Cinnamaldehyde induces apoptosis and enhances anti-colorectal cancer activity via covalent binding to HSPD1.

Colorectal cancer (CRC) is a common malignant tumor with high morbidity and mortality rates worldwide. Although surgical resection and adjuvant radiotherapy/chemotherapy are the mainstays of CRC treatment, the efficacy is unsatisfactory due to several limitations, including high drug resistance. Accordingly, there is a dire need for new drugs or a novel combination approach to treat this patient population. Herein, we found that cinnamaldehyde (CA) could exert an antitumor effect in HCT-116 cell lines. Target fishing, molecular imaging, and live-cell tracing using an alkynyl-CA probe revealed that the heat shock 60 kDa protein 1 (HSPD1) protein was the target of CA. The covalent binding of CA with HSPD1 altered its stability. Furthermore, our results demonstrated that CA could induce cell apoptosis by inhibiting the PI3K/Akt signaling pathway and enhanced anti-CRC activity both in vitro and in vivo. Meanwhile, CA combined with different chemotherapeutic agents was beneficial to patients resistant to anti-CRC drug therapy.

PRMT5 promotes ovarian cancer growth through enhancing Warburg effect by methylating ENO1.

Protein arginine methyltransferase 5 (PRMT5) is a major type II enzyme responsible for symmetric dimethylation of arginine (SDMA), and plays predominantly roles in human cancers, including in ovarian cancer. However, the exactly roles and underlying mechanisms of PRMT5 contributing to the progression of ovarian cancer mediated by reprogramming cell metabolism remain largely elusive. Here, we report that PRMT5 is highly expressed and correlates with poor survival in ovarian cancer. Knockdown or pharmaceutical inhibition of PRMT5 is sufficient to decrease glycolysis flux, attenuate tumor growth, and enhance the antitumor effect of Taxol. Mechanistically, we find that PRMT5 symmetrically dimethylates alpha-enolase (ENO1) at arginine 9 to promotes active ENO1 dimer formation, which increases glycolysis flux and accelerates tumor growth. Moreover, PRMT5 signals high glucose to increase the methylation modification of ENO1. Together, our data reveal a novel role of PRMT5 in promoting ovarian cancer growth by controlling glycolysis flux mediated by methylating ENO1, and highlights that PRMT5 may represent a promising therapeutic target for treating ovarian cancer.

Discovery of novel CDK2 inhibitors using multistage virtual screening and in vitro melanoma cell lines.

Cyclin-dependent kinases 2 (CDK2) is a serine/threonine-protein kinase, which plays a key role in the regulation of cell cycle and is related to the occurrence and development of melanoma. In this study, we identified potent inhibitors for CDK2 by combining a multistage virtual screening strategy with bioassay validations. The biochemical activity of compounds was validated with ADP-Glo™ Kinase assay in vitro, and the results indicated that the biochemical activity of compound 1 (C1) was better than other selected compounds. Cell viability assay showed that the minimum inhibition concentration of C1 for CDK2 was lower than 4 µM. Further functional test results showed that C1 exerted significant antiproliferative, pro-apoptosis, and anti-migration activity in melanoma cell lines (A375 cells, WM35 cells, and A875 cells). Our findings suggested that the C1, virtually screened from compound libraries, as the novel inhibitor of CDK2, may be further developed as an effective therapeutic agent in the treatment of melanoma lines.

Icotinib derivatives as tyrosine kinase inhibitors with anti-esophageal squamous carcinoma activity.

Previous report showed that a variety of icotinib derivatives bearing different 1,2,3-triazole moieties, which could be readily prepared via copper (I)-catalyzed cycloaddition (CuAAC) reaction between icotinib and different azides, exhibited interesting activity against different lung cancer cell lines such as H460, H1975, H1299, A549 or PC-9. To further expand the application scope of the compounds and to validate the function of triazole groups in drug design, the anti-cancer activity of these compounds against esophageal squamous carcinoma (ESCC) cells was tested herein. Preliminary MTT experiments suggested that these compounds were active against different ESCC cell lines such as KYSE70, KYSE410, or KYSE450 as well as their drug-resistant ones. Especially, compound 3l showed interesting anticancer activity against these cell lines. The mode of action was studied via molecular docking, SPR experiments and other biochemical studies, and 3l exhibited higher binding potential to wild-type EGFR than icotinib did. In vivo anticancer study showed that 3l could inhibit tumor growth of cell-line-derived xenografts in ESCC. Study also suggested that 3l was a potent inhibitor for EGFR-TK pathway. Combining these results, 3l represents a promising lead compound for the design of anti-cancer drugs against ESCC.

Cinnamaldehyde Regulates the Generation of γ-aminobutyric Acid to Exert Sedation via Irreversible Inhibition of ENO1 in the Cerebellar Granular Layer.

SCOPE: Glutamate (Glu) and γ-aminobutyric acid (GABA) are the major excitatory and inhibitory neurotransmitters that control information flow in the brain. GABA dysfunction is a general vulnerability factor for mental illness. Cinnamaldehyde (CA) is found to have sedation in a mental illness model. However, the specific targets and molecular mechanisms related to the sedative effects of CA have not been elucidated. METHODS AND RESULTS: Metabolomics analysis and target fishing showed CA could increase the expression of GABA in vivo, and α-enolase (ENO1) is the primary target protein of CA associated with sedation. CA mainly binds with ENO1 in the cerebellar granular layer of brain, which influences the first transformations of the input signals arriving in the cerebellar cortex. The α,ß-unsaturated aldehyde group of CA blocks the hydroxy group of Ser40, which induces a loss in ENO1 activation. CA also disturbs the glycolysis pathway and influences the tricarboxylic acid cycle and oxidative phosphorylation, which activate gluconeogenesis to provide energy to the brain. This mechanism is verified in zebrafish with ENO1 or glutamic acid decarboxylase (GAD) deficiency. CONCLUSIONS: CA demonstrates sedation and alleviates GABA dysfunction via covalent binding ENO1, which shows the potential to improve the therapy of mental illness.

Elastase Inhibitor Cyclotheonellazole A: Total Synthesis and In Vivo Biological Evaluation for Acute Lung Injury.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is one of the most common complications in COVID-19. Elastase has been recognized as an important target to prevent ALI/ARDS in the patient of COVID-19. Cyclotheonellazole A (CTL-A) is a natural macrocyclic peptide reported to be a potent elastase inhibitor. Herein, we completed the first total synthesis of CTL-A in 24 linear steps. The key reactions include three-component MAC reactions and two late-stage oxidations. We also provided seven CTL-A analogues and elucidated preliminary structure-activity relationships. The in vivo ALI mouse model further suggested that CTL-A alleviated acute lung injury with reductions in lung edema and pathological deterioration, which is better than sivelestat, one approved elastase inhibitor. The activity of CTL-A against elastase, along with its cellular safety and well-established synthetic route, warrants further investigation of CTL-A as a candidate against COVID-19 pathogeneses.

Identification of Novel Antagonists Targeting Cannabinoid Receptor 2 Using a Multi-Step Virtual Screening Strategy.

The endocannabinoid system plays an essential role in the regulation of analgesia and human immunity, and Cannabinoid Receptor 2 (CB2) has been proved to be an ideal target for the treatment of liver diseases and some cancers. In this study, we identified CB2 antagonists using a three-step "deep learning-pharmacophore-molecular docking" virtual screening approach. From the ChemDiv database (1,178,506 compounds), 15 hits were selected and tested by radioligand binding assays and cAMP functional assays. A total of 7 out of the 15 hits were found to exhibit binding affinities in the radioligand binding assays against CB2 receptor, with a pKi of 5.15-6.66, among which five compounds showed antagonistic activities with pIC50 of 5.25-6.93 in the cAMP functional assays. Among these hits, Compound 8 with the 4H-pyrido[1,2-a]pyrimidin-4-one scaffold showed the best binding affinity and antagonistic activity with a pKi of 6.66 and pIC50 of 6.93, respectively. The new scaffold could serve as a lead for further development of CB2 drugs. Additionally, we hope that the model in this study could be further utilized to identify more novel CB2 receptor antagonists, and the developed approach could also be used to design potent ligands for other therapeutic targets.

Discovery of novel dual adenosine A1/A2A receptor antagonists using deep learning, pharmacophore modeling and molecular docking.

Adenosine receptors (ARs) have been demonstrated to be potential therapeutic targets against Parkinson's disease (PD). In the present study, we describe a multistage virtual screening approach that identifies dual adenosine A1 and A2A receptor antagonists using deep learning, pharmacophore models, and molecular docking methods. Nineteen hits from the ChemDiv library containing 1,178,506 compounds were selected and further tested by in vitro assays (cAMP functional assay and radioligand binding assay); of these hits, two compounds (C8 and C9) with 1,2,4-triazole scaffolds possessing the most potent binding affinity and antagonistic activity for A1/A2A ARs at the nanomolar level (pKi of 7.16-7.49 and pIC50 of 6.31-6.78) were identified. Further molecular dynamics (MD) simulations suggested similarly strong binding interactions of the complexes between the A1/A2A ARs and two compounds (C8 and C9). Notably, the 1,2,4-triazole derivatives (compounds C8 and C9) were identified as the most potent dual A1/A2A AR antagonists in our study and could serve as a basis for further development. The effective multistage screening approach developed in this study can be utilized to identify potent ligands for other drug targets.

Ursolic acid reduces hepatocellular apoptosis and alleviates alcohol-induced liver injury via irreversible inhibition of CASP3 in vivo.

Alcoholic liver disease (ALD) is one of the pathogenic factors of chronic liver disease with the highest clinical morbidity worldwide. Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid, has shown many health benefits including antioxidative, anti-inflammatory, anticancer, and hepatoprotective activities. We previously found that UA was metabolized in vivo into epoxy-modified UA containing an epoxy electrophilic group and had the potential to react with nucleophilic groups. In this study we prepared an alkynyl-modified UA (AM-UA) probe for tracing and capturing the target protein of UA from liver in mice, then investigated the mode by which UA bound to its target in vivo. By conducting proteome identification and bioinformatics analysis, we identified caspase-3 (CASP3) as the primary target protein of UA associated with liver protection. Molecule docking analysis showed that the epoxy group of the UA metabolite reacted with Cys-163 of CASP3, forming a covalent bond with CASP3. The binding mode of the UA metabolites (UA, CM-UA, and EM-UA) was verified by biochemical evaluation, demonstrating that the epoxy group produced by metabolism played an important role in the inhibition of CASP3. In alcohol-treated HepG2 cells, pretreatment with the UA metabolite (10 µM) irreversibly inhibited CASP3 activities, and subsequently decreased the cleavage of PARP and cell apoptosis. Finally, pre-administration of UA (20-80 mg· kg-1 per day, ig, for 1 week) dose-dependently alleviated alcohol-induced liver injury in mice mainly via the inhibition of CASP3. In conclusion, this study demonstrates that UA is a valuable lead compound for the treatment of ALD.

Myricetin ameliorates bleomycin-induced pulmonary fibrosis in mice by inhibiting TGF-ß signaling via targeting HSP90ß.

Idiopathic pulmonary fibrosis is a progressive-fibrosing lung disease with high mortality and limited therapy, which characterized by myofibroblasts proliferation and extracellular matrix deposition. Myricetin, a natural flavonoid, has been shown to possess a variety of biological characteristics including anti-inflammatory and anti-tumor. In this study we explored the potential effect and mechanisms of myricetin on pulmonary fibrosis in vivo and vitro. The in vivo studies showed that myricetin effectively alleviated bleomycin (BLM)-induced pulmonary fibrosis. KEGG analysis of RNA-seq data indicated that myricetin could regulate the transforming growth factor (TGF)-ß signaling pathway. In vitro studies indicated that myricetin could dose-dependently suppress TGF-ß1/Smad signaling and attenuate TGF-ß1-induced fibroblast activation and epithelial-mesenchymal transition (EMT). Molecular docking indicated that heat shock protein (HSP) 90ß may be a potential target of myricetin, and MST assay demonstrated that the dissociation constant (Kd) of myricetin and HSP90ß was 331.59 nM. We demonstrated that myricetin interfered with the binding of HSP90ß and TGF-ß receptor II and impeded fibroblast activation and EMT. In conclusion, myricetin impedes TGF-ß1-induced lung fibroblast activation and EMT via targeting HSP90ß and attenuates BLM-induced pulmonary fibrosis in mice.

Identification of new potent A1 adenosine receptor antagonists using a multistage virtual screening approach.

The use of antagonists for each adenosine receptor (AR) subtype as potent clinical candidates is of growing interest due to their involvement in the treatment of various diseases. The recent resolution of several A1 and A2A ARs X-ray structures provides opportunities for structure-based drug design. In this study, we describe the discovery of novel A1AR antagonists by applying a multistage virtual screening approach, which is based on random forest (RF), e-pharmacophore modeling and docking methods. A multistage virtual screening approach was applied to screen the ChemDiv library (1,492,362 compounds). Among the final hits, 22 compounds were selected for further radioligand binding assay analysis against human A1AR, and 18 compounds (81.82% success) exhibited nanomolar or low micromolar binding potency (Ki). Then, we selected six compounds (pKi > 6) to further evaluate their antagonist profile in a cAMP functional assay, and we found that they had low micromolar antagonistic activity (pIC50 = 5.51-6.38) for the A1AR. Particularly, four of six compounds (pKi > 6) showed very good affinity (pKi = 6.11-7.13) and selectively (>100-fold) for A1AR over A2AAR. Moreover, the novelty analysis suggested that four of six compounds (pKi > 6) were dissimilar to existing A1AR antagonists and hence represented novel A1AR antagonists. Further molecular docking and molecular dynamics (MD) studies showed that the three selective compounds 15, 20 and 22 were stabilized (RMSlig value ≤ 2 Å) inside the binding pocket of A1AR with similar orientations to the docking pose in 100-ns MD simulations, whereas they escaped from the binding area of A2AAR with larger values of RMSlig (RMSlig ≥ 2 Å). We hope that these findings provide new insights into the discovery of drugs targeting A1AR and facilitate research on new drugs and treatments for A1AR-related human pathologies.

The effect of dimerization on the activation and conformational dynamics of adenosine A1 receptor.

The adenosine A1 receptor (A1R) is one of four adenosine receptors in humans, which are involved in the function of the cardiovascular, respiratory and central nervous systems. Experimental results indicate that A1R can form a homodimer and that the protomer-protomer interaction in the A1R dimer is related to certain pharmacological characteristics of A1R activation. In this work, we performed docking, metadynamics simulation, conventional molecular dynamics simulations, Gaussian-accelerated molecular dynamics simulations, potential of mean force calculations, dynamic cross-correlation motions analysis and community network analysis to study the binding mode of 5'-N-ethylcarboxamidoadenosine (NECA) to A1R and the effect of dimerization on the activation of A1R. Our results show that NECA binds to A1R in a similar mode to adenosine in the A1R crystal structure and NECA in the A2AR crystal structure. The A1R homodimer can be activated by one or two agonists with NECA occupying its orthosteric pockets in one (which we call the NECA-A1R system) or both protomers (which we call the dNECA-A1R system). In the NECA-A1R system, activation is predicated in the protomer without NECA bound. In the dNECA-A1R system, only one protomer achieves the active state. These findings suggest an asymmetrical activation mechanism of the homodimer and a negative cooperativity between the two protomers. We envision that our results may further facilitate the drug development of A1R.