Cullin-RING E3 ligases (CRLs) are the major components of ubiquitin-proteasome system, responsible for ubiquitylation and subsequent degradation of thousands of cellular proteins. CRLs play vital roles in the regulation of multiple cellular processes, including cell cycle, cell apoptosis, DNA replication, signalling transduction among the others, and are frequently dysregulated in many human cancers. The discovery of specific neddylation inhibitors, represented by MLN4924, has validated CRLs as promising targets for anti-cancer therapies with a growing market. Recent studies have focused on the discovery of the CRLs inhibitors by a variety of approaches, including high through-put screen, virtual screen or structure-based drug design. The field is, however, still facing the major challenging, since CRLs are a large multi-unit protein family without typical active pockets to facilitate the drug design, and enzymatic activity is mainly dependent on undruggable protein-protein interactions and dynamic conformation changes. Up to now, most reported CRLs inhibitors are aiming at targeting the F-box family proteins (e.g., SKP2, β-TrCP and FBXW7), the substrate recognition subunit of SCF E3 ligases. Other studies reported few small molecule inhibitors targeting the UBE2M-DCN1 interaction, which specifically inhibits CRL3/CRL1 by blocking the cullin neddylation. On the other hand, several CRL activators have been reported, such as plant auxin and immunomodulatory imide drugs, thalidomide. Finally, proteolysis-targeting chimeras (PROTACs) has emerged as a new technology in the field of drug discovery, specifically targeting the undruggable protein-protein interaction. The technique connects the small molecule that selectively binds to a target protein to a CRL E3 via a chemical linker to trigger the degradation of target protein. The PROTAC has become a hotspot in the field of E3-ligase-based anti-cancer drug discovery.
Antineoplastic Agents ; pharmacology ; therapeutic use ; Drug Design ; Drug Discovery ; Enzyme Inhibitors ; pharmacology ; therapeutic use ; Humans ; Neoplasms ; enzymology ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; drug effects

Two new 2-carboxymethyl-3-hexyl-maleic anhydride derivatives, arthrianhydride A (1) and B (2), along with three known compounds 3-5, were isolated from the fermentation broth of a grasshopper-associated fungus Arthrinium sp. NF2410. The structures of new compounds 1 and 2 were determined based on the analysis of the HR-ESI-MS and NMR spectroscopic data. Furthermore, compounds 1 and 2 were evaluated on inhibitory activity against the enzyme SHP2 and both of them showed moderate inhibitory activity against SHP2.
Anhydrides/pharmacology* ; Animals ; Biological Products/pharmacology* ; Enzyme Inhibitors/pharmacology* ; Fungi/chemistry* ; Grasshoppers/microbiology* ; Molecular Structure ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors* ; Secondary Metabolism

Macrophages show significant heterogeneity in function and phenotype, which could shift into different populations of cells in response to exposure to various micro-environmental signals. These changes, also termed as macrophage polarization, of which play an important role in the pathogenesis of many diseases. Numerous studies have proved that Hesperidin (HDN), a traditional Chinese medicine, extracted from fruit peels of the genus citrus, play key roles in anti-inflammation, anti-tumor, anti-oxidant and so on. However, the role of HDN in macrophage polarization has never been reported. Additional, because of its poor water solubility and bioavailability. Our laboratory had synthesized many hesperidin derivatives. Among them, hesperidin derivatives-12 (HDND-12) has better water solubility and bioavailability. So, we evaluated the role of HDND-12 in macrophage polarization in the present study. The results showed that the expression of Arginase-1 (Arg-1), interleukin-10 (IL-10), transforming growth factor β (TGF-β) were up-regulated by HDND-12, whereas the expression of inducible Nitric Oxide Synthase (iNOS) was down-regulated in LPS- and IFN-γ-treated (M1) RAW264.7 cells. Moreover, the expression of p-JAK2 and p-STAT3 were significantly decreased after stimulation with HDND-12 in M1-like macrophages. More importantly, when we taken AG490 (inhibitor of JAK2/STAT3 signaling), the protein levels of iNOS were significantly reduced in AG490 stimulation group compare with control in LPS, IFN-γ and HDND-12 stimulation cells. Taken together, these findings indicated that HDND-12 could prevent polarization toward M1-like macrophages, at least in part, through modulating JAK2/STAT3 pathway.
Animals ; Cytokines ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Hesperidin ; chemistry ; pharmacology ; Inflammation ; genetics ; metabolism ; Janus Kinase 2 ; antagonists & inhibitors ; metabolism ; Macrophages ; drug effects ; immunology ; metabolism ; Medicine, Chinese Traditional ; Mice ; Molecular Structure ; Phosphorylation ; drug effects ; RAW 264.7 Cells ; STAT3 Transcription Factor ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects

The flower buds of Lonicera macranthoides (Shan Yin-Hua), represent an important traditional Chinese medicine and food ingredient. A phytochemical investigation of the 70% EtOH extract of the flower buds of L. macranthoides resulted in the isolation of 12 triterpenoids (1-12), including two new ursane-type nortriterpenes, 2α, 24-dihydroxy-23-nor-ursolic acid (1) and 2α, 4α-dihydroxy-23-nor-ursolic acid (2). Their structures were established by multiple spectroscopic methods and comparison with literature data. All isolated compounds were evaluated for their anti-inflammatory effects in LPS-activated RAW264.7 cells. Compounds 1 and 2 exhibited inhibitory effects on iNOS at the concentration of 30 μmol·L.
Animals ; Anti-Inflammatory Agents ; chemistry ; pharmacology ; Drugs, Chinese Herbal ; chemistry ; Enzyme Inhibitors ; chemistry ; pharmacology ; Ethanol ; chemistry ; Flowers ; chemistry ; Lonicera ; chemistry ; Macrophages ; drug effects ; metabolism ; Mice ; Molecular Structure ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; Plant Extracts ; chemistry ; Plants, Edible ; chemistry ; RAW 264.7 Cells ; Triterpenes ; chemistry ; pharmacology

OBJECTIVE: To investigate the effects of millimeter wave (MMW) exposure on apoptosis of human melanoma A375 cells and explore the mechanisms. METHODS: Through electromagnetic field calculation we simulated MMW exposure in cells and calculated the specific absorption rate (SAR). The optimal irradiation parameters were determined according to the uniformity and intensity of the SAR. A375 cells were then exposed to MMV for 15, 30, 60, or 90 min, with or without pretreatment with the caspase-3 inhibitor AC-DEVD-fmk (10 μmol/L) for 1 h at 90 min before the exposure. CCK-8 assay was used to assess the changes in the viability and Annexin-V/ PI staining was used to detect the apoptosis of the cells following the exposures; Western blotting was used to detect the expression of caspase-3 in the cells. RESULTS: The results of electromagnetic field calculation showed that for optimal MMV exposure, the incident field needed to be perpendicular to the bottom of the plastic Petri dish with the antenna placed below the dish. CCk-8 assay showed that MMW exposure significantly inhibited the cell viability in a time-dependent manner ( < 0.05); exposures for 15, 30, 60, and 90 min all resulted in significantly increased apoptosis of the cells ( < 0.05). The cells with MMW exposure showed significantly increased expression of caspase-3. The inhibitory effect of MMW on the cell viability was antagonized significantly by pretreatment of the cells with AC-DEVD-fmk ( < 0.05), which increased the cell viability rate from (36.7±0.09)% to (59.8±0.06)% ( < 0.05). CONCLUSIONS 35.2 GHz millimeter wave irradiation induces apoptosis in A375 cells by activating the caspase-3 protein.
Apoptosis ; Caspase 3 ; metabolism ; Caspase Inhibitors ; pharmacology ; Cell Line, Tumor ; Cell Survival ; Electromagnetic Fields ; Enzyme Activation ; Humans ; Magnetic Field Therapy ; Melanoma ; enzymology ; pathology ; therapy ; Time Factors

Rifamycins, a group of bacterial RNA polymerase inhibitors, are the firstline antimicrobial drugs to treat tuberculosis. In light of the emergence of rifamycinresistant bacteria, development of new RNA polymerase inhibitors that kill rifamycinresistant bacteria with high bioavailability is urgent. Structural analysis of bacterial RNA polymerase in complex with inhibitors by crystallography and cryo-EM indicates that RNA polymerase inhibitors function through five distinct molecular mechanisms:inhibition of the extension of short RNA; competition with substrates; inhibition of the conformational change of the'bridge helix'; inhibition of clamp opening;inhibition of clamp closure. This article reviews the research progress of these five groups of RNA polymerase inhibitors to provide references for the modification of existing RNA polymerase inhibitors and the discovery of new RNA polymerase inhibitors.
Antitubercular Agents ; therapeutic use ; Bacteria ; drug effects ; enzymology ; DNA-Directed RNA Polymerases ; metabolism ; Drug Discovery ; trends ; Drug Resistance, Bacterial ; Enzyme Activation ; drug effects ; Enzyme Inhibitors ; pharmacology ; Humans ; RNA, Bacterial ; Tuberculosis ; drug therapy ; enzymology

The aim of this study was to investigate the inhibitory effect and the underlying mechanism of ethacrynic acid (EA) on the contraction in mice. BL-420S force measuring system was used to measure the tension of mouse tracheal rings. The whole cell patch clamp technique was utilized to record the channel currents of airway smooth muscle (ASM) cells. The calcium imaging system was used to determine the intracellular Ca concentration ([Ca]) in ASM cells. The results showed that EA significantly inhibited the high K (80 mmol/L) and acetylcholine (ACh, 100 µmol/L)-induced contraction of mouse tracheal rings in a dose-dependent manner. The maximal relaxation percentages were (97.02 ± 1.56)% and (85.21 ± 0.03)%, and the median effective concentrations were (40.28 ± 2.20) μmol/L and (56.22 ± 7.62) μmol/L, respectively. EA decreased the K and ACh-induced elevation of [Ca] from 0.40 ± 0.04 to 0.16 ± 0.01 and from 0.50 ± 0.01 to 0.39 ± 0.01, respectively. In addition, EA inhibited L-type voltage-dependent calcium channel (LVDCC) and store-operated calcium channel (SOCC) currents in ASM cells, and Ca influx. Moreover, EA decreased the resistance of the respiratory system (Rrs) in vivo in mice. These results indicated that EA inhibits LVDCC and SOCC, which results in termination of Ca influx and decreases of [Ca], leading to relaxation of ASM. Taken together, EA might be a potential bronchodilator.
Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; Enzyme Inhibitors ; pharmacology ; Ethacrynic Acid ; pharmacology ; Mice ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; Respiratory System ; cytology ; drug effects

Valproic acid (VPA), an agent that is used to treat epileptic seizures, can cause spatial memory impairment in adults and children. This effect is thought to be due to the ability of VPA to inhibit neurogenesis in the hippocampus, which is required for learning. We have previously used an animal model to show that VPA significantly impairs hippocampal-spatial working memory and inhibits neuronal generation in the sub-granular zone of the dentate gyrus. As there are patient reports of improvements in memory after discontinuing VPA treatment, the present study investigated the recovery of both spatial memory and hippocampal neurogenesis at two time points after withdrawal of VPA. Male Wistar rats were given intraperitoneal injections of 0.9% normal saline or VPA (300 mg/kg) twice a day for 10 d. At 1, 30, or 45 d after the drug treatment, the novel object location (NOL) test was used to examine spatial memory; hippocampal cell division was counted using Ki67 immunohistochemistry, and levels of brain-derived neurotrophic factor (BDNF) and Notch1 were measured using western immunoblotting. Spatial working memory was impaired 1 and 30 d after the final administration, but was restored to control levels by 45 d. Cell proliferation had increased to control levels at 30 and 45 d. Both markers of neurogenesis (BDNF and Notch1 levels) had returned to control levels at 45 d. These results demonstrate that memory recovery occurs over a period of six weeks after discontinuing VPA treatment and is preceded by a return of hippocampal neurogenesis to control levels.
Animals ; Brain-Derived Neurotrophic Factor/metabolism* ; Cell Proliferation ; Cognition/drug effects* ; Dentate Gyrus/drug effects* ; Enzyme Inhibitors/pharmacology* ; Hippocampus/metabolism* ; Immunohistochemistry ; Male ; Memory Disorders/therapy* ; Memory, Short-Term/drug effects* ; Neurogenesis/drug effects* ; Neurons/metabolism* ; Rats ; Rats, Wistar ; Receptor, Notch1/metabolism* ; Spatial Memory/drug effects* ; Valproic Acid/pharmacology*

Increasing evidence suggests that spinal microglia regulate pathological pain in males. In this study, we investigated the effects of several microglial and astroglial modulators on inflammatory and neuropathic pain following intrathecal injection in male and female mice. These modulators were the microglial inhibitors minocycline and ZVEID (a caspase-6 inhibitor) and the astroglial inhibitors L-α-aminoadipate (L-AA, an astroglial toxin) and carbenoxolone (a connexin 43 inhibitor), as well as U0126 (an ERK kinase inhibitor) and D-JNKI-1 (a c-Jun N-terminal kinase inhibitor). We found that spinal administration of minocycline or ZVEID, or Caspase6 deletion, reduced formalin-induced inflammatory and nerve injury-induced neuropathic pain primarily in male mice. In contrast, intrathecal L-AA reduced neuropathic pain but not inflammatory pain in both sexes. Intrathecal U0126 and D-JNKI-1 reduced neuropathic pain in both sexes. Nerve injury caused spinal upregulation of the astroglial markers GFAP and Connexin 43 in both sexes. Collectively, our data confirmed male-dominant microglial signaling but also revealed sex-independent astroglial signaling in the spinal cord in inflammatory and neuropathic pain.
2-Aminoadipic Acid ; toxicity ; Animals ; Anti-Inflammatory Agents ; therapeutic use ; Astrocytes ; pathology ; Carbenoxolone ; pharmacology ; Caspase 6 ; deficiency ; metabolism ; Connexin 43 ; metabolism ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Male ; Mice ; Mice, Transgenic ; Microglia ; pathology ; Minocycline ; therapeutic use ; Neuralgia ; chemically induced ; drug therapy ; pathology ; Pain Measurement ; Phenylurea Compounds ; pharmacology ; Sex Characteristics ; Spinal Cord ; pathology ; Time Factors

OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05). CONCLUSIONS Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.
Aldehyde Dehydrogenase, Mitochondrial ; antagonists & inhibitors ; metabolism ; Animals ; Animals, Newborn ; Benzamides ; pharmacology ; Benzodioxoles ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Culture Media ; Enzyme Inhibitors ; pharmacology ; Glucose ; administration & dosage ; pharmacology ; Isoflavones ; pharmacology ; Mitochondria, Heart ; enzymology ; Myocytes, Cardiac ; drug effects ; metabolism ; NFATC Transcription Factors ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley