Design, synthesis, and bioactivities of tasiamide B derivatives as cathepsin D inhibitors.

Cathepsin D (Cath D) is overexpressed and hypersecreted by malignant tumors and involved in the progress of tumor invasion, proliferation, metastasis, and apoptosis. Cath D has been considered as a potential target to treat cancer. Our previous studies revealed that tasiamide B derivatives TB-9 and TB-11 exhibited high potent inhibition against Cath D and other aspartic proteases, but their molecular weights are still high, and the role of each residue is unknown yet. Based on this, two series of tasiamide B derivatives have been designed, synthesized, and evaluated for their inhibitory activity against Cath D/Cath E/BACE1. Enzymatic assays revealed that the target compound 1 with lower molecule weight showed good inhibitory activity against Cath D with IC50 of 3.29 nM and satisfactory selectivity over Cath E (72-fold) and BACE1 (295-fold), which could be a valuable template for the design of highly potent and selective Cath D inhibitors.

Cyanobacterial peptides as a prototype for the design of cathepsin D inhibitors.

Cathepsin D (Cath D) is overexpressed and secreted in a number of solid tumors and involved in the progress of tumor invasion, proliferation, metastasis, and apoptosis. Inhibition of Cath D is regarded as an attractive pathway for the development of novel anticancer drugs. Our previous studies revealed that tasiamide B, a cyanobacterial peptide that contained a statine-like unit, exhibited good inhibition against Cath D and other aspartic proteases. Using this natural product as prototype, we designed and synthesized three new analogs, which bear isophthalic acid fragment at the N-terminus and isobutyl amine (1), cyclopropyl amine (2), or 3-methoxybenzyl amine (3) moiety at the C-terminus. Enzymatic assays revealed that all these three compounds showed moderate-to-good inhibition against Cath D, with IC50 s of 15, 884, and 353 nM, respectively. Notably, compound 1 showed extreme selectivity for Cath D with 576-fold over Cath E and 554-fold over BACE1, which could be a valuable template for the design of highly potent and selective Cath D inhibitors. Additionally, compound 1 showed moderated activity against HeLa cell lines with IC50 of 41.8 µM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Crotamiton derivative JM03 extends lifespan and improves oxidative and hypertonic stress resistance in Caenorhabditis elegans via inhibiting OSM-9.

While screening our in-house 1072 marketed drugs for their ability to extend the lifespan using Caenorhabditis elegans (C. elegans) as an animal model, crotamiton (N-ethyl-o-crotonotoluidide) showed anti-aging activity and was selected for further structural optimization. After replacing the ortho-methyl of crotamiton with ortho-fluoro, crotamiton derivative JM03 was obtained and showed better activity in terms of lifespan-extension and stress resistance than crotamiton. It was further explored that JM03 extended the lifespan of C. elegans through osmotic avoidance abnormal-9 (OSM-9). Besides, JM03 improves the ability of nematode to resist oxidative stress and hypertonic stress through OSM-9, but not osm-9/capsaicin receptor related-2 (OCR-2). Then the inhibition of OSM-9 by JM03 reduces the aggregation of Q35 in C. elegans via upregulating the genes associated with proteostasis. SKN-1 signaling was also found to be activated after JM03 treatment, which might contribute to proteostasis, stress resistance and lifespan extension. In summary, this study explored a new small molecule derived from crotamiton, which has efficient anti-oxidative, anti-hypertonic, and anti-aging effects, and could further lead to promising application prospects.

Anti-aging effects of chlorpropamide depend on mitochondrial complex-II and the production of mitochondrial reactive oxygen species.

Sulfonylureas are widely used oral anti-diabetic drugs. However, its long-term usage effects on patients' lifespan remain controversial, with no reports of influence on animal longevity. Hence, the anti-aging effects of chlorpropamide along with glimepiride, glibenclamide, and tolbutamide were studied with special emphasis on the interaction of chlorpropamide with mitochondrial ATP-sensitive K+ (mitoK-ATP) channels and mitochondrial complex II. Chlorpropamide delayed aging in Caenorhabditis elegans, human lung fibroblast MRC-5 cells and reduced doxorubicin-induced senescence in both MRC-5 cells and mice. In addition, the mitochondrial membrane potential and ATP levels were significantly increased in chlorpropamide-treated worms, which is consistent with the function of its reported targets, mitoK-ATP channels. Increased levels of mitochondrial reactive oxygen species (mtROS) were observed in chlorpropamide-treated worms. Moreover, the lifespan extension by chlorpropamide required complex II and increased mtROS levels, indicating that chlorpropamide acts on complex II directly or indirectly via mitoK-ATP to increase the production of mtROS as a pro-longevity signal. This study provides mechanistic insight into the anti-aging effects of sulfonylureas in C. elegans.

Cucurbitacin B-induced G2/M cell cycle arrest of conjunctival melanoma cells mediated by GRP78-FOXM1-KIF20A pathway.

Conjunctival melanoma (CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B (CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16, CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a K d value of 0.11 µmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.

Fangchinoline suppresses conjunctival melanoma by directly binding FUBP2 and inhibiting the homologous recombination pathway.

Conjunctival melanoma (CM) is a rare and fatal ocular tumour with poor prognosis. There is an urgent need of effective therapeutic drugs against CM. Here, we reported the discovery of a novel potential therapeutic target for CM. Through phenotypic screening of our in-house library, fangchinoline was discovered to significantly inhibit the growth of CM cells including CM-AS16, CRMM1, CRMM2 and CM2005.1. Further mechanistic experiments indicated that fangchinoline suppressed the homologous recombination (HR)-directed DNA repair by binding with far upstream element binding protein 2 (FUBP2) and downregulating the expression of HR factors BRCA1 and RAD51. In vitro and in vivo antitumour experiments revealed that fangchinoline increased the efficacy of cisplatin by blocking HR factors and reduced the drug dose and toxicity. In conclusion, our work provides a promising therapeutic strategy for the treatment of CM that is worthy of extensive preclinical investigation.

Antiaging Effects of Vicatia thibetica de Boiss Root Extract on Caenorhabditis elegans and Doxorubicin-Induced Premature Aging in Adult Mice.

The roots of Vicatia thibetica de Boiss are a kind of Chinese herb with homology of medicine and food. This is the first report showing the property of the extract of Vicatia thibetica de Boiss roots (HLB01) to extend the lifespan as well as promote the healthy parameters in Caenorhabditis elegans (C. elegans). For doxorubicin- (Doxo-) induced premature aging in adult mice, HLB01 counteracted the senescence-associated biomarkers, including P21 and γH2AX. Interestingly, HLB01 promoted the expression of collagen in C. elegans and mammalian cell systemically, which might be one of the essential factors to exert the antiaging effects. In addition, HLB01 was also found as a scavenger of free radicals, thereby performing the antioxidant ability. Lifespan extension by HLB01 was also dependent on DAF-16 and HSF-1 via oxidative stress resistance and heat stress resistance. Taken together, overall data suggested that HLB01 could extend the lifespan and healthspan of C. elegans and resist Doxo-induced senescence in mice via promoting the expression of collagen, antioxidant potential, and stress resistance.

Verapamil extends lifespan in Caenorhabditis elegans by inhibiting calcineurin activity and promoting autophagy.

Previous evidence has revealed that increase in intracellular levels of calcium promotes cellular senescence. However, whether calcium channel blockers (CCBs) can slow aging and extend lifespan is still unknown. In this study, we showed that verapamil, an L-type calcium channel blocker, extended the Caenorhabditis elegans (C. elegans) lifespan and delayed senescence in human lung fibroblasts. Verapamil treatment also improved healthspan in C. elegans as reflected by several age-related physiological parameters, including locomotion, thrashing, age-associated vulval integrity, and osmotic stress resistance. We also found that verapamil acted on the α1 subunit of an L-type calcium channel in C. elegans. Moreover, verapamil extended worm lifespan by inhibiting calcineurin activity. Furthermore, verapamil significantly promoted autophagy as reflected by the expression levels of LGG-1/LC3 and the mRNA levels of autophagy-related genes. In addition, verapamil could not further induce autophagy when tax-6, calcineurin gene, was knocked down, indicating that verapamil-induced lifespan extension is mediated via promoting autophagy processes downstream of calcineurin. In summary, our study provided mechanistic insights into the anti-aging effect of verapamil in C. elegans.

Lactose induced redox-dependent senescence and activated Nrf2 pathway.

Lactose is a disaccharide found in milk and thus a part of our daily food intake. Upon ingestion, it is hydrolyzed to glucose and galactose by the enzyme lactase and absorbed in the small intestine. People who suffer from lactose intolerance are unable to completely digest it due to deficiency of lactase, leading to intestinal problems such as diarrhoea, and bloating. Various studies have focused on treating these symptoms. However, the effects of lactose that diffuses passively into cells, on cellular senescence have largely remained unknown. Thus, the present study investigated the effects and mechanisms of lactose on senescence both in vitro and in vivo. The study was conducted in MRC-5 cells. The cellular senescence was estimated by determining the expression of SA-ß-gal and p16ink4a. The cell viability of MRC-5 cells was determined by the CCK-8 Assay. Activity of intracellular reactive oxygen species was estimated by measuring the levels of superoxide dismutase (SOD), glutathione (GHS), and reactive oxygen species (ROS). The mechanism of lactose on cellular senescence was explored by western blotting. We also studied the effect of lactose on the lifespan of Caenorhabditis elegans. Increased activities of SA-ß-gal and p16ink4a revealed the ability of lactose to induce senescence in MRC-5 cells. The elevated intracellular ROS level and decreased GSH and SOD levels in these cells were indicative of cellular oxidative stress induced by lactose. Furthermore, western blotting analysis of Nrf2 and mRNA expression of its downstream genes suggested the Nrf2/ARE pathway was involved in the oxidative stress induced by lactose. These results were further validated by the shortened lifespan of C. elegans after lactose supplement. Moreover, the lactose-induced senescence could be alleviated by an antioxidant, N-Acetyl-L-cysteine (NAC), both in vitro and in vivo. The present study observed a positive correlation between lactose and cellular oxidative stress, suggesting the latter to be an underlying mechanism of lactose-induced senescence.

Rational Design of Novel Selective Dual-Target Inhibitors of Acetylcholinesterase and Monoamine Oxidase B as Potential Anti-Alzheimer's Disease Agents.

Multifunctional agents aiming at cholinesterases (ChEs) and monoamine oxidases (MAOs) are promising therapy for Alzheimer's disease (AD). Herein, a series of novel propargylamine-modified pyrimidinylthiourea derivatives (1-4) were designed and synthesized as dual inhibitors of ChEs and MAOs with other functions against AD. Most of these derivatives inhibited ChEs and MAOs with IC50 values in the micro- or nanomolar ranges. Compound 1c displayed the dual functional profile of targeting the AChE (IC50 = 0.032 ± 0.007 µM) and MAO-B (IC50 = 2.117 ± 0.061 µM), along with the improved blood-brain barrier (BBB) permeability, antioxidant ability, and good copper chelating property in vitro. Animal studies showed that compound 1c·HCl could inhibit the cerebral AChE/MAO-B activities and alleviate scopolamine-induced cognitive impairment in mice. Combined with good oral bioavailability ( F = 45.55%), these findings demonstrated that compound 1c may be a potent brain permeable multifunctional candidate for the treatment of AD.

Design, Synthesis, and Biological Evaluation of Orally Available First-Generation Dual-Target Selective Inhibitors of Acetylcholinesterase (AChE) and Phosphodiesterase 5 (PDE5) for the Treatment of Alzheimer's Disease.

Through drug discovery strategies of repurposing and redeveloping existing drugs, a series of novel tadalafil derivatives were rationally designed, synthesized, and evaluated to seek dual-target AChE/PDE5 inhibitors as good candidate drugs for Alzheimer's disease (AD). Among these derivatives, 1p and 1w exhibited excellent selective dual-target AChE/PDE5 inhibitory activities and improved blood-brain barrier (BBB) penetrability. Importantly, 1w·Cit (citrate of 1w) could reverse the cognitive dysfunction of scopolamine-induced AD mice and exhibited an excellent effect on enhancing cAMP response element-binding protein (CREB) phosphorylation in vivo, a crucial factor in memory formation and synaptic plasticity. Moreover, the molecular docking simulations of 1w with hAChE and hPDE5A confirmed that our design strategy was rational. In summary, our research provides a potential selective dual-target AChE/PDE5 inhibitor as a good candidate drug for the treatment of AD, and it could also be regarded as a small molecule probe to validate the novel AD therapeutic approach in vivo.

Screening of microalgae for integral biogas slurry nutrient removal and biogas upgrading by different microalgae cultivation technology.

The microalgae-based technology has been developed to reduce biogas slurry nutrients and upgrade biogas simultaneously. In this work, five microalgal strains named Chlorella vulgaris, Scenedesmus obliquus, Selenastrum capricornutum, Nitzschia palea, and Anabaena spiroides under mono- and co-cultivation were used for biogas upgrading. Optimum biogas slurry nutrient reduction could be achieved by co-cultivating microalgae (Chlorella vulgaris, Scenedesmus obliquus, and Nitzschia palea) with fungi using the pelletization technology. In addition, the effects of different ratio of mixed LED light wavelengths applying mixed light-emitting diode during algae strains and fungi co-cultivation on CO2 and biogas slurry nutrient removal efficiency were also investigated. The results showed that the COD (chemical oxygen demand), TN (total nitrogen), and TP (total phosphorus) removal efficiency were 85.82 ± 5.37%, 83.31 ± 4.72%, and 84.26 ± 5.58%, respectively at red: blue = 5:5 under the co-cultivation of S. obliquus and fungi. In terms of biogas upgrading, CH4 contents were higher than 90% (v/v) for all strains, except the co-cultivation with S. obliquus and fungi at red: blue = 3:7. The results indicated that co-cultivation of microalgae with fungi under mixed light wavelengths treatments was most successful in nutrient removal from wastewater and biogas upgrading.