Structural Optimization and Structure-Activity Relationship of 1H-Pyrazole-4-carboxylic Acid Derivatives as DNA 6mA Demethylase ALKBH1 Inhibitors and Their Antigastric Cancer Activity.

DNA N6-methyladenine (6mA) demethylase ALKBH1 plays an important role in various cellular processes. Dysregulation of ALKBH1 is associated with the development of some cancer types, including gastric cancer, implicating a potential therapeutic target. However, there is still a lack of potent ALKBH1 inhibitors. Herein, we report the discovery of a highly potent ALKBH1 inhibitor, 1H-pyrazole-4-carboxylic acid derivative 29. The structure-activity relationship of this series of compounds was also discussed. Because of the poor cell membrane permeability of 29, we prepared a prodrug of 29 (29E), which showed excellent cellular activities. In gastric cancer cell lines HGC27 and AGS, 29E treatment significantly increased the abundance of 6mA, inhibited cell viability, and upregulated the AMP-activated protein kinase (AMPK) signaling pathway. In addition, the hydrolysis product 29 showed high exposure in mice after administration of 29E. Collectively, this research provides a new potent ALKBH1 inhibitor, which could serve as a lead compound for subsequent drug development.

Loss of ADAR1 induces ferroptosis of breast cancer cells.

Adenosine deaminases acting on RNA 1(ADAR1), an RNA editing enzyme that converts adenosine to inosine by deamination in double-stranded RNAs, plays an important role in occurrence and progression of various types of cancer. Ferroptosis has emerged as a hot topic of cancer research in recent years. We have previously reported that ADAR1 promotes breast cancer progression by regulating miR-335-5p and METTL3. However, whether ADAR1 has effects on ferroptosis in breast cancer cells is largely unknown. In this study, we knocked down ADAR1 using CRISPR-Cas9 technology or over-expressed ADAR1 protein using plasmid expressing ADAR1 in MCF-7 and MDA-MB-231 breast cancer cell lines, then detected cell viability, and levels of ROS, MDA, GSH, Fe2+, GPX4 protein and miR-335-5p. We showed that the cell proliferation was inhibited, levels of ROS, MDA, Fe2+, and miR-335-5p were increased, while GSH and GPX4 levels were decreased after loss of ADAR1, compared to the control group. The opposite effects were observed after ADAR1 overexpression in the cells. Further, we demonstrated that ADAR1-controlled miR-335-5p targeted Sp1 transcription factor of GPX4, a known ferroptosis molecular marker, leading to inhibition of ferroptosis by ADAR1 in breast cancer cells. Moreover, RNA editing activity of ADAR1 is not essential for inducing ferroptosis. Collectively, loss of ADAR1 induces ferroptosis in breast cancer cells by regulating miR-335-5p/Sp1/GPX4 pathway. The findings may provide insights into the mechanism by which ADAR1 promotes breast cancer progression via inhibiting ferroptosis.

Identification of a new class of activators of the Hippo pathway with antitumor activity in vitro and in vivo.

The Hippo pathway is a key regulator of tissue growth, organ size, and tumorigenesis. Activating the Hippo pathway by gene editing or pharmaceutical intervention has been proven to be a new therapeutic strategy for treatment of the Hippo pathway-dependent cancers. To now, a number of compounds that directly target the downstream effector proteins of Hippo pathway, including YAP and TEADs, have been disclosed, but very few Hippo pathway activators are reported. Here, we discovered a new class of Hippo pathway activator, YL-602, which inhibited CTGF expression in cells irrespective of cell density and the presence of serum. Mechanistically, YL-602 activates the Hippo pathway via MST1/2, which is different from known activators of Hippo pathway. In vitro, YL-602 significantly induced tumor cell apoptosis and inhibited colony formation of tumor cells. In vivo, oral administration of YL-602 substantially suppressed the growth of cancer cells by activation of Hippo pathway. Overall, YL-602 could be a promising lead compound, and deserves further investigation for its mechanism of action and therapeutic applications.

An integrative pan-cancer bioinformatics analysis of MSRB1 and its association with tumor immune microenvironment, prognosis, and immunotherapy.

Methionine sulfoxide reductase B1 (MSRB1) is involved in the development and immune regulation of multiple tumors. However, the role of MSRB1 in the tumor microenvironment and its potential as a therapeutic target remain largely unknown. In this study, MSRB1 expression patterns were evaluated using pan-cancer RNA sequencing data from multiple cell lines, tissues, and single cells. The pan-cancer prognostic role of MSRB1 was assessed and the association between MSRB1 expression and certain cancer characteristics was analyzed. We showed that MSRB1 expression levels were increased in several types of cancer (P < 0.05) and in certain cell types (macrophages, dendritic cells, and malignant tumor cells). The upregulation of MSRB1 expression was due to DNA copy number amplification. Furthermore, MSRB1 was significantly associated with the activation of immune pathways (P < 0.05, NES > 0), immune cell infiltration, and expression of immune checkpoint molecules. In addition, high expression of MSRB1 was found in a series of in vivo and in vitro immunotherapy response models (P < 0.05), and showed resistance to most targeted drugs. Our results indicated that MSRB1 may regulate the tumor immune microenvironment through an immunoresponse and potentially influence cancer development. This could make it a promising predictive biomarker and therapeutic target for precise tumor immunotherapy.

From Hit to Lead: Structure-Based Optimization of Novel Selective Inhibitors of Receptor-Interacting Protein Kinase 1 (RIPK1) for the Treatment of Inflammatory Diseases.

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of cellular necroptosis, which is considered as an important therapeutic target for necroptosis-related indications. Herein, we report the structural optimization and structure-activity relationship investigations of a series of eutectic 5-substituted-indole-3-carboxamide derivatives. The prioritized compound 10b exhibited low nanomolar IC50 values against RIPK1 and showed good kinase selectivity. Based on its eutectic structure, 10b occupied both the allosteric and ATP binding pockets of RIPK1, making it a potent dual-mode inhibitor of RIPK1. In vitro, 10b had a potent protective effect against necroptosis in cells. Compound 10b also provided robust protection in a TNFα-induced systemic inflammatory response syndrome (SIRS) model and imiquimod (IMQ)-induced psoriasis model. It also showed good pharmacokinetic properties and low toxicity. Overall, 10b is a promising lead compound for drug discovery targeting RIPK1 and warrants further study.

Discovery of a Hidden Pocket beneath the NES Groove by Novel Noncovalent CRM1 Inhibitors.

Protein localization is frequently manipulated to favor tumor initiation and progression. In cancer cells, the nuclear export factor CRM1 is often overexpressed and aberrantly localizes many tumor suppressors via protein-protein interactions. Although targeting protein-protein interactions is usually challenging, covalent inhibitors, including the FDA-approved drug KPT-330 (selinexor), were successfully developed. The development of noncovalent CRM1 inhibitors remains scarce. Here, by shifting the side chain of two methionine residues and virtually screening against a large compound library, we successfully identified a series of noncovalent CRM1 inhibitors with a stable scaffold. Crystal structures of inhibitor-protein complexes revealed that one of the compounds, B28, utilized a deeply hidden protein interior cavity for binding. SAR analysis guided the development of several B28 derivatives with enhanced inhibition on nuclear export and growth of multiple cancer cell lines. This work may benefit the development of new CRM1-targeted therapies.

Repression of LSD1 potentiates homologous recombination-proficient ovarian cancer to PARP inhibitors through down-regulation of BRCA1/2 and RAD51.

Poly (ADP-ribose) polymerase inhibitors (PARPi) are selectively active in ovarian cancer (OC) with homologous recombination (HR) deficiency (HRD) caused by mutations in BRCA1/2 and other DNA repair pathway members. We sought molecular targeted therapy that induce HRD in HR-proficient cells to induce synthetic lethality with PARPi and extend the utility of PARPi. Here, we demonstrate that lysine-specific demethylase 1 (LSD1) is an important regulator for OC. Importantly, genetic depletion or pharmacological inhibition of LSD1 induces HRD and sensitizes HR-proficient OC cells to PARPi in vitro and in multiple in vivo models. Mechanistically, LSD1 inhibition directly impairs transcription of BRCA1/2 and RAD51, three genes essential for HR, dependently of its canonical demethylase function. Collectively, our work indicates combination with LSD1 inhibitor could greatly expand the utility of PARPi to patients with HR-proficient tumor, warranting assessment in human clinical trials.

Ligand recognition and G-protein coupling of trace amine receptor TAAR1.

Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders.

A New Type of Endometrial Cancer Models in Mice Revealing the Functional Roles of Genetic Drivers and Exploring their Susceptibilities.

Endometrial cancer (EC) is the most common female reproductive tract cancer and its incidence has been continuously increasing in recent years. The underlying mechanisms of EC tumorigenesis remain unclear, and efficient target therapies are lacking, for both of which feasible endometrial cancer animal models are essential but currently limited. Here, an organoid and genome editing-based strategy to generate primary, orthotopic, and driver-defined ECs in mice is reported. These models faithfully recapitulate the molecular and pathohistological characteristics of human diseases. The authors names these models and similar models for other cancers as organoid-initiated precision cancer models (OPCMs). Importantly, this approach can conveniently introduce any driver mutation or a combination of driver mutations. Using these models,it is shown that the mutations in Pik3ca and Pik3r1 cooperate with Pten loss to promote endometrial adenocarcinoma in mice. In contrast, the Kras G12D mutati led to endometrial squamous cell carcinoma. Then, tumor organoids are derived from these mouse EC models and performed high-throughput drug screening and validation. The results reveal distinct vulnerabilities of ECs with different mutations. Taken together, this study develops a multiplexing approach to model EC in mice and demonstrates its value for understanding the pathology of and exploring the potential treatments for this malignancy.

Pharmacological inhibition of LSD1 suppresses growth of hepatocellular carcinoma by inducing GADD45B.

Lysine-specific histone demethylase 1 (LSD1) is an attractive target for malignancies therapy. Nevertheless, its role in hepatocellular carcinoma (HCC) progression and the potential of its inhibitor in HCC therapy remains unclear. Here, we show that LSD1 overexpression in human HCC tissues is associated with HCC progression and poor patient survival. ZY0511, a highly selective and potent inhibitor of LSD1, suppressed human HCC cell proliferation in vitro and tumor growth in cell-derived and patient-derived HCC xenograft models in vivo. Mechanistically, ZY0511 induced mRNA expression of growth arrest and DNA damage-inducible gene 45beta (GADD45B) by inducing histone H3 at lysine 4 (H3K4) methylation at the promoter of GADD45B, a novel target gene of LSD1. In human HCC tissues, LSD1 level was correlated with a decreased level of GADD45B, which was associated with HCC progression and predicted poor patient survival. Moreover, co-administration of ZY0511 and DTP3, which specifically enhanced the pro-apoptotic effect of GADD45B, effectively inhibited HCC cell proliferation both in vitro and in vivo. Collectively, our study revealed the potential value of LSD1 as a promising target of HCC therapy. ZY0511 is a promising candidate for HCC therapy through upregulating GADD45B, thereby providing a novel combinatorial strategy for treating HCC.

Tumor PKCδ instigates immune exclusion in EGFR-mutated non-small cell lung cancer.

BACKGROUND: The recruitment of a sufficient number of immune cells to induce an inflamed tumor microenvironment (TME) is a prerequisite for effective response to cancer immunotherapy. The immunological phenotypes in the TME of EGFR-mutated lung cancer were characterized as non-inflamed, for which immunotherapy is largely ineffective. METHODS: Global proteomic and phosphoproteomic data from lung cancer tissues were analyzed aiming to map proteins related to non-inflamed TME. The ex vivo and in vivo studies were carried out to evaluate the anti-tumor effect. Proteomics was applied to identify the potential target and signaling pathways. CRISPR-Cas9 was used to knock out target genes. The changes of immune cells were monitored by flow cytometry. The correlation between PKCδ and PD-L1 was verified by clinical samples. RESULTS: We proposed that PKCδ, a gatekeeper of immune homeostasis with kinase activity, is responsible for the un-inflamed phenotype in EGFR-mutated lung tumors. It promotes tumor progression by stimulating extracellular matrix (ECM) and PD-L1 expression which leads to immune exclusion and assists cancer cell escape from T cell surveillance. Ablation of PKCδ enhances the intratumoral penetration of T cells and suppresses the growth of tumors. Furthermore, blocking PKCδ significantly sensitizes the tumor to immune checkpoint blockade (ICB) therapy (αPD-1) in vitro and in vivo model. CONCLUSIONS: These findings revealed that PKCδ is a critical switch to induce inflamed tumors and consequently enhances the efficacy of ICB therapy in EGFR-mutated lung cancer. This opens a new avenue for applying immunotherapy against recalcitrant tumors.

Discovery of Small Molecule Agonist of Gonadotropin-Releasing Hormone Receptor (GnRH1R).

The gonadotrophin-releasing hormone (GnRH) is a central regulator of the human reproductive system and exerts physiological effects by binding to GnRH1R. The GnRH-GnRH1R system is a promising therapeutic target for the maintenance of reproductive function. There are several GnRH1R agonists on the market, but like GnRH, they are all peptide compounds and are limited by their way of administration (subcutaneous or intramuscular injection). To date, no published GnRH1R small molecule agonists have been reported. In this paper, the HTRF-based screening method has been used to screen our in-house chemical library, and we found and confirmed CD304 as a hit compound. Subsequently, structure optimization led to the discovery of compound 6d, exhibited with a certain GnRH1R activation activity (EC50: 1.59 ± 0.38 µM). Further molecular dynamics simulation experiments showed that 6d can well bind to the orthosteric site of GnRH1R through forming a hydrogen-bonding interaction with Y2836.51. Binding of 6d further induces conformational changes in TM6 and TM7, promoting the formation of a continuous water channel in GnRH1R, thereby promoting GnRH1R activation. This well-characterized hit compound will facilitate the further development of novel small molecule agonists of GnRH1R.

RNA Editing Enzyme ADAR1 Regulates METTL3 in an Editing Dependent Manner to Promote Breast Cancer Progression via METTL3/ARHGAP5/YTHDF1 Axis.

A-to-I RNA editing and m6A modification are two of the most prevalent types of RNA modifications controlling gene expression in mammals and play very important roles in tumorigenesis and tumor progression. However, the functional roles and correlations of these two RNA modifications remain to be further investigated in cancer. Herein, we show that ADAR1, an A-to-I RNA-editing enzyme, interacts with METTL3 and increases its protein level to promote the proliferation, migration and invasion of breast cancer cells through a mechanism connecting ADAR1, METTL3 and YTHDF1. We show that both ADAR1 and METTL3 are upregulated in breast cancer samples, and ADAR1 positively correlates with METTL3; ADAR1 edits METTL3 mRNA and changes its binding site to miR532-5p, leading to increased METTL3 protein, which further targets ARHGAP5, recognized by YTHDF1. Additionally, we show that loss of ADAR1 significantly inhibits breast cancer growth in vivo. Collectively, our findings identify the ADAR1-METTL3 axis as a novel, important pathway that connects A-to-I editing and m6A RNA modifications during breast cancer progression.

Discovery of 2-vinyl-10H-phenothiazine derivatives as a class of ferroptosis inhibitors with minimal human Ether-a-go-go related gene (hERG) activity for the treatment of DOX-induced cardiomyopathy.

Ferroptosis was an iron-dependent, nonapoptotic form of regulated cell death. In our previous study, we discovered a potent ferroptosis inhibitor with phenothiazine scaffold (1), but subsequent investigation showed that this compound had potent hERG binding affinity. Herein, we report the discovery of a series of 2-vinyl-10H-phenothiazine derivatives as new class of ferroptosis inhibitors. Structure-activity relationship (SAR) analyses led to the identification of compound 7j, which exhibited significantly reduced hERG inhibition (IC50 > 30 µM) while maintaining high ferroptosis inhibitory activity (EC50 = 0.001 µM on the erastin-induced HT1080 cell ferroptosis model). Further studies confirmed 7j acted as a ROS scavenger and could relieve DOX-induced cardiomyopathy. 7j also displayed favorable pharmacokinetic properties and exhibited no obvious toxicity in vivo and vitro. Overall, this study provides a promising lead compound for drug discovery targeting ferroptosis.

Loss of PRMT7 reprograms glycine metabolism to selectively eradicate leukemia stem cells in CML.

Our group has reported previously on the role of various members of the protein arginine methyltransferase (PRMT) family, which are involved in epigenetic regulation, in the progression of leukemia. Here, we explored the role of PRMT7, given its unique function within the PRMT family, in the maintenance of leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). Genetic loss of Prmt7, and the development and testing of a small-molecule specific inhibitor of PRMT7, showed that targeting PRMT7 delayed leukemia development and impaired self-renewal of LSCs in a CML mouse model and in primary CML CD34+ cells from humans without affecting normal hematopoiesis. Mechanistically, loss of PRMT7 resulted in reduced expressions of glycine decarboxylase, leading to the reprograming of glycine metabolism to generate methylglyoxal, which is detrimental to LSCs. These findings link histone arginine methylation with glycine metabolism, while suggesting PRMT7 as a potential therapeutic target for the eradication of LSCs in CML.

Discovery of a potent, selective and cell active inhibitor of m6A demethylase ALKBH5.

AlkB homolog 5 (ALKBH5) is an RNA m6A demethylase involved in the regulation of genes transcription, translation and metabolism and has been considered as a promising therapeutic target for various human diseases, especially cancers. However, there is still a lack of potent and selective ALKBH5 inhibitors. Herein, we report a new class of ALKBH5 inhibitors containing the 1-aryl-1H-pyrazole scaffold, which were obtained through fluorescence polarization-based screening, structural optimization and structure-activity relationship analysis. Among these compounds, 20m was the most potent one, which showed an IC50 value of 0.021 µM in fluorescence polarization assay. Compound 20m exhibited high selectivity towards ALKBH5 versus FTO as well as other AlkB subfamily members, indicating good selectivity for ALKBH5. Cellular thermal shift assay (CETSA) analysis showed that 20m could efficiently stabilize ALKBH5 in HepG2 cells. Dot blot assay demonstrated that 20m could increase m6A level in intact cells. Collectively, 20m is a potent, selective and cell active ALKBH5 inhibitor and could be used as a versatile chemical probe to explore the biological function of ALKBH5.

Discovery of benzo[d]oxazol-2(3H)-one derivatives as a new class of TNIK inhibitors for the treatment of colorectal cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancer types and Traf2- and Nck-interacting kinase (TNIK) has been thought as a potential target for CRC treatment. Herein we report the discovery and structure-activity relationship (SAR) of benzo[d]oxazol-2(3H)-one derivatives as a new class of TNIK inhibitors. The most potent compound 8g showed an IC50 value of 0.050 µM against TNIK. It effectively suppressed proliferation and migration of colorectal cancer cells. Western blot analysis indicated that 8g could inhibit aberrant transcription activation of Wnt signaling. Collectively, this study provides a potential lead compound for subsequent drug discovery targeting TNIK.

Discovery and structural optimization of potent epidermal growth factor receptor (EGFR) inhibitors against L858R/T790M/C797S resistance mutation for lung cancer treatment.

The C797S mutation in EGFR is a leading mechanism of clinically acquired resistance against osimertinib for non-small cell lung cancer (NSCLC). In this study, we identified a potent and oral EGFRL858R/T790M/C797S tyrosine kinase inhibitor, 14aj with a novel chemical scaffold. Compound 14aj showed low nanomolar activity against EGFRL858R/T790M/C797S mutant with IC50 value as 0.010 µM. In vitro assays, compound 14aj exhibited high potency against NSCLC cells harboring EGFRL858R/T790M/C797S and induced tumor cell cycle arrest and cell apoptosis. 14aj inhibited cellular phosphorylation of EGFR. In vivo xenograft mouse model, oral administration of compound 14aj led to significant tumor regression without obvious toxicity. In addition, this compound showed good pharmacokinetics.

KMT2C deficiency promotes small cell lung cancer metastasis through DNMT3A-mediated epigenetic reprogramming.

Small cell lung cancer (SCLC) is notorious for its early and frequent metastases, which contribute to it as a recalcitrant malignancy. To understand the molecular mechanisms underlying SCLC metastasis, we generated SCLC mouse models with orthotopically transplanted genome-edited lung organoids and performed multiomics analyses. We found that a deficiency of KMT2C, a histone H3 lysine 4 methyltransferase frequently mutated in extensive-stage SCLC, promoted multiple-organ metastases in mice. Metastatic and KMT2C-deficient SCLC displayed both histone and DNA hypomethylation. Mechanistically, KMT2C directly regulated the expression of DNMT3A, a de novo DNA methyltransferase, through histone methylation. Forced DNMT3A expression restrained metastasis of KMT2C-deficient SCLC through repressing metastasis-promoting MEIS/HOX genes. Further, S-(5'-adenosyl)-L-methionine, the common cofactor of histone and DNA methyltransferases, inhibited SCLC metastasis. Thus, our study revealed a concerted epigenetic reprogramming of KMT2C- and DNMT3A-mediated histone and DNA hypomethylation underlying SCLC metastasis, which suggested a potential epigenetic therapeutic vulnerability.

Discovery of a potent and highly selective inhibitor of ataxia telangiectasia mutated and Rad3-Related (ATR) kinase: Structural activity relationship and antitumor activity both in vitro and in vivo.

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is an important regulator of the DNA damage response (DDR), especially in response to replication stress (RS). Tumor cells with ataxia-telangiectasia mutated (ATM) kinase loss of function or DDR defects that promote replicative stress are often more reliant on ATR for survival, highlighting ATR as a good antitumor target under the principle of synthetic lethality. Herein we report the discovery of a potent and highly selective ATR inhibitor, SKLB-197, which was obtained through structural optimization and structure-activity relationship (SAR) studies towards a hit compound (Cpd-1). SKLB-197 showed an IC50 value of 0.013 µM against ATR but very weak or no activity against other 402 protein kinases. It displayed potent antitumor activity against ATM-deficent tumors both in vitro and in vivo. In addition, this compound exhibited good pharmacokinetic properties. Overall, SKLB-197 could be a promising lead compound for drug discovery targeting ATR and deserves further in-depth studies.