PPIA dictates NRF2 stability to promote lung cancer progression.

Nuclear factor erythroid 2-related factor 2 (NRF2) hyperactivation has been established as an oncogenic driver in a variety of human cancers, including non-small cell lung cancer (NSCLC). However, despite massive efforts, no specific therapy is currently available to target NRF2 hyperactivation. Here, we identify peptidylprolyl isomerase A (PPIA) is required for NRF2 protein stability. Ablation of PPIA promotes NRF2 protein degradation and blocks NRF2-driven growth in NSCLC cells. Mechanistically, PPIA physically binds to NRF2 and blocks the access of ubiquitin/Kelch Like ECH Associated Protein 1 (KEAP1) to NRF2, thus preventing ubiquitin-mediated degradation. Our X-ray co-crystal structure reveals that PPIA directly interacts with a NRF2 interdomain linker via a trans-proline 174-harboring hydrophobic sequence. We further demonstrate that an FDA-approved drug, cyclosporin A (CsA), impairs the interaction of NRF2 with PPIA, inducing NRF2 ubiquitination and degradation. Interestingly, CsA interrupts glutamine metabolism mediated by the NRF2/KLF5/SLC1A5 pathway, consequently suppressing the growth of NRF2-hyperactivated NSCLC cells. CsA and a glutaminase inhibitor combination therapy significantly retard tumor progression in NSCLC patient-derived xenograft (PDX) models with NRF2 hyperactivation. Our study demonstrates that targeting NRF2 protein stability is an actionable therapeutic approach to treat NRF2-hyperactivated NSCLC.

Remodeling the immune microenvironment for gastric cancer therapy through antagonism of prostaglandin E2 receptor 4.

Gastric cancer is highly prevalent among digestive tract tumors. Due to the intricate nature of the gastric cancer immune microenvironment, there is currently no effective treatment available for advanced gastric cancer. However, there is promising potential for immunotherapy targeting the prostaglandin E2 receptor subtype 4 (EP4) in gastric cancer. In our previous study, we identified a novel small molecule EP4 receptor antagonist called YY001. Treatment with YY001 alone demonstrated a significant reduction in gastric cancer growth and inhibited tumor metastasis to the lungs in a mouse model. Furthermore, administration of YY001 stimulated a robust immune response within the tumor microenvironment, characterized by increased infiltration of antigen-presenting cells, T cells, and M1 macrophages. Additionally, our research revealed that YY001 exhibited remarkable synergistic effects when combined with the PD-1 antibody and the clinically targeted drug apatinib, rather than fluorouracil. These findings suggest that YY001 holds great promise as a potential therapeutic strategy for gastric cancer, whether used as a standalone treatment or in combination with other drugs.

Naphthalene Diimide-Based Metallacage as an Artificial Ion Channel for Chloride Ion Transport.

Developing synthetic molecular devices for controlling ion transmembrane transport is a promising research field in supramolecular chemistry. These artificial ion channels provide models to study ion channel diseases and have huge potential for therapeutic applications. Compared with self-assembled ion channels constructed by intermolecular weak interactions between smaller molecules or cyclic compounds, metallacage-based ion channels have well-defined structures and can exist as single components in the phospholipid bilayer. A naphthalene diimide-based artificial chloride ion channel is constructed through efficient subcomponent self-assembly and its selective ion transport activity in large unilamellar vesicles and the planar lipid bilayer membrane by fluorescence and ion-current measurements is investigated. Molecular dynamics simulations and density functional theory calculations show that the metallacage spans the entire phospholipid bilayer as an unimolecular ion transport channel. This channel transports chloride ions across the cell membrane, which disturbs the ion balance of cancer cells and inhibits the growth of cancer cells at low concentrations.

Subtle Structural Changes across the Boundary between A2AR/A2BR Dual Antagonism and A2BR Antagonism: A Novel Class of 2-Aminopyrimidine-Based Derivatives.

Aberrantly elevated adenosine in the tumor microenvironment exerts its immunosuppressive functions through adenosine receptors A2AR and A2BR. Antagonism of A2AR and A2BR has the potential to suppress tumor growth. Herein, we report a systemic assessment of the effects of an indole modification at position 4, 5, 6, or 7 on both A2AR/A2BR activity and selectivity of novel 2-aminopyrimidine compounds. Substituting indole at the 4-/5-position produced potent A2AR/A2BR dual antagonism, whereas the 6-position of indole substitution gave highly selective A2BR antagonism. Molecular dynamics simulation showed that the 5-cyano compound 7ai had a lower binding free energy than the 6-cyano compound 7aj due to water-bridged hydrogen bond interactions with E169 or F168 in A2AR. Of note, dual A2AR/A2BR antagonism by compound 7ai can profoundly promote the activation and cytotoxic function of T cells. This work provided a strategy for obtaining novel dual A2AR/A2BR or A2BR antagonists by fine-tuning structural modification.

Discovery of a Highly Potent Oxysterol Receptor GPR183 Antagonist Bearing the Benzo[d]thiazole Structural Motif for the Treatment of Inflammatory Bowel Disease (IBD).

Accumulating evidence has demonstrated a critical pathological role of oxysterol receptor GPR183 in various inflammatory and autoimmune diseases, including inflammatory bowel disease (IBD). However, the currently reported GPR183 antagonists are very limited and not qualified for in vivo studies due to their inferior druglike properties. Herein, we conducted a structural elaboration focusing on improving its PK and safety profile based on a reference antagonist NIBR189. Of note, compound 33, bearing an aminobenzothiazole motif, exhibited reduced hERG inhibition, improved PK properties, and robust antagonistic activity (IC50 = 0.82 nM) with high selectivity against GPR183. Moreover, compound 33 displayed strong in vitro antimigration and anti-inflammatory activity in monocytes. Oral administration of compound 33 effectively improved the pathological symptoms of DSS-induced experimental colitis. All of these findings demonstrate that compound 33 is a novel and promising GPR183 antagonist suitable for further investigation to treat IBD.

Structural insight into the dual-antagonistic mechanism of AB928 on adenosine A2 receptors.

The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A2AR complexed with AB928 and a selective A2AR antagonist 2-118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2-118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy.

Structurally-informed human interactome reveals proteome-wide perturbations by disease mutations.

Human genome sequencing studies have identified numerous loci associated with complex diseases. However, translating human genetic and genomic findings to disease pathobiology and therapeutic discovery remains a major challenge at multiscale interactome network levels. Here, we present a deep-learning-based ensemble framework, termed PIONEER (Protein-protein InteractiOn iNtErfacE pRediction), that accurately predicts protein binding partner-specific interfaces for all known protein interactions in humans and seven other common model organisms, generating comprehensive structurally-informed protein interactomes. We demonstrate that PIONEER outperforms existing state-of-the-art methods. We further systematically validated PIONEER predictions experimentally through generating 2,395 mutations and testing their impact on 6,754 mutation-interaction pairs, confirming the high quality and validity of PIONEER predictions. We show that disease-associated mutations are enriched in PIONEER-predicted protein-protein interfaces after mapping mutations from ~60,000 germline exomes and ~36,000 somatic genomes. We identify 586 significant protein-protein interactions (PPIs) enriched with PIONEER-predicted interface somatic mutations (termed oncoPPIs) from pan-cancer analysis of ~11,000 tumor whole-exomes across 33 cancer types. We show that PIONEER-predicted oncoPPIs are significantly associated with patient survival and drug responses from both cancer cell lines and patient-derived xenograft mouse models. We identify a landscape of PPI-perturbing tumor alleles upon ubiquitination by E3 ligases, and we experimentally validate the tumorigenic KEAP1-NRF2 interface mutation p.Thr80Lys in non-small cell lung cancer. We show that PIONEER-predicted PPI-perturbing alleles alter protein abundance and correlates with drug responses and patient survival in colon and uterine cancers as demonstrated by proteogenomic data from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium. PIONEER, implemented as both a web server platform and a software package, identifies functional consequences of disease-associated alleles and offers a deep learning tool for precision medicine at multiscale interactome network levels.

Berberrubine is a novel and selective IMPDH2 inhibitor that impairs the growth of colorectal cancer.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting reaction in the de novo synthesis pathway of guanine nucleotides that is highly required for cancer cell outgrowth. Herein, we found that IMPDH isoform 2 (IMPDH2) is highly expressed in colorectal cancer (CRC) and is correlated with poor patient prognosis. Via structure-based virtual screening, we identified berberrubine, a critical ingredient of the medical plant Coptis chinensis, as a novel, selective, and competitive inhibitor of IMPDH2, which demonstrated over 15-fold selectivity to IMPDH2 than IMPDH1. Besides, we also confirmed the interaction between berberrubine and IMPDH2. Of note, berberrubine treatment significantly impairs the growth of human CRC cells in a dose-dependent manner, which can be rescued by supplementing with guanosine. Furthermore, oral administration of berberrubine remarkably reduced tumor volume and weight in a human cell line-derived xenograft model. Importantly, the anti-cancer activity of berberrubine was also confirmed by using the azoxymethane (AOM) / dextran sulfate sodium (DSS)-induced spontaneous CRC mouse model. Taken together, our study highlights that berberrubine acts as a novel IMPDH2 inhibitor, suppressing the growth of CRC in vitro and in vivo, providing a fresh perspective for its potential application in the treatment of CRC.

Blockade of de novo pyrimidine biosynthesis triggers autophagic degradation of oncoprotein FLT3-ITD in acute myeloid leukemia.

The internal tandem duplication of the FMS-like tyrosine kinase 3 (FLT3-ITD) is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Limited and transient clinical benefit of FLT3 kinase inhibitors (FLT3i) emphasizes the need for alternative therapeutic options for this subset of myeloid malignancies. Herein, we showed that FLT3-ITD mutant (FLT3-ITD+) AML cells were susceptible toward inhibitors of DHODH, a rate-limiting enzyme of de novo pyrimidine biosynthesis. Genetic and pharmacological blockade of DHODH triggered downregulation of FLT3-ITD protein, subsequently suppressed activation of downstream ERK and STAT5, and promoted cell death of FLT3-ITD+ AML cells. Mechanistically, DHODH blockade triggered autophagy-mediated FLT3-ITD degradation via inactivating mTOR, a potent autophagy repressor. Notably, blockade of DHODH synergized with an FDA-approved FLT3i quizartinib in significantly impairing the growth of FLT3-ITD+ AML cells and improving tumor-bearing mice survival. We further demonstrated that DHODH blockade exhibited profound anti-proliferation effect on quizartinib-resistant cells in vitro and in vivo. In summary, this study demonstrates that the induction of degradation of FLT3-ITD protein by DHODH blockade may offer a promising therapeutic strategy for AML patients harboring FLT3-ITD mutation.

Discovery of 2H-Indazole-3-carboxamide Derivatives as Novel Potent Prostanoid EP4 Receptor Antagonists for Colorectal Cancer Immunotherapy.

Nowadays, small-molecule drugs have become an indispensable part of tumor immunotherapy. Accumulating evidence has indicated that specifically blocking PGE2/EP4 signaling to induce robust antitumor immune response represents an attractive immunotherapy strategy. Herein, a 2H-indazole-3-carboxamide containing compound 1 was identified as a EP4 antagonist hit by screening our in-house small-molecule library. Systematic structure-activity relationship exploration leads to the discovery of compound 14, which displayed single-nanomolar EP4 antagonistic activity in a panel of cell functional assays, high subtype selectivity, and favorable drug-like profiles. Moreover, compound 14 profoundly inhibited the up-regulation of multiple immunosuppression-related genes in macrophages. Oral administration of compound 14, either as monotherapy or in combination with an anti-PD-1 antibody, significantly impaired tumor growth via enhancing cytotoxic CD8+ T cell-mediated antitumor immunity in a syngeneic colon cancer model. Thus, these results demonstrate the potential of compound 14 as a candidate for developing novel EP4 antagonists for tumor immunotherapy.

Discovery of Pyridinone Derivatives as Potent, Selective, and Orally Bioavailable Adenosine A2A Receptor Antagonists for Cancer Immunotherapy.

Recent studies and clinical evidence have strongly supported the development of adenosine A2A receptor (A2AR) antagonists as novel approaches for cancer immunotherapy. By screening our in-house compound library, a pyridinone hit compound (1) with weak A2AR antagonistic activity was identified. Further structure-activity relationship studies revealed a series of pyridinone derivatives with strong potency. Compound 38 stood out with a potent A2AR antagonistic activity (IC50 = 29.0 nM), good mouse liver microsomal metabolic stability (t1/2 = 86.1 min), and excellent oral bioavailability (F = 86.1%). Of note, 38 effectively enhanced the activation and killing ability of T cells in vitro by down-regulation of immunosuppressive molecules (LAG-3 and TIM-3) and up-regulation of effector molecules (GZMB, IFNG, and IL-2). Moreover, 38 exhibited excellent in vivo antitumor activity with a tumor growth inhibition (TGI) of 56.0% in the MC38 tumor model via oral administration, demonstrating its potential as a novel A2AR antagonist candidate for cancer immunotherapy.

Drug repurposing of ilepcimide that ameliorates experimental autoimmune encephalomyelitis via restricting inflammatory response and oxidative stress.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS) that remains incurable. Herein, we demonstrated that ilepcimide (Antiepilepsirine), an antiepileptic drug used for decades, protects mice from experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Our studies found that ilepcimide treatment effectively ameliorates demyelination, blood-brain barrier leakage and infiltration of CD4+ and CD8+ T cells in EAE mice. On the one hand, ilepcimide can inhibit dihydroorotate dehydrogenase (DHODH), an important therapeutic target for MS. Computer molecular docking, thermal shift and fluorescence quenching assay demonstrated the directly interaction between ilepcimide and DHODH. Accordingly, ilepcimide observably repressed T cell proliferation in mixed lymphocyte reaction (MLR) assay and concanavalin A (Con-A) model in a DHODH-dependent manner. On the other hand, ilepcimide exhibited neuroprotective effect possibly through activating NRF2 antioxidant pathway in mouse neural crest-derived Neuro2a cells. Collectively, our findings have revealed the therapeutic potential of ilepcimide in EAE mouse model via restricting inflammatory response and oxidative stress, offering a potential opportunity for repurposing existing drug ilepcimide for MS therapy.

Modality-DTA: Multimodality Fusion Strategy for Drug-Target Affinity Prediction.

Prediction of the drug-target affinity (DTA) plays an important role in drug discovery. Existing deep learning methods for DTA prediction typically leverage a single modality, namely simplified molecular input line entry specification (SMILES) or amino acid sequence to learn representations. SMILES or amino acid sequences can be encoded into different modalities. Multimodality data provide different kinds of information, with complementary roles for DTA prediction. We propose Modality-DTA, a novel deep learning method for DTA prediction that leverages the multimodality of drugs and targets. A group of backward propagation neural networks is applied to ensure the completeness of the reconstruction process from the latent feature representation to original multimodality data. The tag between the drug and target is used to reduce the noise information in the latent representation from multimodality data. Experiments on three benchmark datasets show that our Modality-DTA outperforms existing methods in all metrics. Modality-DTA reduces the mean square error by 15.7% and improves the area under the precisionrecall curve by 12.74% in the Davis dataset. We further find that the drug modality Morgan fingerprint and the target modality generated by one-hot-encoding play the most significant roles. To the best of our knowledge, Modality-DTA is the first method to explore multimodality for DTA prediction.

Cryo-EM structure of the human adenosine A2B receptor-Gs signaling complex.

The human adenosine A2B receptor (A2BR) is a class A G protein-coupled receptor that is involved in several major physiological and pathological processes throughout the body. A2BR recognizes its ligands adenosine and NECA with relatively low affinity, but the detailed mechanism for its ligand recognition and signaling is still elusive. Here, we present two structures determined by cryo-electron microscopy of A2BR bound to its agonists NECA and BAY60-6583, each coupled to an engineered Gs protein. The structures reveal conserved orthosteric binding pockets with subtle differences, whereas the selectivity or specificity can mainly be attributed to regions extended from the orthosteric pocket. We also found that BAY60-6583 occupies a secondary pocket, where residues V2506.51 and N2737.36 were two key determinants for its selectivity against A2BR. This study offers a better understanding of ligand selectivity for the adenosine receptor family and provides a structural template for further development of A2BR ligands for related diseases.

Activation of orphan receptor GPR132 induces cell differentiation in acute myeloid leukemia.

Blocked cellular differentiation is a critical pathologic hallmark of acute myeloid leukemia (AML). Here, we showed that genetic activation of the orphan GPCR GPR132 significantly induced cell differentiation of AML both in vitro and in vivo, indicating that GPR132 is a potential trigger of myeloid differentiation. To explore the therapeutic potential of GPR132 signaling, we screened and validated a natural product 8-gingerol (8GL) as a GPR132 agonist. Notably, GPR132 activation by 8GL promoted differentiation and reduced colony formation in human AML cell lines with diverse genetic profiles. Mechanistic studies revealed that 8GL treatment inhibits the activation of the mammalian target of rapamycin (mTOR), a regulator of AML cell differentiation blockade, via activating GPR132-Gs-PKA pathway. We further showed that the combination of 8GL and an mTOR inhibitor synergistically elicited AML cell differentiation in vitro. Importantly, 8GL alone or in combination with an mTOR inhibitor remarkably impaired tumor growth and extended mouse survival in an AML xenograft model accompanied by enhanced cell differentiation. Notably, genetic or pharmacological activation of GPR132 triggered the differentiation of human primary AML cells. In summary, this study demonstrated that activation of orphan GPR132 represents a potential strategy for inducing myeloid differentiation in AML patients.

METTL3 promotes non-small cell lung cancer (NSCLC) cell proliferation and colony formation in a m6A-YTHDF1 dependent way.

BACKGROUND: N6-methyladenosine (m6A) is the most common RNA modification, which plays a pivotal role in tumor development and progression. In this study, we assessed the role of m6A methyltransferase METTL3 in FRAS1-involved cell proliferation and colony formation of non-small cell lung cancer (NSCLC) cell lines. METHODS: Cell viability was analyzed by Cell Counting Kit (CCK-8) and colony formation. M6A RNA immunoprecipitation (IP), Ribosomal immunoprecipitation, RNA immunoprecipitation (RIP) were performed to verify the relationship between METTL3, FRAS1 and YTHDF1. Rescue experiments to confirm the regulatory mechanism of METTL3-FRAS1 promoted NSCLC cell proliferation through CDON by cooperating YTHDF1. RESULTS: We found that FRAS1 was correlated with poor prognosis in NSCLC patients, of which the transcript undergoes m6A modification regulated by METTL3. METTL3 silence reduced cell viability of NSCLC cells HCC827 and NCI-H1975, which could be restored by FRAS1 overexpression. The m6A modification of FRAS1 could be recognized by YTHDF1. FRAS1 silence or YTHDF1 silence could rescue the elevated NSCLC cell proliferation, colony formation, and tumor growth induced by METTL3 overexpression in vitro and in vivo. CONCLUSIONS: Our study reveals that METTL3-FRAS1 plays a crucial role in NSCLC cell proliferation, colony formation, and tumor growth through the regulation of CDON by cooperating YTHDF1.

Scaffold Hopping Strategy to Identify Prostanoid EP4 Receptor Antagonists for Cancer Immunotherapy.

Cancer cells can effectively suppress the natural immune response in humans, and prostaglandin E2 (PGE2) is a key mediator in the development of tumor cell resistance to immunotherapy. As a major contributor to PGE2-elicited immunosuppressive activity, the EP4 receptor promotes tumor development and progression in the tumor microenvironment, and the development of selective and potent EP4 receptor antagonists should have promising potential for tumor immunotherapy. Aiming at improving the drug-like properties, a series of 4,7-dihydro-5H-thieno[2,3-c]pyran derivatives were designed and synthesized through a scaffold hopping strategy. The most promising compound 47 exhibited good EP4 antagonistic activity and excellent subtype selectivity, as well as favorable drug-like properties. It effectively suppressed the expression of multiple immunosuppression-related genes in macrophages. Meanwhile, oral administration of compound 47, alone or in combination with anti-PD-1 antibody, significantly enhanced the antitumor immune response and inhibited tumor growth in the mouse CT26 colon carcinoma model.

A novel prostaglandin E receptor 4 (EP4) small molecule antagonist induces articular cartilage regeneration.

Articular cartilage repair and regeneration is an unmet clinical need because of the poor self-regeneration capacity of the tissue. In this study, we found that the expression of prostaglandin E receptor 4 (PTGER4 or EP4) was largely increased in the injured articular cartilage in both humans and mice. In microfracture (MF) surgery-induced cartilage defect (CD) and destabilization of the medial meniscus (DMM) surgery-induced CD mouse models, cartilage-specific deletion of EP4 remarkably promoted tissue regeneration by enhancing chondrogenesis and cartilage anabolism, and suppressing cartilage catabolism and hypertrophy. Importantly, knocking out EP4 in cartilage enhanced stable mature articular cartilage formation instead of fibrocartilage, and reduced joint pain. In addition, we identified a novel selective EP4 antagonist HL-43 for promoting chondrocyte differentiation and anabolism with low toxicity and desirable bioavailability. HL-43 enhanced cartilage anabolism, suppressed catabolism, prevented fibrocartilage formation, and reduced joint pain in multiple pre-clinical animal models including the MF surgery-induced CD rat model, the DMM surgery-induced CD mouse model, and an aging-induced CD mouse model. Furthermore, HL-43 promoted chondrocyte differentiation and extracellular matrix (ECM) generation, and inhibited matrix degradation in human articular cartilage explants. At the molecular level, we found that HL-43/EP4 regulated cartilage anabolism through the cAMP/PKA/CREB/Sox9 signaling. Together, our findings demonstrate that EP4 can act as a promising therapeutic target for cartilage regeneration and the novel EP4 antagonist HL-43 has the clinical potential to be used for cartilage repair and regeneration.

PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis.

Prostaglandin E2 (PGE2), a major cyclooxygenase-2 (COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis (OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts (EP4LysM) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4LysM mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of platelet-derived growth factor-BB (PDGF-BB) was also lower in the EP4LysM mice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4LysM mice. Finally, we showed that the Gαs/PI3K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.