Acta Pharmaceutica Sinica B

Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs.

A novel STING agonist CDGSF unilaterally modified with phosphorothioate and fluorine was synthesized. CDGSF displayed better STING activity over dithio CDG. Immunization of SARS-CoV-2 Spike protein with CDGSF as an adjuvant elicited an exceptional high antibody titer and a robust T cell response, which were better than the group using aluminium hydroxide as a adjuvant. These results highlighted the adjuvant potential of STING agonist in SARS-CoV-2 vaccine preparation for the first time.

Immunotherapy has emerged as a powerful strategy for combating cancer by harnessing the patients immune system to recognize and eliminate malignant cells. Major histocompatibility complex class I (MHC-I) plays a pivotal role by presenting neoantigens to CD8+ T cells, triggering T cell-mediated killing. However, cancer cells often evade detection by downregulating MHC-I surface expression, hindering the immune response. This resistance mechanism offers an opportunity to bolster MHC-I surface expression via therapeutic interventions. We conducted a comprehensive evaluation of previously purported small molecule MHC-I inducers and identified heat shock protein 90 (Hsp90) inhibitors as privileged enhancers. Using a core scaffold, we employed an in situ click chemistry-based derivatization strategy to generate 380 novel compounds. New agents showed high induction levels, with one triazole-based analog, CliMB-325, also enhancing T cell activation and exhibiting lower toxicity. Altogether, we demonstrated the potential of click chemistry-based diversification for discovering small molecules to counter immune evasion.

Ginseng has been known as a "cure-all" traditional medicine to treat various illnesses and as an adaptogen to relieve stress. However, the known active compounds of ginseng are small-molecule metabolites. Here we report ginsentides, which are disulfide-dense, super-stable and cell-penetrating peptides with 31-33 amino acids, as active compounds and adaptogens that restore homeostasis in response to stress. Using mass spectrometry-based target identification and functional studies, we show that ginsentides promote vasorelaxation by producing nitric oxide through endothelial cells via the PI3K/Akt signaling pathway. Ginsentides were also found to alleviate 1-adrenergic receptor overactivity by reversing phenylephrine-induced constriction of the aorta, decrease monocyte adhesion to endothelial cells via CD166/ESAM/CD40, inhibit P2Y12 receptors, reduce platelet aggregation, and thrombus formation in the lung. Orally administered ginsentides were effective in anti-stress behavior using animal models of tail suspension and forced swimming tests. Together, these results suggest that ginsentides interact with multiple systems to restore homeostasis by reversing stress-induced physiological changes and provide new insights into the panacea medicinal effects of ginseng.

Despite the prevalence of obesity and related health consequences around the globe, effective treatments for inducing healthy weight loss are still lacking. Celastrol is a pentacyclic triterpene that was recently identified as a potent anti-obesity agent. Celastrol increases sensitivity to leptin, but the molecular target of celastrol is unknown. Therefore, the mechanisms by which this agent exerts its anti-obesity effect remain elusive. Using tissue-specific ABPP (activity-based protein profiling), we found that PFKM, a rate-limiting enzyme for glycolysis in skeletal muscle, is a direct target of celastrol. Celastrol inhibited PFKM enzymatic activity, and Pfkm knockout mice were resistant to a high fat diet, were hypersensitive to exogenous leptin, and were unresponsive to celastrol. PFKM inhibition led to activation of AMPK and inactivation of ACC in cultured myotubes and mouse skeletal muscle. Specific loss of AMPK in muscle significantly attenuated the anti-obesity effects of celastrol. Further, PFKM inhibition and subsequent activation of the AMPK/ACC signaling pathway reduced levels of free fatty acids by switching energy expenditure and consequently decreasing levels of SOCS1 expression, which are both required for leptin sensitization in 293t/hLepRb cells and mice. Finally, using a high throughput compound screen we identified an alternative PFKM inhibitor, 3-79, which exhibits a strong anti-obesity effect and non-covalent binding capacity. This compound is a promising agent for treating obesity in the clinic.

Upregulation of protein SUMOylation is associated with various diseases, and SUMOylation inhibitors are promising drug candidates. We performed the first virtual screening of SUMO E1 covalent allosteric inhibitors (CAIs) and identified two SUMO E1 CAIs with new scaffolds and covalent warheads. We further demonstrated that these new CAIs perturbed the SUMOylation pathway and protein SUMOylation. Specifically, these CAIs affected the SUMOylation of the methionine adenosyltransferases MAT2A. The inhibition of MAT2A SUMOylation unexpectedly stimulated polyamine synthesis. Lastly, we showed that the combination of SUMO E1 CAIs with a polyamine synthesis inhibitor had synergistic effects in inhibiting T47D cells. Our work demonstrated the first cost-effective virtual screening of SUMO E1 CAIs, found that the downregulation of MAT2A SUMOylation increases polyamine synthesis, and SUMO E1 CAIs can synergize with polyamine synthesis inhibitors. This work would be of great value to the study of SUMOylation, covalent/allosteric drugs, and the polyamine metabolism network.

Fusion with host cell membrane is the main mechanism of infection of SARS-CoV-2. Here, we propose a new strategy to double block SARS-CoV-2 membrane fusion by using Harringtonine (HT), a small-molecule antagonist. By using cell membrane chromatography (CMC), we found that HT specifically targeted the SARS-CoV-2 S protein and host cell TMPRSS2, and then confirmed that HT can inhibit pseudotyped virus membrane fusion. Furthermore, HT successfully blocked SARS-CoV-2 infection, especially in the delta and Omicron mutant. Since HT is a small-molecule antagonist, it is minimally affected by the continuous variation of SARS-CoV-2. Our findings show that HT is a potential small-molecule antagonist with a new mechanism of action against SARS-CoV-2 infection, and thus HT mainly targets the S protein, and thus, greatly reduces the damage of the S proteins autotoxicity to the organ system, has promising advantages in the clinical treatment of COVID-19.

Ulcerative colitis and Crohns disease are inflammatory bowel diseases (IBD) that lead to chronic inflammations of the gastrointestinal tract due to an abnormal response of the immune system. Finding new effective drugs to tackle IBD represents a major therapeutic concern since IBD incidence and prevalence is increasing worldwide. Recent studies positioned Cortistatin (CST) as a candidate for IBD treatment due to its anti-inflammatory and immunomodulatory activity. Here, we studied the structural properties of CST using NMR and synthesized and characterized new analogs displaying enriched populations of some native conformations. One of them, Analog 5, preserved the activity against IBD with an increased half-life in serum, overcoming the native hormone limitation and opening the door for the use of CST analogs as therapeutic agents. This work represents a new approach to the rational design of molecules to treat IBD and a possibility for patients that fail to respond to other therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/839787v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@b84405org.highwire.dtl.DTLVardef@179d0bdorg.highwire.dtl.DTLVardef@775736org.highwire.dtl.DTLVardef@fea7cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

SARS-CoV-2 is the etiological agent responsible for the COVID-19 outbreak in Wuhan. Specific antiviral drug are urgently needed to treat COVID-19 infections. The main protease (Mpro) of SARS-CoV-2 is a key CoV enzyme that plays a pivotal role in mediating viral replication and transcription, which makes it an attractive drug target. In an effort to rapidly discover lead compounds targeting Mpro, two compounds (11a and 11b) were designed and synthesized, both of which exhibited excellent inhibitory activity with an IC50 value of 0.05 M and 0.04 M respectively. Significantly, both compounds exhibited potent anti-SARS-CoV-2 infection activity in a cell-based assay with an EC50 value of 0.42 M and 0.33 M, respectively. The X-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a and 11b were determined at 1.5 [A] resolution, respectively. The crystal structures showed that 11a and 11b are covalent inhibitors, the aldehyde groups of which are bound covalently to Cys145 of Mpro. Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity which is promising drug leads with clinical potential that merits further studies.

Ovarian cancer patients usually develops peritoneal metastasis and malignant ascites in the advanced stages, which form immuno-suppressive tumor microenvironments that limit the efficacy of immuno-therapies. However, during our previous research trying to develop a {gamma}{delta} T cell-based cell therapy, we noticed that the malignant ascites may enhance the cytotoxicity of {gamma}{delta} T cells towards ovarian cancer cells. Herein, in this work we showed that the phenomenon is real and the low molecular weight components in the ascites act on the cancer to promote the killing by {gamma}{delta} T cells. Transcriptome analysis and in vitro experiments revealed that the malignant ascites induce the secretion of chemokines CXCL2 and CXCL8 by ovarian cancer cells, which recruits {gamma}{delta} T cells through the chemokine receptors CXCR1 and CXCR2, to enhance the cytotoxicity of {gamma}{delta} T cells. Metabolomics analysis discovered compounds that are responsible for the enhancement of {gamma}{delta} T cell cytotoxicity, one of which follows the aforementioned mechanism, while other compounds reflect undiscovered mechanisms. Overall, we presented the positive side of the malignant ascites in anti-tumor immunity, revealed the underlining mechanisms and at least partially interpreted the molecular basis. Our work thus provides new insights into the development of cell therapies for ovarian cancer.

Sphingosine 1-phosphate receptor 1 (S1PR1), a G protein-coupled receptor, is required for lymphocyte trafficking, and is a promising therapeutic target in inflammatory diseases. We synthesized a competitive antagonist, KSI-6666, that persistently inhibits S1PR1 activity and effectively suppresses pathogenic inflammation. Metadynamics simulation proposed that the interaction of KSI-6666 with a methionine residue in the ligand-binding pocket of S1PR1 may inhibit the dissociation of KSI-6666 from S1PR1. Consistently, in vitro functional and mutational analyses revealed that KSI-6666 causes pseudoirreversible inhibition of S1PR1, dependent on the methionine residue of the protein and substituents on the distal benzene ring of KSI-6666. Moreover, in vivo study suggested that this pseudoirreversible inhibition is responsible for the persistent activity of KSI-6666. In this study, we discover that KSI-6666 is a potent S1PR1 antagonist, achieving pseudoirreversible inhibition of S1PR1 activity through interacting with a specific methionine residue.

The angiotensin II (AngII) type 1 receptor (AT1R) is a key prototypical G protein-coupled receptor in cardiovascular regulation. Biased agonists that activate G protein or {beta}-arrestin pathways provide promising therapeutic potential, but the molecular determinants for this signaling bias and its physiological implications remain poorly understood. This study profiles AngII analogs with modifications at the C-terminal Phe8, revealing that analogs 11, 12, and 29a exhibit varying degrees of Gq engagement while maintaining potent {beta}-arrestin recruitment. Notably, 12 enhances left ventricular ejection fraction with minimal pressor responses in normotensive rats, while other analogs with variable Gq activity do not promote inotropy. Molecular modeling indicates that the unique profile of 12 results from its flexible long side chain engaging a deep allosteric pocket within AT1R. This study demonstrates that engineering AngIIs C-terminus enables selective tuning of AT1R signaling to control arterial versus cardiac responses, providing strategies for developing improved cardiovascular therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/670122v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@52b49corg.highwire.dtl.DTLVardef@1cf92dcorg.highwire.dtl.DTLVardef@b2c103org.highwire.dtl.DTLVardef@19db799_HPS_FORMAT_FIGEXP M_FIG C_FIG

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 M and EC50 of 1.27 M, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the -methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.

Uncontrolled inflammation is central to the development of diseases such as sepsis, and autophagy has emerged as a critical regulatory mechanism in this process. The ethanol extract of Cinnamomum cassia (EECC) was identified as a potent autophagy inducer through high-throughput LC3 reporter screening. EECC enhanced autophagic flux, as confirmed by RFP-GFP-LC3 imaging and immunoblotting. It also suppressed Toll-like receptor signaling and reduced pro-inflammatory cytokine production in macrophages. EECC inhibited nuclear factor-{kappa}B signaling in an autophagy-dependent manner, as this effect was reversed by autophagy inhibitors. To identify active constituents, 24 compounds were isolated from EECC, including six novel structures. Among the novel compounds, Cassitamine F exhibited dual activity as an autophagy inducer and inflammation suppressor. In a lipopolysaccharide-induced sepsis model, Cassitamine F significantly reduced serum levels of pro-inflammatory cytokines. These findings suggest that EECC and Cassitamine F may hold therapeutic potential for autophagy-targeted treatment of sepsis and other inflammation-related disorders. Highlights- Ethanol extract of Cinnamomum cassia (EECC) identified as a potent autophagy inducer via high-throughput LC3 HiBiT screening. - EECC suppresses Toll-like receptor signaling and pro-inflammatory cytokines through autophagy-dependent pathways. - Twenty-four compounds were isolated from EECC, including six novel structures (Cassitamine A-F). - Cassitamine F exhibits dual activity: induction of autophagy and inhibition of inflammation. - Cassitamine F significantly reduces serum cytokine levels in an LPS-induced sepsis model, suggesting therapeutic potential.

Inflammation serves as a vital physiological process essential for preserving health and countering illness. Yet, persistent inflammation drives osteoclastogenesis and ongoing bone erosion in rheumatoid arthritis (RA), mainly via macrophage activation and overproduction of pro-inflammatory cytokines like TNF-, IL-1{beta}, and IL-6. Limitations of prolonged conventional treatments underscore the need for safer small-molecule inhibitors that address both inflammation and osteoclast formation. Chalcones, natural plant defense compounds, exhibit diverse pharmacological properties including anti-inflammatory, anticancer, antibacterial, antifungal, and antiparasitic actions, owing to their characteristic reactive , {beta}- unsaturated carbonyl moiety. This study assessed chalcone derivative 7a for its anti-inflammatory effects in vitro and in vivo, alongside its capacity to modulate osteoclast differentiation, offering the inaugural demonstration of its dual anti-inflammatory and anti-osteoclastogenic properties. In LPS-stimulated macrophages, 7a substantially curtailed nitric oxide production, curbed pro-inflammatory cytokines (TNF-, IL-1{beta}, IL-6), and concentration-dependently diminished iNOS and COX-2 expression while inhibiting reactive oxygen species levels. In vivo, oral 7a dosing potently alleviated carrageenan-evoked paw swelling and restored serum lactate dehydrogenase and C-reactive protein to normalcy. In LPS-exposed mice, it further lowered systemic cytokines and rectified dysregulated biomarkers such as LDH, ALP, ALT, AST, creatinine, and urea. Moreover, in RANKL-stimulated osteoclast cultures, 7a markedly suppressed osteoclastogenesis by downregulating pivotal markers like tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). Derivative 7a also enhances antioxidant defense--superoxide dismutase and catalase--via blockade of NF-{kappa}B and MAPK pathways. Overall, chalcone derivative 7a displays robust anti-inflammatory and anti-osteoclastogenic activity, positioning it as a compelling candidate for RA therapy.

Epigenetics regulate cell-cycle kinetics, differentiation, apoptosis, and migration. Nuclear receptor-binding SET Domain (NSD) histone methyltransferases represent a family of oncoproteins with aberrant expression in cancer. Emerging reports suggest that NSD1 could be an attractive target as its expression is correlated with poor prognosis and tumorigenesis. Previously, we reported the target validation and structure-based virtual screening against NSD1, leading to the selection of several hit molecules with relatively high docking and MMGBSA delta G Bind scores. One of the best-fit molecules identified was 5-O-sulfamoyl adenosine (5-SA) and was compared with the S-Adenosyl-l-Cysteine (SAC), a structural analog of S-Adenosyl-l-Methionine (SAM) for its inhibitory activity against NSD1. IC50 values for 5-SA and SAC against NSD1 were 53.819 {micro}M and 115.003 {micro}M respectively. 5-SA significantly reduced the viability of DU145 and HepG2 cells with IC50 values calculated as 198{micro}M and 168.3 {micro}M respectively. It also reduced the RNA and protein expression levels of NSD1 and subsequently prevented dimethylation of lysine 36 on histone H3 (H3K36me2). Furthermore, 5-SA impeded proliferation, and migration, altered the cell cycle phase, and induced cell apoptosis. Interestingly, 5-SA potentiated the anticancer activity of 5-Fluorouracil (5-FU) against cancer cells. The xenograft model of prostate cancer also showed that 5-SA significantly reduced the tumor growth kinetics. However, the combination of 5-SA and 5-FU synergistically reduced tumor growth and improved survival of animals. To the best of our knowledge, we report for the first time that 5-SA mediated inhibition of NSD1 enhanced the tumor sensitivity to 5-FU and thereby, improved the tumor growth and progression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/731397v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@e2f0bdorg.highwire.dtl.DTLVardef@12b2ca6org.highwire.dtl.DTLVardef@1807613org.highwire.dtl.DTLVardef@c7f8f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Vinigrol is a natural diterpenoid with unprecedented chemical structure, driving great efforts into its total synthesis and the chemical analogs in the past decades. Despite its pharmacological efficacies reported on anti-hypertension and anti-clot, comprehensive functional investigations on Vinigrol and the underlying molecular mechanisms are entirely missing. In this study, we carried out a complete functional prediction of Vinigrol using a transcriptome-based strategy, Connectivity Map, and identified "anti-cancer" as the most prominent biofunction ahead of anti-hypertension and anti-depression/psychosis. A broad cytotoxicity was subsequently confirmed on multiple cancer types. Further mechanistic investigation on MCF7 cells revealed that its anti-cancer effect is mainly through activating PERK/eIF2 arm of unfolded protein response (UPR) and subsequent upregulation of p53/p21 to halt the cell cycle. The other two branches of UPR, IRE1 and ATF6, are functionally irrelevant to Vinigrol-induced cell death. CRISPR/Cas9-based gene activation, repression, and knockout systems identified essential contribution of ATF4/DDIT3 not ATF6 to the death process. This study unraveled a broad anti-cancer function of Vinigrol and its underlying targets and regulatory mechanisms, and also paved the way for further inspection on the structure-efficacy relationship of the whole compound family, making them a novel cluster of chemical hits for cancer therapy.

The tumor microenvironment (TME) is a pro-cancerous niche harboring immunosuppressive factors that are secreted by cancer cells and the surrounding cancer-supportive tissue, such as kynurenine, prostaglandin E2 and transforming growth factor {beta} (TGF{beta}). These factors dampen the activity of cytotoxic lymphocytes like natural killer (NK) cells, allowing evasion of immune cell-mediated killing. To identify small molecules that counteract the immunosuppressive effect of the TME and restore NK cell-mediated cytotoxicity, we developed a phenotypic co-culture assay of cancer cells and primary lymphocytes suitable for medium-throughput screening. We discovered small molecules that restore NK cell-mediated cytotoxicity through diverse mechanisms. The potent TGF{beta} type I receptor (TGF{beta}R-1) inhibitor, RepSox, stood out as superior to other TGF{beta}R-1 inhibitors due to its ability to abolish the effects of both inhibitory factors used in our setup. This mode of action goes beyond TGF{beta}R-1 inhibition and is related to the simultaneous abrogation of cyclooxygenase 1 (COX1) activity.

The antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) is a desirable therapeutic target for a broad range of pathologies, including chronic diseases of the lung and liver, and autoimmune, neurodegenerative, and cardiovascular disorders. However, current Nrf2 activators are limited by unwanted effects due to non-specificity, and systemic distribution and action. Here we report that a 1,2-dicarbonyl moiety masks the electrophilic reactivity of the Nrf2 activator monomethyl fumarate (MMF), otherwise responsible for its non-specific effects. The 1,2-dicarbonyl compound is highly susceptible to Baeyer-Villiger oxidation, with generation of MMF specifically on exposure to pathological levels of hydrogen peroxide or peroxynitrite. Oral treatment with the MMF generating 1,2-dicarbonyl compound reversed chronic neuropathic and osteoarthritis pain in mice, and selectively activated Nrf2 at sites of oxidative stress. This 1,2-dicarbonyl platform may be used to treat additional disorders of oxidative stress, and to selectively target other therapeutics to sites of redox imbalance.

The rate-limiting enzyme of salvage pathway for NAD+ synthesis, NAMPT, is aberrantly overexpressed in a variety of tumor cells and is a poor prognosis factor for patient survival. NAMPT plays a major role in tumor cell proliferation, acting concurrently as an NAD+ synthase and unexpectedly, an extracellular ligand for several tumor-promoting signaling pathways. While previous efforts to modulate NAMPT activity were limited to enzymatic inhibitors with low success in clinical studies, protein degradation offers a possibility to simultaneously disrupt NAMPTs enzyme activity and ligand capabilities. Here, we report the development of two highly selective NAMPT-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent NAMPT degradation in a cereblon-dependent manner in multiple tumor cell lines. Notably, both PROTAC degraders outperform a clinical candidate, FK866, in killing effect on hematological tumor cells. These results emphasize the importance and feasibility of applying PROTACs as a better strategy for targeting proteins like NAMPT with dual tumor-promoting functions, which are not easily achieved by conventional enzymatic inhibitors.