kg/d was highly protective against lethal EV-A71 challenge in mice, potentially reducing mortality from 100% to 20% as well as alleviating symptoms. These results suggested that AcTU could be a potent clinical candidate for the treatment of enterovirus infections.

Anticancer or carcinogenic? The role of estrogen receptor ß in breast cancer progression.

Estrogen receptor ß (ERß) is closely related to breast cancer (BC) progression. Traditional concepts regard ERß as a tumor suppressor. As studies show the carcinogenic effect of ERß, some people have come to a new conclusion that ERß serves as a tumor suppressor in estrogen receptor α (ERα)-positive breast cancer, while it is a carcinogen in ERα-negative breast cancer. However, we re-examine the role of ERß and find this conclusion to be misleading based on the last decade's research. A large number of studies have shown that ERß plays an anticancer role in both ERα-positive and ERα-negative breast cancers, and its carcinogenicity does not depend solely on the presence of ERα. Herein, we review the anticancer and oncogenic effects of ERß on breast cancer progression in the past ten years, discuss the mechanism respectively, analyze the main reasons for the inconsistency and update ERß selective ligand library. We believe a detailed and continuously updated review will help correct the one-sided understanding of ERß, promoting ERß-targeted breast cancer therapy.

Estrogen receptor ß-targeted hypoxia-responsive near-infrared fluorescence probes for prostate cancer study.

Aberrant expression of estrogen receptor ß (ERß) and tumor hypoxia have been observed in castration-resistant prostate cancer (CRPC); therefore, hypoxia-responsive labeling of ERß will be beneficial for the early diagnosis and treatment of CRPC. Herein, we report the first ERß-targeted hypoxia-responsive near-infrared fluorescent probes, which showed superior ERß selectivity and favorable optical properties. These two probes exhibited excellent hypoxia responsiveness and specific mitochondrial ERß imaging ability in CRPC cells. In addition, P1 displayed strong anti-interference ability and good tumor imaging capacity in vivo, contributing to effective diagnosis of CRPC. Mechanistic studies, including high resolution mass spectrometry (HRMS) and density functional theory (DFT) calculations, showed that the introduction of a nitro group quenched the probe fluorescence by inducing a PET effect, while in the hypoxic tumor microenvironment, reduction of the nitro group blocked the PET effect and turned on the probe fluorescence. These novel ERß-targeted hypoxia-responsive near-infrared fluorescent probes may promote the study of prostate cancer.

Discovery of Novel Bicyclic Phenylselenyl-Containing Hybrids: An Orally Bioavailable, Potential, and Multiacting Class of Estrogen Receptor Modulators against Endocrine-Resistant Breast Cancer.

Breast cancer (BC) is a multifactorial disease and is prone to drug resistance during treatment. In this study, we described a new class of multifunctional estrogen receptor (ER) modulators ground on a prerogative indirect antagonism skeleton (OBHS, oxabicycloheptene sulfonate) of ER containing a phenylselenyl group. Compound 34b showed significant antiproliferative activities against tamoxifen-sensitive (MCF-7) and -resistant (LCC2) cells. Moreover, hexokinase 1 (HK1) was identified as a direct target of 34b. Further mechanism investigations proved that 34b induced apoptosis, which was associated with mitochondrial dysfunction caused by the synergistic effects of downregulating mitochondrial-bound HK1 protein and promoting reactive oxygen species generation. In vivo, 34b had a favorable pharmacokinetic profile with a bioavailability of 23.20% and exhibited more potent tumor suppression than tamoxifen both in MCF-7 and LCC2 tumor xenograft models. Collectively, our studies showed that 34b is a promising new multifunctional candidate compound for ERα+ BC treatment, particularly for tamoxifen-resistant BC.

Discovery of aminothiazole derivatives as novel human enterovirus A71 capsid protein inhibitors.

Enterovirus A71 (EV-A71), one of the major pathogens that causes hand, foot and mouth disease (HFMD), has seriously threatened the health and safety of young children. In this study, aminothiazole derivatives were synthesized and screened against EV-A71 in Rhabdomyosarcoma (RD) cells. The best compound (12s), with a biphenyl group, showed activity against EV-A71 (EC50: 0.27 µM) but also against a series of different human enteroviruses without significant cytotoxicity (CC50 > 56.2 µM). Mechanistic studies including time-of-drug-addition assays, viral entry assays and microscale thermophoresis (MST) experiments, showed that 12s binds to EV-A71 capsid and blocks the binding between the viral protein VP1 and the relevant human scavenger receptor class B member 2 (hSCARB2).

Discovery of Aryl Benzoyl Hydrazide Derivatives as Novel Potent Broad-Spectrum Inhibitors of Influenza A Virus RNA-Dependent RNA Polymerase (RdRp).

Influenza A viruses possess a high antigenic shift, and the approved anti-influenza drugs are extremely limited, which makes the development of novel anti-influenza drugs for the clinical treatment and prevention of influenza outbreaks imperative. Herein, we report a series of novel aryl benzoyl hydrazide analogs as potent anti-influenza agents. Particularly, analogs 10b, 10c, 10g, 11p, and 11q exhibited potent inhibitory activity against the avian H5N1 flu strain with EC50 values ranging from 0.009 to 0.034 µM. Moreover, compound 11q exhibited nanomolar antiviral effects against both the H1N1 virus and Flu B virus and possessed good oral bioavailability and inhibitory activity against influenza A virus in a mouse model. Preliminary mechanistic studies suggested that these compounds exert anti-influenza virus effects mainly by interacting with the PB1 subunit of RNA-dependent RNA polymerase (RdRp). These results revealed that 11q has the potential to become a potent clinical candidate to combat seasonal influenza and influenza pandemics.

Design and synthesis of marine sesterterpene analogues as novel estrogen receptor α degraders for breast cancer treatment.

Targeted protein degradation using small molecules is an intriguing strategy for drug development. The marine sesterterpene compound MHO7 had been reported to be a potential ERα degradation agent. In order to further improve its biological activity, two series of novel MHO7 derivatives with long side chains were designed and identified as novel selective estrogen receptor down-regulators (SERDs). The growth inhibition activity of the novel SERD compounds were significantly affected by the type and length of the side chain. Most of the derivatives were significantly more potent than MHO7 against both drug-sensitive and drug-resistant breast cancer cells. Among them, compound 16a, with IC50 values of 0.41 µM against MCF-7 cell lines and 9.6-fold stronger than MHO7, was the most potential molecule. A whole-genome transcriptomic analysis of MCF-7 cells revealed that the mechanism of 16a against MCF-7 cell was similar with that of MHO7. The estrogen signaling pathway was the most affected among the disturbed genes, but the ERα degradation activity of 16a was observed higher than that of MHO7. Other effects of 16a were confirmed similar with MHO7, which means that the basic mechanisms of the derivatives are the same with the ophiobolin backbone, i.e. the degradation of ERα is mediated via proteasome-mediated process, the induction of apoptosis and the cell cycle arrest at the G1 phase. Meanwhile, a decrease of mitochondrial membrane potential and an increase of cellular ROS were also detected. Based on these results, as a novel modified ophiobolin derived compound, 16a may warrant further exploitation as a promising SERD candidate agent for the treatment of breast cancer.

Estrogen Receptor ß-Targeted Near-Infrared Inherently Fluorescent Probe: A Potent Tool for Estrogen Receptor ß Research.

Estrogen receptor ß (ERß) is associated with many diseases, and ERß probes can help to reveal the complex role of ERß and promote the development of ERß-targeted therapy. Herein, we designed and synthesized the first ERß-targeted near-infrared (NIR) inherently fluorescent probe P5, which showed the advantages of high ERß selectivity, good optical properties, and excellent ERß imaging capability in living cells. The probe was successfully utilized to explore ERß motion characteristic, and for the first time, the diffusion coefficient of ERß was obtained. Moreover, P5 was also successfully applied to the in vivo imaging of ERß in the prostate cancer mice model. Therefore, this ERß-targeted NIR probe might be employed as a potential tool for the research of ERß and related diseases.

Discovery of oseltamivir-based novel PROTACs as degraders targeting neuraminidase to combat H1N1 influenza virus.

Annual and sporadic influenza outbreaks pose a great threat to human health and the economy worldwide. Moreover, the frequent mutation of influenza viruses caused by antigen drift complicates the application of antiviral therapeutics. As such, there is an urgent need for novel antiviral agents to tackle the problem of insufficient efficacy of licensed drugs. Inspired by the success of the newly emerged PROTACs (PROteolysis TArgeting Chimeras) strategy, we report herein the design and synthesis of novel PROTAC molecules based on an oseltamivir scaffold to combat severe annual influenza outbreaks. Among these, several compounds showed good anti-H1N1 activity and efficient influenza neuraminidase (NA) degradation activity. The best compound, 8e, effectively induced influenza NA degradation in a dose-dependent manner and relied on the ubiquitin-proteasome pathway. Moreover, Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain (H1N1, H274Y). A molecular docking study demonstrated that Compound 8e had good hydrogen-bonding and hydrophobic interactions with both the active sites of NA and Von Hippel-Lindau (VHL) proteins, which could effectively drive the favorable interaction of these two proteins. Thus, as the first report of a successful anti-influenza PROTAC, this proof of concept will greatly widen the application range of the PROTAC technique to antiviral drug discovery.