Scavenger receptor CD36 could bind and endocytose oxLDL into macrophages which were then differentiated into foam cells that constitute the atherosclerotic lesion core, and was considered to be a potential target to treat atherosclerosis. In the establishment of the compound library of berberine (BBR, 1) analogues, we discovered that 13-hexylberberine (2) showed an antagonistic activity against CD36. Taking 2 as the lead compound, 21 derivatives were synthesized and their antagonistic activities were evaluated via an ELISA-like high-throughput screening (HTS) model. The primary structure-activity relationships were studied. It was indicated that the introduction of suitable groups at the 2- and 3-position of the aromatic ring A or at the 9-position of the aromatic ring D could enhance the activity. Among the 21 studied compounds, 7g bearing a benzyloxyl group at the 9-position provided a highest CD36 antagonistic activity with the IC50 value of 7.7 micromol L(-1). Besides, its antagonistic activity was further verified with Sf9 insect cell HTS model. So berberine analogues are a new family of CD36 receptor antagonists and worthy to be studied further.

ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI/CLA-1) are the key proteins in reverse cholesterol transport (RCT). The high expression of ABCA1 and SR-BI/CLA-1 can decrease the danger of atherosclerosis. The purpose of the study is to find ABCA1 and CLA-1up-regulators for treating atherosclerosis by using cell-based high throughput screening models. Among 20 000 compounds screened, E0869 [1-(3, 4-dimethylphenyl)-1-oxopropan-2-yll4-((methylsulfonyl)methyl)benzoate] was found as the positive hit. The up-regulated activities of E0869 in ABCAl1-LUC and bCA-l1-LUC HepG2 cell were 160% and 175%, respectively. The EC50 values of E0869 in ABCAl1-LUC and CLA-l1-LUC HepG2 cell were 3.79 and 1.42 pμol- x ,(-1) respectively. E0869 could upregulate the mRNA and protein levels of ABCA1, SR-BI/CLA-1 and ABCGJ1genes in HepG2 and RAW264.7 cells by Real-Time Quantitative PCR and Western blotting analysis, but could not influence the expression of FAS, SREBP-l1 and CD36. Foam cell assay showed that E0869 could inhibit lipids accumulation in mouse peritoneal macrophages RAW264.7. Cholesterol efflux assay showed that E0869 could induce HDL-mediated cholesterol efflux in mouse peritoneal macrophages RAW264.7. In conclusion, E0869 could up-regulate ABCA1 and CLA-1 activity, and had good anti-atherosclerotic activity in vitro.

In the previous study, a high-throughput screening method was established to find the antagonists of CD36. In the present study, a new compound named IMB-1680 was found using this method. The anti-atherosclerotic activities of IMB-1680 were then evaluated. Dose-dependent activities of IMB-1680 were detected by using Sf9 [hCD36] and CHO [hCD36] models. Fluorescence microscopic photography and flow cytometry were used to analyze uptake of mLDL. Foam cell test with RAW264.7 macrophages was used to examine lipid accumulation. The results showed that IMB-1680 inhibited CD36 activity with IC50 of 2.80 and 8.79 micromol x L(-1) in Sf9[hCD36] and CHO [hCD36] cells, respectively. Fluorescence microscopic photography and flow cytometry revealed that IMB-1680 could significantly reduce DiI-AcLDL uptake. Meanwhile, IMB-1680 also could reduce lipids accumulation in RAW264.7 macrophages. In all, the data indicated that IMB-1680 might be a potent effective anti-atherosclerotic leading compound.

This study is to explore new lead compounds by inhibition of Pin1 for anticancer therapy using temperature sensitive mutants. As Pin1 is conserved from yeast to human, we established a high-throughput screening method for Pin1 inhibitors, which employed yeast assay. This method led to the identification of one potent hits, 8-11. In vitro, 8-11 inhibited purified Pin1 enzyme activity with IC50 of (10.40 +/- 1.68) micromol x L(-1), induced G1 phase arrest and apoptosis, showed inhibitory effects on a series of cancer cell proliferation, reduced Cyclin D1 expression, was defined as reciprocally matched for protein-ligand complex in virtual docking analysis and reduced cell migration ability. In vivo, we could observe reduction of tumor volume after treatment with 8-11 in xenograft mice compared with vehicle DMSO treatment. Altogether, these results provide for the first time the involvement of 8-11 in the anticancer activity against Pin1.

Liver X receptor (LXR) plays an important role in reverse cholesterol transport (RCT), and activation of LXR could reduce atherosclerosis. In the present study we used a cell-based screening method to identify new potential LXRβ agonists. A novel benzofuran-2-carboxylate derivative was identified with LXRβ agonist activity: E17110 showed a significant activation effect on LXRβ with an EC50 value of 0.72 μmol/L. E17110 also increased the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) in RAW264.7 macrophages. Moreover, E17110 significantly reduced cellular lipid accumulation and promoted cholesterol efflux in RAW264.7 macrophages. Interestingly, we found that the key amino acids in the LXRβ ligand-binding domain had distinct interactions with E17110 as compared to TO901317. These results suggest that E17110 was identified as a novel compound with LXRβ agonist activity in vitro via screening, and could be developed as a potential anti-atherosclerotic lead compound.

In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e⁻⁷±7.9e⁻⁸ and 6.0e⁻⁷±2.8e⁻⁸, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e⁻⁷±7.2e⁻⁸. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.
Carrier Proteins ; Escherichia coli/metabolism* ; Escherichia coli Proteins/metabolism* ; Interferometry ; Membrane Proteins/metabolism*

Gram-negative bacteria have become the main pathogens and cause serious clinical problems with increased morbidity and mortality. However, the slow discovery of new antimicrobial agents is unable to meet the need for the treatment of bacterial infections caused by drug-resistant strains. The interaction of L12 and L10 is essential for ribosomal function and protein synthesis. In this study, a yeast two-hybrid system was established to successfully detect the interaction between L12 and L10 proteins from gram-negative bacteria , which allows us to screen compounds that specifically disrupt this interaction. With this system, we identified two compounds IMB-84 and IMB-87 that block L12-L10 interaction and show bactericidal activity against . We used glutathione--transferase (GST) pull-down and surface plasmon resonance (SPR) assays to demonstrate that these compounds disrupt L12-L10 interaction and the target of compounds was further confirmed by the overexpression of target proteins. Moreover, protein synthesis and elongation factor G-dependent GTPase activities are inhibited by two compounds. Therefore, we have identified two antibacterial agents that disrupt L12-L10 interaction by using yeast two-hybrid system.

Tuberculosis (TB) is one of the deadly diseases caused by Mycobacterium tuberculosis (Mtb), which presents a significant public health challenge. Treatment of TB relies on the combination of several anti-TB drugs to create shorter and safer regimens. Therefore, new anti-TB agents working by different mechanisms are urgently needed. FtsZ, a tubulin-like protein with GTPase activity, forms a dynamic Z-ring in cell division. Most of FtsZ inhibitors are designed to inhibit GTPase activity. In Mtb, the function of Z-ring is modulated by SepF, a FtsZ binding protein. The FtsZ/SepF interaction is essential for FtsZ bundling and localization at the site of division. Here, we established a yeast two-hybrid based screening system to identify inhibitors of FtsZ/SepF interaction in M. tuberculosis. Using this system, we found compound T0349 showing strong anti-Mtb activity but with low toxicity to other bacteria strains and mice. Moreover, we have demonstrated that T0349 binds specifically to SepF to block FtsZ/SepF interaction by GST pull-down, fluorescence polarization (FP), surface plasmon resonance (SPR) and CRISPRi knockdown assays. Furthermore, T0349 can inhibit bacterial cell division by inducing filamentation and abnormal septum. Our data demonstrated that FtsZ/SepF interaction is a promising anti-TB drug target for identifying agents with novel mechanisms.

[This corrects the article DOI: 10.1016/j.apsb.2023.01.022.].

Pancreatic adenocarcinoma (PAAD) is one of the most lethal malignancies. Although gemcitabine (GEM) is a standard treatment for PAAD, resistance limits its application and therapy. Secoemestrin C (Sec C) is a natural compound from the endophytic fungus Emericella, and its anticancer activity has not been investigated since it was isolated. Our research is the first to indicate that Sec C is a broad-spectrum anticancer agent and could exhibit potently similar anticancer activity both in GEM-resistant and GEM-sensitive PAAD cells. Interestingly, Sec C exerted a rapid growth-inhibiting effect (80% death at 6 h), which might be beneficial for patients who need rapid tumor shrinkage before surgery. Liquid chromatography/mass spectrometry and N-acetyl-l-cysteine (NAC) reverse assays show that Sec C sulfates cysteines to disrupt disulfide-bonds formation in endoplasmic reticulum (ER) proteins to cause protein misfolding, leading to ER stress and disorder of lipid biosynthesis. Microarray data and subsequent assays show that ER stress-mediated ER-associated degradation (ERAD) ubiquitinates and downregulates YAP to enhance ER stress via destruction complex (YAP-Axin-GSK-βTrCP), which also elucidates a unique degrading style for YAP. Potent anticancer activity in GEM-resistant cells and low toxicity make Sec C a promising anti-PAAD candidate.