Design and synthesis of dual cathepsin L and S inhibitors and antimetastatic activity evaluation in pancreatic cancer cells.

Currently, the migration and invasion of cancer cells remain the main factors of poor prognosis in the majority of cancer patients. Developing an effective antimetastatic agent is crucial for cancer therapy. Our recent research revealed that Cat L and S are expressed concurrently in metastatic pancreatic cancer cells. Asperphenamate analog ASPER-29, which exhibits dual Cat L and S inhibitory potency, showed a definite antimetastatic effect on pancreatic cancer BxPC-3 and PANC-1 cells. To further improve the antimetastatic ability of asperphenamate-type molecules, 24 derivatives were designed and synthesized by a scaffold-hopping strategy. The cathepsin inhibitory activity assay results showed that most of the derivatives exhibited dual inhibitory effects on Cat L and S. Among all derivatives, Compound B1a showed the strongest inhibitory activity, with IC50 values of 4.10 ± 0.14 µM and 1.79 ± 0.11 µM, which were 1.5-fold and 2.8-fold more potent than those of positive drugs against Cat L and S, respectively. Further wound-healing and transwell chamber assays demonstrated that B1a presented significant antimetastatic ability in vitro.

Discovery of a novel and selective cathepsin L inhibitor with anti-metastatic ability in vitro and in vivo against breast cancer cells.

Asperphenamate is a natural product that has attracted considerable interest from researchers worldwide. In the last decade, aiming to increase the biological activity and improve druggability, modifications of the A-ring moiety in asperphenamate have been performed. Our laboratory has also recently reported functional derivatizations of the A ring and studied its effect on the inhibition of cysteine cathepsin L. However, the functional significance of the B-ring fragment toward cathepsin L has not been evaluated thus far. In this paper, forty-four derivatives of the B-ring substituted with different N-phenylsulfonyl groups were designed and synthesized. Among them, the paratrifluromethyl analog B-2a and the 2, 4-difluoro-5-chloro derivative B-11b showed more potent inhibitory activity against cathepsin L than the control compound, ABR, which displayed the strongest inhibitory effect on cathepsin L and S among all reported asperphenamate derivatives. In particular, compound B-2a showed more pronounced selectivity against cathepsin L than the other derivatives. Molecular docking revealed that the N-phenylsulfonylamide moiety was vital for the interactions between B-2a and cathepsin L. Moreover, B-2a displayed no toxicity against normal cells. Therefore, compound B-2a was selected for further studies. Wound-healing assays, Transwell chamber assays and breast cancer lung metastasis mouse models demonstrated that B-2a exhibited antimetastatic ability in vitro and in vivo.

A novel cathepsin L inhibitor prevents the progression of idiopathic pulmonary fibrosis.

In previous work, the target of asperphenamate as a natural product was successfully determined as cathepsin by the natural product consensus pharmacophore strategy. In order to develop accurate SAR (structure-activity relationship) of asperphenamate-type cathepsin inhibitor, we chose several novel analogs with heterocyclic moiety to perform further study. The molecular simulation showed that 4-pyridyl derivative 3 with the greatest cathepsin inhibitory activity presented new interaction modes with protein utilizing its B-ring moiety. And then molecular dynamics (MD) simulation further revealed that 3 and cathepsin kept stable interaction in the binding site, which validated the molecular docking results. In view that cathepsins play an important role in fibrosis and some cathepsin inhibitors display the therapeutic ability for fibrosis, we investigated the anti-fibrotic effect of 3in vitro and in vivo. The results indicated that 3 displayed the strongest inhibitory effect on the formation of α-SMA and collagen I as the protein markers of fibrosis among all tested derivatives. Further in vivo assay confirmed that 3 indeed showed significant inhibitory ability against pulmonary fibrosis by the method of H&E and Masson staining as well as immunohistochemical staining for characteristic α-SMA proteins.

Discovery of novel cathepsin inhibitors with potent anti-metastatic effects in breast cancer cells.

It is still challenging to determine the potential targets of natural products, which is essential for further drug research and development. Due to its novel mechanism of action of inducing autophagy effects in breast cancer cells, asperphenamate has received our considerable attention. However, its unknown target inevitably impedes further study. In our previous work, the target enzyme of asperphenamate was predicted as cathepsin by the natural product consensus pharmacophore strategy. Then, asperphenamate and its three derivatives were chosen to study in detail by molecular docking calculations with AutoDock 4 suite. The docking results showed the three derivatives interacted more tightly with either cathepsin L or cathepsin S than with asperphenamate. The ortho-benzyloxyl phenylacetyl derivative 1 andp-toluenesulfonyl derivative 3 showed similar interactions with cathepsin L and adopted a better geometric shape within the binding pocket than did the N-CBZ-piperidyl analog 2. On the other hand, compound 2 formed more hydrogen bonds than 1 and 3 to make it tightly bind within cathepsin S. The cathepsin inhibitory activity assay verified the molecular simulation results. Compound 2 was remarkably less active than 1 and 3 against cathepsin L. However, compound 2 showed the strongest potency against cathepsin S with IC50 of 13.12 ±â€¯0.29 µM. Considering that cathepsin S plays a vital role in the process of metastasis in breast cancer cells, the inhibitory effect of 2 on migration and invasion was further studied in human breast cancer MDA-MB-231 cells by wound healing and transwell chamber assays. The results illustrated that 2 exhibited an apparent inhibitory ability to the metastasis of MDA-MB-231 cells. Next, 2 will be chosen as a lead compound to develop novel double functional chemotherapeutic agents with both novel mechanisms of action against apoptosis-resistant cancer cells, such as inducing autophagy and inhibiting cancer metastasis.

ASPER-29 suppresses the metastasis of pancreatic cancer cells by dual inhibition of cathepsin-L and cathepsin-S.

Pancreatic cancer will be the second leading cause of cancer-related mortality worldwide due to its high rate of metastasis. Cathepsins (CATs) are effectors of invasive growth in various cancers. Currently, targeting CATs represents an attractive strategy for the treatment of highly metastatic cancers with high CATs activity, such as pancreatic cancer. To develop a stronger antimetastatic agent, ASPER-29, a novel inhibitor of CATs designed by using the asperphenamate derivative BBP as a lead compound, was synthesized, and its therapeutic potential in pancreatic cancer metastasis was investigated in this study. Molecular docking and enzyme inhibition assays proved that ASPER-29 can inhibit the activity of CAT-L and CAT-S by binding with these enzymes in classical action modes. Furthermore, ASPER-29 significantly inhibited the activity of CAT-L and CAT-S but had no effect on their expression in PANC-1 and BxPC-3 cells. The in vitro antimetastatic activities of ASPER-29 were examined by wound healing and Transwell chamber assays. We found that ASPER-29 inhibited the migration and invasion of PANC-1 and BxPC-3 cells in a concentration-dependent manner. Moreover, the in vivo antimetastatic effects of ASPER-29 were confirmed in a mouse xenotransplantation model. H&E staining and immunohistochemistry assays of Ki67 and CEACAM6 proved that ASPER-29 treatment significantly blocked the metastasis of pancreatic cancer cells to lung and liver tissues. Additionally, the activity of both CAT-L and CAT-S was markedly inhibited in the lung and liver tissues of ASPER-29-administered mice compared with the mice in the model group, suggesting that the metastasis-blocking effect of ASPER-29 should be mediated via inhibition of the activity of CAT-L and CAT-S in pancreatic cancer cells. Together, our results demonstrated that ASPER-29, as a novel inhibitor of CAT-L and CAT-S, possessed the evident ability to block the metastasis of pancreatic cancer cells.

The Fungal Endophyte Penicillium olsonii ML37 Reduces Fusarium Head Blight by Local Induced Resistance in Wheat Spikes.

The fungal endophyte Penicillium olsonii ML37 is a biocontrol agent of Fusarium head blight in wheat (caused by Fusarium graminearum), which has shown a limited direct inhibition of fungal growth in vitro. We used RNA-seq and LC-MS/MS analyses to elucidate metabolic interactions of the three-way system Penicillium-wheat-Fusarium in greenhouse experiments. We demonstrated that P. olsonii ML37 colonises wheat spikes and transiently activates plant defence mechanisms, as pretreated spikes show a faster and stronger expression of the defence metabolism during the first 24 h after pathogen inoculation. This effect was transient and the expression of the same genes was lower in the pathogen-infected spikes than in those infected by P. olsonii alone. This response to the endophyte includes the transcriptional activation of several WRKY transcription factors. This early activation is associated with a reduction in FHB symptoms and significantly lower levels of the F. graminearum metabolites 15-acetyl-DON and culmorin. An increase in the Penicillium-associated metabolite asperphanamate confirms colonisation by the endophyte. Our results suggest that the mode of action used by P. olsonii ML37 is via a local defence activation in wheat spikes, and that this fungus has potential as a novel biological alternative in wheat disease control.

Rapid detection of asperphenamate in a hay batch associated with constipation and deaths in dairy cattle. The application of DART-HRMS to veterinary forensic toxicology.

Direct analysis in real time (DART) coupled to high-resolution mass spectrometry (HRMS) was applied for the first time to veterinary forensic toxicology to investigate the presence of toxic compounds in hay after an episode of acute intoxication in a dairy cattle farm. In addition to gross field necropsy and histological examination, microbial cultures, and heavy metals analysis, the molecular fingerprinting of the suspected hay batch was investigated by DART-HRMS. DART-HRMS revealed a distinct signal of m/z 507.2289 in the hay batch thought to be associated with the digestive complications. A search on chemical structure databases matched the ion with asperphenamate, a toxin produced by Penicillium spp. and Aspergillus spp. Liquid Chromatography-HMRS analysis and electrospray-HRMS-MS/MS of the hay extracts further characterized the structure and confirmed the identification of the compound as asperphenamate. Asperphenamate is fungal metabolite which can have cytotoxic and antitumor activity in humans, and it is classified as acute toxicant and harmful if swallowed.

Mass Spectrometry Guided Discovery and Design of Novel Asperphenamate Analogs From Penicillium astrolabium Reveals an Extraordinary NRPS Flexibility.

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of Penicillium astrolabium, a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.

Design, synthesis and biological evaluation of novel asperphenamate derivatives.

A series of novel asperphenamate derivatives were designed and synthesized, including series I (the A-phenyl group replaced with various aromatic heterocycles) and series II (the acyl group substituted by sulfonyl group). All compounds have been screened for their antiproliferative activity in vitro against MCF-7, HeLa, and BEL-7402 cell lines by the standard MTT method. Structure-activity relationship studies displayed the heterocycle type played an important role in activity. Six-membered ring derivatives displayed more potency than five-membered ring and the sulfonyl group in A-ring region made an important contribution to activity. Among all derivatives, tosyl derivative 8c exhibited the greatest potency in three human cancer cell lines. Especially in MCF-7 cells, the cellular potency of 8c was approximately 3.0-fold more potent than that of cisplatin. Firstly, the mechanism of cell death induced by 8c in MCF-7 cells was investigated. The results showed that the cell death was induced by autophagy instead of apoptosis or cell cycle arrest. Further studies indicated that 8c might induce autophagic cell death in HeLa and BEL-7402 cell lines.

Chemical constituents of Piptadenia gonoacantha (Mart.) J.F. Macbr (pau jacaré)

The phytochemical investigation of Piptadenia gonoacantha (Mart.) J.F. Macbr. (Leguminosae-Mimosoideae), commonly known as "pau jacaré" (alligator stick), afforded sitosterol, campesterol, stigmasterol, the N-benzoylphenylalanine-2-benzoylamide-3-phenylpropyl ester, known as asperphenamate, sitosterol-3-O-β-D-glucopyranoside, besides three flavonoids, apigenin, 5-O-methylapigenin and 7,4'-dihydroxy-3',5-dimethoxyflavone from its branches. From its leaves, the methyl gallate and two flavonoids, vitexin and isovitexin, were isolated. From its bark, a mixture of sitosterol, campesterol, and stigmasterol, besides a mixture of cycloartenone, cycloartan-25-en-3-one, and 24-methylene-cycloartenone, and the pure triterpenes 24-methylenecycloartanol, friedelin, lupeol and lupenone, were isolated. Their structures were established on the basis of spectral analysis, comparison with literature data and GC-MS analysis of the mixtures. The ester, flavonoids and the cycloartanes are been identified for first time in the genus Piptadenia.

O estudo fitoquímico de galhos de Piptadenia gonoacantha (Mart.) J.F. Macbr. (Leguminosae-Mimosoideae), comumente conhecida como "pau jacaré", forneceu sitosterol, estigmasterol, o éster N-benzoilfenilalaninato de 2-N-benzoil-3-fenilpropila, conhecido como asperfenamato, 3-O-β-D-glicopiranosil-sitosterol, além de três flavonóides, apigenina (5,7,4'-triidroxiflavona), apigenina-5-O-metil éter e 7,4'-dihidroxi-3' , 5-dimetoxiflavona. Das folhas isolaram-se galato de metila e dois flavonóides, 8-C-glicopiranosil-5,7,4' -trihidroxiflavona e 6-C-glicopiranosil-5,7,4'-trihidroxiflavona, conhecidas como vitexina e isovitexina. Das cascas desta planta isolaram-se uma mistura de sitosterol, campesterol e estigmasterol; mistura de cicloartenona, cicloartan-25,26-en-3-ona e 24-metileno-cicloartanona, além dos triterpenos, 24-metilenocicloartenol, fridelina, lupeol e lupenona. As estruturas foram estabelecidas através de análise de espectros de IV, RMN ¹H e 13C e massas, além de análise com CG-EM para identificar os componentes das misturas de cicloartanos e esteróides. O éster conhecido como asperfenamato, os flavonóides e os cicloartanos estão sendo registrados pela primeira vez em Piptadenia.