Exploring how lignin structure influences the interaction between carbohydrate-binding module and lignin using AFM.

Nonproductive adsorption of cellulase onto the residual lignin in substrate seriously hinders the enzymatic hydrolysis. To understand how lignin structure affects lignin-cellulase interaction, the carbohydrate-binding module (CBM) functionalized atomic force microscope tip was used to measure CBM-lignin interaction by single-molecule dynamic force spectroscopy in this work. The results showed that sulfonated lignin (SL) has the greatest adhesion force to CBM (4.74 nN), while those of masson pine milled wood lignin (MWL), poplar MWL and herbaceous MWLs were 2.85, 1.03 and 0.27-0.61 nN, respectively. It provides direct quantitative evidence for the significance of lignin structure on lignin-cellulase interaction. The CBM-MWLs interaction decreased sharply to 0.054-0.083 nN while SL was added, indicating the primary mechanism of SL promoting lignocellulose hydrolysis was significantly reducing the nonproductive adsorption of substrate lignin on cellulase. Finally, the "competitive adsorption" mechanism was proposed to interpret why SL effectively promotes the enzymatic hydrolysis of lignin-containing substrates.

Optical effect and pre-forming temperature field of in situ laser-assisted micro-stamping by a diamond indenter.

During in situ laser-assisted micro-stamping (In-LAS) using a diamond indenter, the temperature of the machined area cannot be accurately predicted because of the coupling of the beam with the diamond indenter. This study uses geometrical optics and wave optics to determine the effect of the taper angle on laser direction, the light intensity distribution at the diamond indenter tip, and the temperature field of the machined area. This research provides data support and a theoretical basis for the selection of related process parameters of In-LAS and delivers a feasible theoretical method for a similar in situ laser-assisted machining.

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 a novel cathepsin inhibitor with dual autophagy-inducing and metastasis-inhibiting effects on breast cancer cells.

Drug resistance and cancer cells metastasis have been the leading causes of chemotherapy failure and cancer-associated death in breast cancer patients. In present, various active molecules either exhibiting novel mechanism of action such as inducing autophagy or inhibiting metastasis have been developed to address these problems. However, the compounds exhibiting such dual functions have rarely been described. Previous work in our group showed that TSA, as a synthetic analog of asperphenamate, induced autophagic cell death in breast cancer cells instead of apoptosis. Furthermore, the target enzyme of TSA was predicted to be cathepin L (Cat L) by natural product consensus pharmacophore strategy. Accumulated evidences have shown that cathepsins are closely associated with migration and invasion of breast cancer cells. It seemed likely that TSA-like molecules may possess the dual functions of inducing autophagy and inhibiting metastasis. Therefore, sixty optically active derivatives were firstly designed and synthesized by replacing the A-ring moiety of TSA with other substituted-phenyl sulfonyl groups. Further cathepsin inhibitory activity assay showed that (S, S) and (S, R) isomers displayed no activity against four kinds of cathepsins (L, S, K, B), while all derivatives tested were inactive toward K and B subtypes. Compound 6a with meta-bromo substituent displayed the greatest inhibitory activity, and its inhibitory capability against Cat L and S was 3.9 and 11.5-fold more potent than that of TSA, respectively. Molecular docking also exhibited that 6a formed more hydrogen bonds or π-π contacts with Cat L or S than TSA. In order to determine whether 6a could play dual roles, its anti-cancer mechanism was further investigated. On the one hand, MDC staining experiment and western blotting analysis validated that 6a can induce autophagy in MDA-MB-231 cells. On the other hand, its metastatic inhibitory ability was also confirmed by wound healing and transwell chamber experiment.

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.

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.

Synthesis and in vitro antitumor activity of asperphenamate derivatives as autophagy inducer.

In an effort to improve the aqueous solubility and the antitumor activity of natural product asperphenamate, we have designed and synthesized three series of asperphenamate derivatives, including series I (simplifying molecular skeleton series), series II (introducing a hydroxyl group to A-phenyl ring series) and series III (disrupting molecular planarity series). All derivatives have displayed a significantly increased solubility compared with asperphenamate. Their growth inhibitory activities in vitro were screened by the standard MTT method in MCF-7, HeLa, and BEL-7402 cell lines. With the exception of the derivatives in series I, most of derivatives in series II and series III showed growth inhibitory activity. Among all derivatives, IM23b in series III showed the greatest potency in human breast cancer MCF-7 cells. The cellular potency of IM23b was approximately 1.5-fold more potent than that of cisplatin. The mechanism of cell death induced by IM23b in human breast cancer MCF-7 cells was further investigated. We concluded that the cell death was induced by autophagy instead of apoptosis or cell cycle arrest.