Discovery of selective covalent cathepsin K inhibitors containing novel 4-cyanopyrimidine warhead based on quantum chemical calculations and binding mode analysis.

Cathepsin K (Cat K), mainly expressed by osteoclasts, plays an important role in bone resorption. Covalent Cat K inhibitors will show great potential in the future treatment of osteoporosis. It has been reported that the selectivity of covalent cathepsin K inhibitors was related to the drug's safety. The type of warhead has a crucial influence on the enzyme bioactivity and selectivity of covalent inhibitors. In order to develop novel covalent inhibitors with the selective new warhead, quantum chemical calculations were performed to estimate the reactivity of the nitrile warheads. Moreover, binding mode analysis between ligands and high homology Cat K, S and B revealed differences in non-covalent interactions. Novel covalent Cat K inhibitors containing 4-cyanopyrimidine warhead (11) were determined for the first time. Among them, compound 34 significantly inhibited Cat K (IC50 = 61.9 nM) with excellent selectivity compared to Cat S (>810-fold) and Cat B (>1620-fold), respectively. Binding mode analysis of Cat K-34 complex provided the basis for further optimization. Compound 34 could be a valuable lead compound for further research on safe and effective Cat K inhibitors.

Design, synthesis and biological evaluation of novel triazoloquinazolinone and imidazoquinazolinone derivatives as allosteric inhibitors of SHP2 phosphatase.

ABSTRCTA series of novel triazoloquinolinone and imidazoquinazolinone derivatives were designed and synthesised, and their biological activities against SHP2 protein and melanoma A357 cell line were evaluated in vitro. The results show that some target compounds have moderate to excellent inhibitory activity on SHP2 protein and melanoma A357 cell line. Structure-activity relationships (SARs) showed that both imidazoquinazolinone and triazoloquinazolinone derivatives have good SHP2 protein kinase and melanoma cell line A357 inhibitory activity. The results of molecular docking also showed that the cores of imidazoquinazolinone and triazoloquinazolinone have a certain affinity for SHP2 protein at the same time. Compared with SHP244, the target compounds have quite good liver microsomal stability and has more drug potential. The most promising compound B1 has a strong inhibitory effect on the melanoma cell line A357 at 100 µM (76.15% inhibition).

Design, synthesis, and biological evaluation of 1,3,6,7-tetrahydroxyxanthone derivatives as phosphoglycerate mutase 1 inhibitors.

Phosphoglycerate mutase 1 (PGAM1) is a promising target for cancer treatment. Herein, we found that α-mangostin and γ-mangostin exhibited moderate PGAM1 inhibitory activities, with IC50 of 7.2 µM and 1.2 µM, respectively. Based on α-mangostin, a series of 1,3,6,7-tetrahydroxyxanthone derivatives were designed, synthesized and evaluated in vitro for PGAM1 inhibition. The significant structure-activity relationships (SAR) and a fresh binding mode of this kind of new compounds were also clearly described. This study provides valuable information for further optimization of PGAM1 inhibitors with 1,3,6,7-tetrahydroxyxanthone backbone or de novo design of novel inhibitor.

iTRAQ proteomic analysis of salinity acclimation proteins in the gill of tropical marbled eel (Anguilla marmorata).

Osmoregulation plays an important role in the migration process of catadromous fish. The osmoregulatory mechanisms of tropical marbled eel (Anguilla marmorata), a typical catadromous fish, did not gain sufficient attention, especially at the molecular level. In order to enrich the protein database of A. marmorata, a proteomic analysis has been carried out by iTRAQ technique. Among 1937 identified proteins in gill of marbled eel, the expression of 1560 proteins (80 %) was quantified. Compared with the protein expression level in the gill of marbled eel in freshwater (salinity of 0 ‰), 336 proteins were up-regulated and 67 proteins were down-regulated in seawater (salinity of 25 ‰); 33 proteins were up-regulated and 32 proteins were down-regulated in brackish water (salinity of 10 ‰). These up-regulated proteins including Na(+)/K(+)-ATPase, V-type proton ATPase, sodium-potassium-chloride co-transporter and heat shock protein 90 were enriched in many KEGG-annotated pathways, which are related to different functions of the gill. The up-regulated oxidative phosphorylation and seleno-compound metabolism pathways involve the synthesis and consumption of ATP, which represents extra energy consumption. Another identified pathway is the ribosome pathway in which a large number of up-regulated proteins are involved. It is also more notable that tight junction and cardiac muscle contraction pathways may have correlation with ion transport in gill cells. This is the first report describing the proteome of A. marmorata for acclimating to the change of salinity. These results provide a functional database for migratory fish and point out some possible new interactions on osmoregulation in A. marmorata.

Identification of BAF60b as a Chromatin-Remodeling Checkpoint of Diet-Induced Fatty Liver Disease.

Overnutrition has gradually become the primary causative factor in nonalcoholic fatty liver disease (NAFLD). However, how nutritional signals are integrated to orchestrate the transcriptional programs important for NAFLD progression remains poorly understood. We identified hepatic BAF60b as a lipid-sensitive subunit of the switch/sucrose nonfermentable chromatin-remodeling complex that is negatively associated with liver steatosis in mice and humans. Hepatic BAF60b deficiency promotes high-fat diet (HFD)-induced liver steatosis in mice, whereas transgenic expression of BAF60b in the liver attenuates HFD-induced obesity and NAFLD, both accompanied by a marked regulation of peroxisome proliferator-activated receptor γ (PPARγ) expression. Mechanistically, through motif analysis of liver assay for transposase-accessible chromatin sequencing and multiple validation experiments, we identified C/EBPß as the transcription factor that interacts with BAF60b to suppress Pparγ gene expression, thereby controlling hepatic lipid accumulation and NAFLD progression. This work identifies hepatic BAF60b as a negative regulator of liver steatosis through C/EBPß-dependent chromatin remodeling.

Sappanone A: A natural PDE4 inhibitor with dual anti-inflammatory and antioxidant activities from the heartwood of Caesalpinia sappan L.

ETHNOPHARMACOLOGICAL RELEVANCE: Sumu (Lignum sappan), the dry heartwood of Caesalpinia sappan L., is a traditional Chinese medicine used as an analgesic and anti-inflammatory agent. AIM OF THE STUDY: The study aspired to discover natural phosphodiesterase 4 (PDE4) inhibitors with dual anti-inflammatory and antioxidant activities from Sumu for the treatment of chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: To accurately and efficiently identify natural PDE4 inhibitors from Sumu, molecular docking and molecular dynamics (MD) analysis methods were used for structure-based virtual screening of a self-built database of primary polyphenols in Sumu. According to the previous studies of Sumu and the free radical scavenging mechanism of polyphenols, the reported antioxidant components from Sumu and the potential antioxidants with the antioxidant pharmacophore of catechol and π-conjugated moieties were selected from the potential PDE4 inhibitors predicted by docking. Sappanone A, a potential PDE4 inhibitor with antioxidant activity from Sumu, was selected, calculated and synthesized to evaluate its dual anti-inflammatory and antioxidant functions in vitro and in vivo studies. Herein sappanone A was assayed for its inhibitory effects against PDE4 enzyme activity, tumor necrosis factor-alpha (TNF-α) production induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and malondialdehyde (MDA) production induced by Fe2+ in mouse lung homogenate; sappanone A was also assayed for its abilities of radical (DPPH) scavenging, reducing Fe3+ and complexing Fe2+ in vitro. Additionally, LPS-induced acute lung injury (ALI) in mice was used to evaluate its anti-inflammatory activity as a PDE4 inhibitor in vivo, and the levels of TNF-α and total protein in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in the lung were assayed. RESULTS: The present study predicted and validated that sappanone A was a promising PDE4 inhibitor from Sumu with dual anti-inflammation and antioxidant activities from Sumu. In vitro, sappanone A remarkably inhibited PDE4 enzyme activity and reduced TNF-α production induced by LPS in RAW264.7 macrophages and MDA production induced by Fe2+ in mouse lung homogenate. Meanwhile, it showed outstanding abilities of scavenging DPPH radicals, reducing Fe3+ and complexing Fe2+. In vivo, sappanone A (25 mg/kg and 50 mg/kg, i.p., twice daily for 7 days) distinctly prevented LPS-induced ALI in mice by reducing the levels of TNF-α and total protein in BALF and MPO activity in the lung. CONCLUSION: Sappanone A is a natural PDE4 inhibitor with dual anti-inflammatory and antioxidant activities from the traditional Chinese medicine Sumu, which may be a promising therapeutic agent to prevent the vicious cycle of COPD inflammation and oxidative stress.

Mining and characterization of oxidative stress-related binding proteins of parthenolide in Xanthomonas oryzae pv. oryzae.

BACKGROUND: Lack of control agents and development of bacterial resistance are emergent problems in the chemical control of rice bacterial blight, therefore novel bactericides against Xanthomonas oryzae pv. oryzae (Xoo, the causal agent of rice bacterial blight) are urgently needed. We previously found that parthenolide (PTL) is a potential lead against Xoo, and PTL inhibits Xoo growth via oxidative stress. However, the mechanism of action of PTL against Xoo needs further elucidation. RESULTS: In this study, a biotinylated PTL probe was synthesized, and two important subunits in the respiratory chain (NuoF of complex I and SdhB of complex II) of Xoo were captured with the probe and identified with liquid chromatography tandem mass spectrometry (LC-MS/MS). The binding between them was verified with pull-down and drug affinity responsive target stability technologies. In addition, purified proteins of NuoF and SdhB greatly lowered the antibacterial activity of PTL, and PTL evidently inhibited the enzyme activities of complexes I and II. Moreover, knockout of nuoF and sdhB in Xoo caused elevated reactive oxygen species (ROS) levels and increased sensitivity to PTL. Furthermore, molecular simulations indicated that PTL may form covalent bonds with Cys105 and Cys187 in NuoF and Cys106 in SdhB. CONCLUSION: PTL can directly bind to NuoF and SdhB, which impairs the enzyme functions of complexes I and II in the respiratory chain, leading to ROS accumulation in Xoo. This study will provide deep insight into the mechanism of action of PTL against Xoo. © 2022 Society of Chemical Industry.

Na+/K+-ATPase response to salinity change and its correlation with FXYD11 expression in Anguilla marmorata.

The Na+/K+-ATPase (NKA) is a primary electrogenic protein that promotes ion transport in teleosts. FXYD11 is a putative regulatory subunit of the NKA pump. The regulation of Na +/K + -ATPase and FXYD11 is of critical importance for osmotic homeostasis. To investigate the changes of the two genes under different salinity environments, we first identified NKA (AmNKAα1) and FXYD11 (AmFXYD11) in Anguilla marmorata, and then evaluated the mRNA levels of NKA and FXYD11 as well as the activity of NKA in the gill and kidney at different timepoints (0, 1, 3, 6, 12, 24, 48, 72, 96, and 360 h) under three salinity conditions-0‰ (fresh water: FW), 10‰ (brackish water: BW), and 25‰ (seawater: SW). In the gill, the mRNA levels of AmNKAα1 and AmFXYD11 and the enzyme activity of AmNKAα1 were higher in BW and SW than in FW; the protein abundance was positively correlated with the specific activity of NKA in BW/SW. However, in the kidney, the mRNA level of AmNKAα1 in the BW group was higher than that in the FW group. In addition, AmFXYD mRNA levels in both BW and SW groups were significantly lower than that in the FW control group. These results suggested that AmFXYD11 was tissue specific in response to different salinity environment. Our results clearly demonstrated the important roles of AmNKAα1 and AmFXYD11 in osmotic homeostasis of juvenile A. marmorata under saline environment.

MicroRNA-Sequence Profiling Reveals Novel Osmoregulatory MicroRNA Expression Patterns in Catadromous Eel Anguilla marmorata.

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression by post-transcriptional repression of mRNAs. Recently, several miRNAs have been confirmed to execute directly or indirectly osmoregulatory functions in fish via translational control. In order to clarify whether miRNAs play relevant roles in the osmoregulation of Anguilla marmorata, three sRNA libraries of A. marmorata during adjusting to three various salinities were sequenced by Illumina sRNA deep sequencing methods. Totally 11,339,168, 11,958,406 and 12,568,964 clear reads were obtained from 3 different libraries, respectively. Meanwhile, 34 conserved miRNAs and 613 novel miRNAs were identified using the sequence data. MiR-10b-5p, miR-181a, miR-26a-5p, miR-30d and miR-99a-5p were dominantly expressed in eels at three salinities. Totally 29 mature miRNAs were significantly up-regulated, while 72 mature miRNAs were significantly down-regulated in brackish water (10‰ salinity) compared with fresh water (0‰ salinity); 24 mature miRNAs were significantly up-regulated, while 54 mature miRNAs were significantly down-regulated in sea water (25‰ salinity) compared with fresh water. Similarly, 24 mature miRNAs were significantly up-regulated, while 45 mature miRNAs were significantly down-regulated in sea water compared with brackish water. The expression patterns of 12 dominantly expressed miRNAs were analyzed at different time points when the eels transferred from fresh water to brackish water or to sea water. These miRNAs showed differential expression patterns in eels at distinct salinities. Interestingly, miR-122, miR-140-3p and miR-10b-5p demonstrated osmoregulatory effects in certain salinities. In addition, the identification and characterization of differentially expressed miRNAs at different salinities can clarify the osmoregulatory roles of miRNAs, which will shed lights for future studies on osmoregulation in fish.

Genetic diversity of Odontobutis potamophila from different geographic populations inferred from mtDNA control region.

Odontobutis potamophila is a Chinese endemic species and an economically important fishery resource in the Yangtze River. The genetic variability of O. potamophila was studied based on the sequence analysis of mitochondrial DNA control region from 150 individuals of five geographical populations including from Dangtu (n=30), Sheyang (n=30), Yuyao (n=30), Tai Lake nearby Dongxishan (n=30) and Minjiang (n=30). Among five populations, the genetic distance between Minjiang population and other populations (0.1186-0.1223) was larger than that among four populations except for Minjiang (0.0015-0.0198). In addition, 23 haplotypes were obtained and each population had special haplotypes. The samples from five sites had high haplotype diversity (0.80510) and low nucleotide diversity (0.04028). Through Tajima's D and Fu's F neutral testing and mismatch distribution test among all geographical populations, O. potamophila did not undergo recent population expansion. During the population evolution, O. potamophila experienced a balanced selection function and maintained a stable state and population size. Moreover, the haplotype Neighbor-Joining (NJ) tree was separated two haplotype groups. The NJ tree, TCS network and median-joining network could clearly separate the haplotypes of the specimens from different areas. Analysis of molecular variance and pairwise FST revealed an obvious genetic differentiation among different geographical populations, suggesting that O. potamophila in different geographical populations should be managed and conserved separately.