Discovery of tetrahydroisoquinolineindole derivatives as first dual PRMT5 inhibitors/hnRNP E1 upregulators: Design, synthesis and biological evaluation.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as an important therapeutic target for treating various types of cancer. Upregulation of tumor suppressor hnRNP E1 has also been considered as an effective antitumor therapy. In this study, a series of tetrahydroisoquinolineindole hybrids were designed and prepared, and compounds 3m and 3s4 were found to be selective inhibitors of PRMT5 and upregulators of hnRNP E1. Molecular docking studies indicated that compounds 3m occupied the substrate site of PRMT5 and formed essential interactions with amino acid residues. Furthermore, compounds 3m and 3s4 exerted antiproliferative effects against A549 cells by inducing apoptosis and inhibiting cell migration. Importantly, silencing of hnRNP E1 eliminated the antitumor effect of 3m and 3s4 on the apoptosis and migration in A549 cells, suggesting a regulatory relationship between PRMT5 and hnRNP E1. Additionally, compound 3m exhibited high metabolic stability on human liver microsomes (T1/2 = 132.4 min). In SD rats, the bioavailability of 3m was 31.4%, and its PK profiles showed satisfactory AUC and Cmax values compared to the positive control. These results suggest that compound 3m is the first class of dual PRMT5 inhibitor and hnRNP E1 upregulator that deserves further investigation as a potential anticancer agent.

Cryptotanshinone induces apoptosis of activated hepatic stellate cells via modulating endoplasmic reticulum stress.

BACKGROUND: Cryptotanshinone (CPT) has wide biological functions, including anti-oxidative, antifibrosis, and anti-inflammatory properties. However, the effect of CPT on hepatic fibrosis is unknown. AIM: To investigate the effects of CPT treatment on hepatic fibrosis and its underlying mechanism of action. METHODS: Hepatic stellate cells (HSCs) and normal hepatocytes were treated with different concentrations of CPT and salubrinal. The CCK-8 assay was used to determine cell viability. Flow cytometry was used to measure apoptosis and cell cycle arrest. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses were used to measure mRNA levels and protein expression of endoplasmic reticulum stress (ERS) signaling pathway related molecules, respectively. Carbon tetrachloride (CCL4) was used to induce in vivo hepatic fibrosis in mice. Mice were treated with CPT and salubrinal, and blood and liver samples were collected for histopathological examination. RESULTS: We found that CPT treatment significantly reduced fibrogenesis by modulating the synthesis and degradation of the extracellular matrix in vitro. CPT inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in cultured HSCs. Furthermore, we found that CPT promoted apoptosis of activated HSCs by upregulating expression of ERS markers (CHOP and GRP78) and activating ERS pathway molecules (PERK, IRE1α, and ATF4), which were inhibited by salubrinal. Inhibition of ERS by salubrinal partially eliminated the therapeutic effect of CPT in our CCL4-induced hepatic fibrosis mouse model. CONCLUSION: CPT can promote apoptosis of HSCs and alleviate hepatic fibrosis through modulating the ERS pathway, which represents a promising strategy for treating hepatic fibrosis.

Association between clustering of cardiovascular risk factors and resting heart rate in Chinese population: a cross-sectional study.

BACKGROUND: Epidemiologic studies have explored the association between a single cardiovascular risk factor (CVRF) and resting heart rate (RHR), but the research on the relation of multiple risk factors with RHR remains scarce. This study aimed to explore the associations between CVRFs clustering and the risk of elevated RHR. METHODS: In this cross-sectional study, adults aged 35-75 years from 31 provinces were recruited by the China PEACE Million Persons Projects from September 2015 to August 2020. We focused on seven risk factors: hypertension, diabetes mellitus, dyslipidemia, obesity, smoking, alcohol use, and low physical activity. Multivariate logistic regression was used to calculate odds ratios (OR) for elevated RHR (> 80 beats/min). RESULTS: Among 1,045,405 participants, the mean age was 55.67 ± 9.86 years, and 60.4% of participants were women. The OR (95% CI) for elevated RHR for the groups with 1, 2, 3, 4 and ≥ 5 risk factor were 1.11 (1.08-1.13), 1.36 (1.33-1.39), 1.68 (1.64-1.72), 2.01 (1.96-2.07) and 2.58 (2.50-2.67), respectively (P trend < 0.001). The association between the CVRFs clustering number and elevated RHR was much more pronounced in young males than in other age-sex subgroups. Clusters comprising more metabolic risk factors were associated with a higher risk of elevated RHR than those comprising more behavioral risk factors. CONCLUSIONS: There was a significant positive association between the CVRFs clustering number and the risk of elevated RHR, particularly in young males. Compared clusters comprising more behavioral risk factors, clusters comprising more metabolic risk factors were associated with a higher risk of elevated RHR. RHR may serve as an indicator of the cumulative effect of multiple risk factors.

Synthesis and evaluation of 1,2,4-oxadiazole derivatives as potential anti-inflammatory agents by inhibiting NF-κB signaling pathway in LPS-stimulated RAW 264.7 cells.

In this study, a series of compounds with 1,2,4-oxadiazole core was designed and synthesized for the optimization of JC01, an anti-inflammatory hit identified from our in-house compound library using NF-κB pathway luciferase assay and NO production assay. All the synthetic compounds 1-29 have been screened for their anti-inflammatory effects by evaluating their inhibition against LPS-induced NO release, and compound 17 exhibited the highest activity. Western blotting and immunofluorescence analysis revealed that 17 prominently inhibited LPS-induced activation of NF-κB in RAW264.7 cells and blocked the phosphorylation of p65. Consistent with these results, it was found that 17 prevented the nuclear translocation of NF-κB induced by LPS. These data highlighted 17 as a promising anti-inflammatory agent by inhibiting NF-κB activity.

Simultaneous reduction of nitrate and Cr(VI) by Pseudomonas aeruginosa strain G12 in wastewater.

The interference of toxic heavy metals in the process of microbial aerobic denitrification is a hot issue in industry wastewater treatment in recent years. In this study, a multifunctional aerobic denitrifying bacterium - Pseudomonas aeruginosa G12 isolated from sewage sludge was used to explore the simultaneous removal ability to NO3--N and Cr(VI) in wastewater by a series of batch experiments. The results showed that G12 could effectively remove NO3--N (500 mg L-1) and Cr(VI) (10 mg L-1) by 98% and 93%, respectively. Meanwhile, the study found that the strain G12 had the potential to adapt to the complex external environment, including different carbon resources, nitrogen sources, and the coexisting heavy metals (Mn2+ and Cu2+). The strain G12 also had the considerable tolerance to initial NO3--N (100-700 mg L-1) and Cr(VI) (1-20 mg L-1) concentrations. The instrument analysis methods-Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD), from the molecular level, further confirmed that the strain G12 could remove NO3--N by aerobic denitrification, and the reduced functional groups (amino group, amide group, hydroxyl group and carboxyl group) on the surface of bacteria could transform Cr(VI) to Cr(III) (mainly CrCl3). This study will offer a promising new microbial resource for nitrogen and Cr(VI) removal in industry wastewater treatment.

New cinnamic acid-pregenolone hybrids as potential antiproliferative agents: Design, synthesis and biological evaluation.

A series of new cinnamic acid-pregenolone hybrids (5a-5o) was designed, synthesized and evaluated for their in vitro antiproliferative activity. Some of them showed potential antiproliferative activity and selectivity towards a panel of cancer cell lines, including A549, H157, HepG2, MCF-7, and HL-60. Among these analogs, compound 5f showed the most promising activity with IC50 values ranging from 3.2 to 6.8 µM, and it was taken as a model compound in the following antiproliferative mechanism study. In Hoechst 33258 staining assay, 5f-treated A549 cells displayed significant apoptosis characteristics. Flow cytometry analysis revealed that 5f showed the antiproliferative activity against A549 via G1 cell cycle arrest and inducing apoptosis. Western blotting analysis demonstrated that 5f enhanced apoptosis of A549 cells by down-regulating Bcl-2 and up-regulating Bax protein expression. The present study highlighted this series of cinnamic acid-pregenolone hybrids as a new antiproliferative lead prototype.

Discovery of new multifunctional selective acetylcholinesterase inhibitors: structure-based virtual screening and biological evaluation.

Although the mechanism of Alzheimer's disease (AD) is still not fully understood, the development of multifunctional AChE inhibitors remains a research focus for AD treatment. In this study, 48 AChE candidate inhibitors were picked out from SPECS database through a pharmacophore- and molecular docking-based virtual screening. The biological evaluation results indicated that four compounds 7, 29, 41 and 48 with different scaffolds exhibited potent and selective AChE inhibitory activity, with the best IC50 value of 1.62 ± 0.11 µM obtained for 48. Then their mechanism of action, the inhibition on Aß aggregation, neurotoxicity, and neuroprotective activity against Aß-induced nerve cell injury were well studied. The binding mode of 48 with AChE was also proposed. The present bioassay results indicated that these multifunctional AChE inhibitors were worth for further structural derivatization to make them the anti-AD lead compounds.

Synthesis and evaluation of coumarin/1,2,4-oxadiazole hybrids as selective BChE inhibitors with neuroprotective activity.

A series of new coumarin/1,2,4-oxadiazole hybrids were synthesized and evaluated for cholinesterase inhibitory and neuroprotective activities. Among them, enantiomers 5u and 5v showed potent hBChE inhibitory activity with IC50 values of 8.17 and 9.56 µM, respectively, and also exhibited good selectivity for hBChE over hAChE by 9.49- and 7.58-fold, respectively. In addition, both compounds could protect SH-SY5Y cells against Aß25-35-induced neurotoxicity. The preliminary bioassay results provided a new chemotype for multifunctional anti-Alzheimer's disease agents and continuing investigation into compounds 5u and 5v is warranted.

Molecular-docking-guided design and synthesis of new IAA-tacrine hybrids as multifunctional AChE/BChE inhibitors.

A series of new indole-3-acetic acid (IAA)-tacrine hybrids as dual acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) inhibitors were designed and prepared based on the molecular docking mode of AChE with an IAA derivative (1a), a moderate AChE inhibitor identified by screening our compound library for anti-Alzheimer's disease (AD) drug leads. The enzyme assay results revealed that some hybrids, e.g. 5d and 5e, displayed potent dual in vitro inhibitory activities against AChE/BChE with IC50 values in low nanomolar range. Molecular modeling studies in tandem with kinetic analysis suggest that these hybrids target both catalytic active site and peripheral anionic site of cholinesterase (ChE). Molecular dynamic simulations and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) calculations indicate that 5e has more potent binding affinity than hit 1a, which may explain the stronger inhibitory effect of 5e on AChE. Furthermore, their predicted pharmacokinetic properties and in vitro influences on mouse brain neural network electrical activity were discussed. Taken together, compound 5e can be highlighted as a lead compound worthy of further optimization for designing new anti-AD drugs.

Discovery of new potent protein arginine methyltransferase 5 (PRMT5) inhibitors by assembly of key pharmacophores from known inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as a promising therapeutic target for human cancer. Up to now, two small molecule PRMT5 inhibitors has been put into phase I clinical trial. In the present study, a series of candidate molecules were designed by combining key pharmacophores of formerly reported PRMT5 inhibitors. The in vitro PRMT5 inhibitory testing of compound 4b14 revealed an IC50 of 2.71 µM, exhibiting high selectivity over PRMT1 and PRMT4 (>70-fold selective). As expected, 4b14 exhibited potent anti-proliferative activity against a panel of leukemia and lymphoma cells, including MV4-11, Pfeiffer, SU-DHL-4 and KARPAS-422. Besides, 4b14 showed significant cell cycle arrest and apoptosis-inducing effects, as well as reduced the cellular symmetric arginine dimethylation level of SmD3 protein. Finally, affinity profiling analysis indicated that hydrophobic interactions, π-π stacking and cation-π actions made the major contributions to the overall binding affinity. This scaffold provides a new chemical template for further development of better lead compounds targeting PRMT5.

Identification of 5-benzylidene-2-phenylthiazolones as potent PRMT5 inhibitors by virtual screening, structural optimization and biological evaluations.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as an important therapeutic target for glioblastoma and mantel cell lymphoma. In the present study, 11 novel PRMT5 inhibitors with 5-benzylidene-2-phenylthiazolone scaffold were identified by molecular docking-based virtual screening and structural optimization. Their IC50 values against PRMT5 at enzymatic level were ranging from 0.77 to 23 µM. As expected, the top two active hits (5 and 19) showed potent anti-proliferative activity against MV4-11 cells with EC50 values lower than 10 µM and reduced the cellular symmetric arginine dimethylation levels of SmD3 protein. Besides, 5 and 19 demonstrated the mechanism of cell killing in cell cycle arrest and apoptotic effect. The probable binding modes of the two compounds were explored and further verified by molecular dynamics simulation. The structure-activity relationship (SAR) of this class of structures was also discussed and further demonstrated by molecular docking simulation.

Design, Synthesis, and Antiproliferative Evaluation of Novel Coumarin/2-Cyanoacryloyl Hybrids as Apoptosis Inducing Agents by Activation of Caspase-Dependent Pathway.

A series of novel coumarin/2-cyanoacryloyl hybrids were prepared and evaluated for their in vitro anticancer activity. Among them, two analogs 5p and 5q showed promising antiproliferative activity against a panel of cancer cell lines, including A549, H157, HepG2, MCF7, MG63, and U2OS. Particularly, 5q showed the most potent activity towards MG63 cells with an IC50 value of 5.06 ± 0.25 µM. Morphological observation and 4,6-diamidino-2-phenylindole (DAPI) staining assay showed that 5q-treated MG63 cells displayed significant apoptosis characteristics. Moreover, flow cytometric detection of phosphatidylserine externalization revealed that 5q induced MG63 apoptosis in a dose-dependent manner. Real-time PCR and western blot assay further confirmed that 5q had strong effects to induce MG63 cell apoptosis, suggesting that the action was associated with down-regulation of the anti-apoptotic protein Bcl-2, upregulation of pro-apoptotic protein Bax, and induced activation of caspase-3, 8, and 9. The present results provide a new chemotype for anticancer drug development and continuing investigation into candidates with coumarin/2-cyanoacryloyl scaffold is warranted.

Total Synthesis of Pulmonarin B and Design of Brominated Phenylacetic Acid/Tacrine Hybrids: Marine Pharmacophore Inspired Discovery of New ChE and Aß Aggregation Inhibitors.

A marine natural product, pulmonarin B (1), and a series of related tacrine hybrid analogues were synthesized and evaluated as cholinesterase (ChE) inhibitors. The in vitro ChE assay results revealed that 1 showed moderate dual acetylcholinesterase (AChE)/ butyrylcholinesterase (BChE) inhibitory activity, while the hybrid 12j proved to be the most potent dual inhibitor among the designed derivatives, being almost as active as tacrine. Molecular modeling studies together with kinetic analysis suggested that 12j interacted with both the catalytic active site and peripheral anionic site of AChE. Compounds 1 and 12j could also inhibit self-induced and AChE-induced Aß aggregation. In addition, the cell-based assay against the human hepatoma cell line (HepG2) revealed that 1 and 12j did not show significant hepatotoxicity compared with tacrine and donepezil. Taken together, the present study confirmed that compound 1 was a potential anti-Alzheimer's disease (AD) hit, and 12j could be highlighted as a multifunctional lead compound for anti-AD drug development.

Synthesis and Evaluation of New Potential Benzo[a]phenoxazinium Photosensitizers for Anticancer Photodynamic Therapy.

The use of photodynamic therapy (PDT) and development of novel photosensitizers (PSs) for cancer treatment have received more and more attention nowadays. In the present work, five benzo[a]phenoxazinium derivatives have been prepared and evaluated for their in vitro anticancer photodynamic activity for the first time. They are red light absorbers and show low fluorescence quantum yield. Of these compounds, PS4 exhibited a higher quantum yield for reactive oxygen species (ROS) generation. The assays with cells in vitro showed that PS1 and PS4 were not significantly toxic in the dark, but was robustly toxic against the murine breast adenocarcinoma cells 4T1 and normal murine fibroblast cells NIH-3T3 upon photoactivation. More interestingly, PS5 was particularly selective towards 4T1 cancer cells and nearly non-phototoxic to non-cancerous NIH-3T3 cells. The results described in this report suggest that these new benzo[a]phenoxazinium derivatives are potential candidates as PSs for anticancer PDT. Further investigation of benzo[a]phenoxaziniums for anticancer PDT is warranted.

Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with dual NF-κB inhibitory and anti-proliferative activities.

Twenty-five novel pregnenolone/2-cyanoacryloyl conjugates (6-30) were designed and prepared, with the aim of developing novel anticancer drugs with dual NF-κB inhibitory and anti-proliferative activities. Compounds 22 and 27-30 showed inhibition against TNF-α-induced NF-κB activation in luciferase assay, which was confirmed by Western blotting. Among them, compound 30 showed potent NF-κB inhibitory activity (IC50=2.5µM) and anti-proliferative against MCF-7, A549, H157, and HL-60 cell lines (IC50=6.5-36.2µM). The present study indicated that pregnenolone/2-cyanoacryloyl conjugate I can server asa novel scaffold for developing NF-κB inhibitors and anti-proliferative agents in cancer chemotherapy.

Preventive effect of Lactobacillus fermentum Lee on activated carbon-induced constipation in mice.

The aim of this study was to investigate the effects of Lactobacillus fermentum Lee (LF-Lee) on activated carbon-induced constipation in ICR mice. ICR mice were orally administered lactic acid bacteria for nine days. Body weight, dietary and water intake, defecation status, gastrointestinal (GI) transit and defecation time, as well as levels of motilin (MTL), gastrin (Gas), endothelin (ET), somatostatin (SS), acetylcholinesterase (AChE), substance P (SP) and vasoactive intestinal peptide (VIP) in serum were measured to evaluate the preventive effects of LF-Lee on constipation. Bisacodyl, a laxative drug, was administered as a positive control. The time taken until the first defecation of a black stool for normal, control, bisacodyl- (100 mg/kg, oral administration), Lactobacillus bulgaricus (LB)-, LF-Lee low dose (L)- and LF-Lee high dose (H)-treated mice was 90, 218, 117, 180, 161 and 151 min, respectively. Following the consumption of LB, LF-Lee (L) or LF-Lee (H), or the oral administration of bisacodyl, the GI transit was reduced to 55.2, 65.8, 73.1 and 94.6%, respectively, of the transit in normal mice. The serum levels of MTL, Gas, ET, AChE, SP and VIP were significantly increased and those of SS were reduced in the mice treated with LF-Lee compared with those in the untreated control mice (P<0.05). These results demonstrate that lactic acid bacteria have preventive effects on constipation in mice and that LF-Lee has superior functional activity.