Rho-associated coiled-coil-containing kinase 2 (ROCK2) is a key regulator of cellular motility and invasion and represents a promising therapeutic target for metastatic breast cancer. Through systematic structure-activity relationship (SAR) studies, we designed and synthesized a novel series of 1-methyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione derivatives as selective ROCK2 inhibitors. Strategic deuterium incorporation during structural optimization was employed to enhance pharmacokinetic profiles. This effort led to the identification of compound 22d, a highly potent deuterated analog with excellent ROCK2 inhibitory activity (IC50 = 13 nM) and remarkable selectivity (≥769-fold over ROCK1). In functional assays, 22d exhibited robust antimetastatic activity in vitro and significantly suppressed tumor metastasis in MDA-MB-231 breast cancer xenograft models. Mechanistically, its antimetastatic effects were attributed to selective inhibition of STAT3 phosphorylation at tyrosine 705 (Y705). Collectively, these findings establish 22d as a novel lead compound that impairs breast cancer metastasis through targeted disruption of the ROCK2-STAT3 signaling axis, providing a promising foundation for further therapeutic development against metastatic breast cancer.

Dysregulation of RNA N6-methyladenosine (m6A) readers has been linked to various diseases, but the therapeutic potential of small-molecule inhibitors targeting them is of interest. Here, we reported the identification and characterization of a potent and selective first-in-class inhibitor (YL-5092) of YTHDC1, a nuclear RNA m6A reader. We provided a high-resolution cocrystal structure of the YTHDC1-YL-5092 complex. In acute myeloid leukemia (AML) models, YL-5092 blocked the binding of YTHDC1 to its m6A substrates and reduced mRNA stability, resulting in apoptosis of AML cells and myeloid differentiation. In multiple xenograft models of AML representing disease heterogeneity, YL-5092 alone or in combination with standard AML therapy eliminated leukemia and extended survival. Moreover, YL-5092 functionally impaired leukemia stem cells yet spared normal hematopoietic counterparts. Collectively, our work demonstrates the efficacy of a selective YTHDC1 inhibitor and suggests that targeting of m6A readers is a potential strategy in the treatment of hematologic cancers.

Ferroptosis, a regulated form of cell death driven by iron-dependent lipid peroxidation, contributes to diverse pathological conditions. However, the clinical translation of ferroptosis inhibitors has been hampered by the limited efficacy or suboptimal pharmacokinetic profiles. Here, we report the discovery of diazepine derivatives as a new structural class of ferroptosis inhibitors. Through systematic structure-activity relationship optimization, we identified YL3147 as the most potent analogue, demonstrating exceptional cellular potency with an EC50 of 0.8 nM. Mechanistically, YL3147 functions as a radical-trapping antioxidant, directly halting the propagation of lipid peroxidation and thereby blocking ferroptosis. This compound also exhibits favorable drug-like pharmacokinetic properties. In vivo, YL3147 provided substantial protection against doxorubicin-induced cardiomyopathy in both acute and chronic murine models, with no detectable toxicity. Together, these findings establish YL3147 as a promising lead compound for the treatment of ferroptosis-related diseases, warranting further preclinical development.

ABSTRACT Protein histidine N1‐methylation (1‐methylhistidine, 1‐MH) is a prevalent yet underexplored post‐translational modification in mammals. The methyltransferase METTL9 acts as an important enzyme catalyzing 1‐MH of histidine in diverse protein substrates, with mounting evidence suggesting its involvement in tumor progression. Despite its potential significance, no inhibitors targeting METTL9 have been previously identified. Here, we introduce compound METTL9i as a first‐in‐class, highly potent, and selective METTL9 inhibitor. METTL9i inhibits METTL9 with a half‐maximum inhibitory concentration (IC 50 ) of 0.067 ± 0.009 µM and exhibits selectivity over other methyltransferases. Structural analysis via crystallography reveals that METTL9i binds within the S‐adenosylmethionine (SAM) binding pocket. In cells, METTL9i engages METTL9 and leads to a reduction in global 1‐MH levels. These results support METTL9i as a useful tool compound for investigating METTL9 biology and the functions of histidine 1‐MH, while also serving as a promising lead compound for drug discovery targeting METTL9.

The PDZ domain of neuronal nitric oxide synthase (nNOS-PDZ) plays a crucial role in regulating serotonin signaling in the forebrain and has emerged as a promising target for developing rapid-acting antidepressants. Here, we report the identification of N24, a potent and selective small-molecule inhibitor of nNOS-PDZ. N24 induces a substantial thermal shift (ΔTm) of 5.44 °C in a differential scanning fluorimetry (DSF) assay and displays an IC50 of 0.76 ± 0.07 μM in a fluorescence polarization (FP) assay. The cocrystal structure of the nNOS-PDZ-N24 complex reveals key binding interactions. In vivo, N24 produces rapid, dose-dependent antidepressant effects in mouse models of depression induced by chronic unpredictable mild stress (CUMS) and chronic corticosterone treatment, with no evidence of addictive potential or motility-related side effects. Together, these results establish N24 as a potent and selective nNOS-PDZ inhibitor, providing a promising lead compound for the development of antidepressants.

G protein-coupled receptor 68 (GPR68), a proton-sensing GPCR, has emerged as a key player in inflammatory diseases. Its expression is substantially upregulated in the inflamed intestinal mucosa of inflammatory bowel disease (IBD) patients, and pharmacological inhibition of GPR68 has been shown to ameliorate colitis in preclinical models, highlighting GPR68 as a promising therapeutic target. Herein, we report the discovery of diphenylethane derivatives as a novel class of potent GPR68 antagonists. Structure-activity relationship (SAR) of these compounds was analyzed, which led to the identification of a potent GPR68 antagonist (18l) with an IC50 value of 0.081 ± 0.006 μM. The lead compound demonstrated significant inhibition of GPR68-mediated signaling and reduced the production of key pro-inflammatory cytokines. In a dextran sulfate sodium (DSS)-induced mouse model of IBD, 18l effectively alleviated disease symptoms. It also showed good pharmacokinetic properties and a commendable safety profile. Overall, compound 18l could be a promising lead compound for the treatment of IBD and deserves further in-depth studies.

Antivirals have provided important protection against COVID-19, however, the emergence of SARS-CoV-2 variants and drug-resistant mutants calls for the development of novel anti-coronavirus drugs with alternative mechanisms of action. The nonstructural protein 13 (nsp13) of SARS-CoV-2 plays a conserved role in the replication of coronaviruses and has been identified as a promising target. In this study, we report a series of 4-((quinolin-8-ylthio)methyl)benzamide derivatives as inhibitors of SARS-CoV-2 nsp13. Through structure-activity relationship (SAR) analyses, we identified compound 6r, which demonstrated potent inhibition of nsp13 with an IC50 value of 0.28 ± 0.11 μM. Collectively, we discovered a new potent SARS-CoV-2 nsp13 inhibitor, which could be taken as a promising lead compound for further drug development targeting SARS-CoV-2 nsp13.

Inflammatory programmed cell death mediated by NLRP3 inflammasome activation is one of the most representative forms of pyroptosis, involving multiple autoinflammatory diseases. In this investigation, we report the discovery of 3-pyridazinesulfonyl derivatives as a new class of inhibitors against NLRP3 inflammasome-dependent pyroptosis. We initially performed a phenotypic screening against NLRP3-dependent pyroptosis and discovered compound 1 (Hit-1), which showed moderate anti-pyroptotic activity (EC50 = 10.977 ± 2.122 μM). Further structure-activity relationship (SAR) studies resulted in a novel potent compound 32 (N102), which exhibited an EC50 of 0.029 ± 0.010 μM against cell pyroptosis induced by nigericin. N102 displayed remarkable inhibitory activity against NLRP3-dependent activation of caspase-1 and the release of IL-1β in human THP-1 cell-derived macrophages. Mechanistically, N102 disturbed the interaction of NLRP3 with the adaptor protein ASC and inhibited ASC oligomerization. Moreover, N102 possesses favorable HLM stability (T1/2 > 120 min), low CYP3A4 inhibition (IC50 > 10 μM) and good permeability (Papp = 9.063 × 10-5 cm s-1). Overall, we discovered a new potent small molecular inhibitor against NLRP3 inflammasome-dependent pyroptosis with potent cellular activity, favorable human-derived metabolic stability and permeability in vitro, which could be a good lead compound and deserves further in-depth studies.

Ferroptosis is an iron-dependent regulated cell death, which has been implicated in the onset and progression of numerous diseases. Ferroptosis inhibitors are thought as potential agents for treating these related diseases. However, the majority of currently available ferroptosis inhibitors are antioxidants or iron chelators (called classical ferroptosis inhibitors), which might have potential risks of side effects during clinical use. Herein, we report the discovery of phenazine derivatives as a new class of non-classical ferroptosis inhibitors. Structure-activity relationship of these series compounds led to the discovery of the most active compound 13l with an EC50 value of 0.0007 μM. Mechanistically, 13l could inhibit NCOA4-mediated ferritinophagy, hence protecting cells from ferroptosis. Notably, in the acetaminophen-induced acute liver injury model, 13l showed an excellent therapeutic effect. Overall, this compound reported here could be a promising lead compound for drug discovery targeting ferroptosis.

DNA N( 6-)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.

AlkB homolog 2 (ALKBH2) is a Fe (II) and 2-oxoglutarate (2OG)-dependent DNA demethylase. It has been reported to be highly expressed in many cancers including glioblastoma (GBM) and affected disease progression by regulating gene expression. Small molecule inhibitors of ALKBH2 might be used as disease intervention reagents or chemical tools for bio-functional studies of ALKBH2, but currently no potent and selective ALKBH2 inhibitors are reported. We herein disclose a new potent and selective ALKBH2 inhibitor (AH2-15c), which showed an IC50 value of 0.031 ± 0.001 μM in a fluorescence polarization (FP) assay and exhibited more than 200-fold selectivity towards ALKBH2 versus other AlkB subfamily members. Since AH2-15c showed very low cellular activity due to its poor cell membrane permeability originating from the carboxyl group, we investigated the un-hydrolyzed counterpart AH2-14c. AH2-14c could directly bind to ALKBH2 and increase the abundance of DNA N3-methylcytosine (3meC) modifications in GBM U87 cells, with a superior effect to AH2-15c. In addition, AH2-14c exhibited much better activities of anti-viability, anti-proliferation and anti-migration against U87 cells. Collectively, we discovered the first potent and selective ALKBH2 inhibitor, which could be taken as a foundation for future drug development and mechanism of action studies.

Although the SARS-CoV-2 pandemic has ended, multiple sporadic cases still exist, posing a request for more antivirals. The main protease (Mpro) of SARS-CoV-2, a key enzyme for viral replication, is an attractive target for drug development. Here, we report the discovery of a new potent α-ketoamide-containing Mpro inhibitor, N-((R)-1-cyclohexyl-2-(((R)-3-methoxy-1-oxo-1-((1-(2-oxo-2-((thiazol-2-ylmethyl)amino)acetyl)cyclobutyl)amino)propan-2-yl)amino)-2-oxoethyl)-4,4-difluorocyclohexane-1-carboxamide (20j). This compound presented promising enzymatic inhibitory activity against SARS-CoV-2 Mpro with an IC50 value of 19.0 nM, and an excellent antiviral activity in cell-based assay with an EC50 value of 138.1 nM. This novel covalent inhibitor may be used as a lead compound for subsequent drug discovery against SARS-CoV-2.

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 μM and 1.39 ± 0.13 μM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 μM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.

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.

GPR34 is a rhodopsin-like class G protein-coupled receptor (GPCR) that is involved in the development and progression of several diseases. Despite its importance, effective targeting strategies are lacking. We herein report a series of (S)-3-(4-(benzyloxy)phenyl)-2-(2-phenoxyacetamido)propanoic acid derivatives as a new class of GPR34 antagonists. Structure-activity relationship (SAR) studies led to the identification of the most potent compound, 5e, which displayed an IC50 value of 0.680 mu M in the GloSensor cAMP assay and 0.059 mu M in the Tango assay. 5e demonstrated low cytotoxicity and high selectivity in vitro, and it was able to dose-dependently inhibit Lysophosphatidylserine-induced ERK1/2 phosphorylation in CHO cells expressing GPR34. Furthermore, 5e displayed excellent efficacy in a mouse model of neuropathic pain without any apparent signs of toxicity. Collectively, this study has identified a promising compound, which shows great potential in the development of potent antagonists with a new chemical scaffold targeting GPR34.

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.

Influenza pandemics have emerged as a significant global public health and security concern. PB2, a crucial subunit of the influenza RNA-dependent RNA polymerase (RdRP), has been identified as a promising target for influenza treatment. We herein report the discovery of a potent novel PB2 inhibitor, 7-51A, with a KD value of 1.64 nM as determined by ITC. The high activity of 7-51A was elucidated by the co-crystal structure of the PB27-51A complex, and comparative analysis revealed unique interactions that had never been observed before. The preliminary pharmacological evaluation indicated that 7-51A exhibited commendable cellular safety, hepatic microsomal metabolic safety and stability. Collectively, 7-51A was found to be an effective PB2 inhibitor and could be used as a lead compound for further studies.

Ataxia-telangiectasia mutated (ATM) is an atypical serine/threonineprotein kinase which is implicated in the repair of DNA double-strandbreaks. Numerous reports have shown that ATM inhibition is an attractivetarget for radiotherapy and chemotherapy sensitization. Herein wereport a new series of ATM kinase inhibitors containing the 1H-[1,2,3]-triazolo-[4,5-c]-quinoline scaffold,which was obtained by virtual screening, structural optimization,and structure-activity relationship studies. Among the inhibitors, A011 was one of the most potent, with an IC50 valueof 1.0 nM against ATM. In colorectal cancer cells (SW620 and HCT116), A011 was able to inhibit activation of ATM signaling inducedby irinotecan (CPT-11) and ionizing radiation and thenincreased the sensitivity of colorectal cancer cells to irinotecanand ionizing radiation through increasing G2/M arrest and inducingapoptosis. In the SW620 human colorectal adenocarcinoma tumor xenograftmodel, A011 sensitized SW620 to CPT-11 byinhibiting ATM activity. Collectively, this work has identified apromising lead in the discovery of potent inhibitors against ATM.