Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing

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天然产物在新药开发中的应用--HMG-CoA还原酶抑制剂新药的发现对天然产物研究与开发的启示

自1973年发现第一个作用于HMG-CoA还原酶的化合物mevastatin以来,多个具有HMG-CoA还原酶抑制活性的抗高血脂新药被开发成功,并成为治疗高血脂症的首选药物。在此简单介绍该类新药的发现与开发过程,并就天然产物的研究与开发提出一点个人见解。     

Antifeedant Activity toward Larvae of Pieris rapae crucivora of Aromatic Carbonyl Compounds Related to Capillin Isolated from Artemisia capillaris

The antifeedant activity toward larvae of Pieris rapae crucivora of aromatic carbonyl compounds was studied. When an H atom of aldehyde group of aromatic aldehydes was replaced by a CH₃ group, it was observed that arylmethylketones were more active toward larvae than aromatic aldehydes.     

The Isolation and Characterization of the Acid-Soluble Nucleotides from the Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The acid-soluble nucleotides were extracted from the tubers of Jerusalem artichoke with percbloric acid, and separated and purified by means of adsorption on and elution from active charcoal, repeated chromatography on columns of Dowex I (Cl^-), followed by paper chromatography. The following nucleotides have been characterized and/or identified: 5′-AMP, 3′-AMP, ADP, ATP, 5′-GMP, 2′-GMP, 3′-GMP, 2′,3′-cyclic GMP, GDP, GTP, 5′-UMP, UDP, UTP, NADP, UDP-glucose, UDP-galactose, UDP-fructose, UDP-N-acetylhexosamine and GDP-mannose.^** Neither cytosine ribonucleotides nor deoxyribonucleotides have been detected. The significance of these observations is discussed.     

Azetidines and spiro azetidines as novel P2 units in hepatitis C virus NS3 protease inhibitors

Herein, we report the synthesis and structure–activity relationship studies of new analogs of boceprevir 1 and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyrrolidine moiety of 1 and 2 led to the discovery of a potent hepatitis C protease inhibitor 37c (EC₅₀ = 0.8 μM).     

A CRYGC gene mutation associated with autosomal dominant pulverulent cataract

Purpose

To describe at molecular level a family with pulverulent congenital cataract associated with a CRYGC gene mutation.

Methods

One family with several affected members with pulverulent congenital cataract and 230 healthy controls were examined. Genomic DNA from leukocytes was isolated to analyze the CRYGA-D cluster, CX46, CX50 and MIP genes through high-resolution melting curve and DNA sequencing.

Results

DNA sequencing in the affected members revealed the c.143G>A mutation (p.R48H) in exon 2 of the CRYGC gene; 230 healthy controls and ten healthy relatives were also analyzed and none of them showed the c.143G>A mutation. No other polymorphisms or mutations were found to be present.

Conclusion

In the present study, we described a family with pulverulent congenital cataract that segregated the c.143G>A mutation (p.R48H) in the CRYGC gene. A few mutations have been described in the CRYGC gene in autosomal dominant cataract, none of them with pulverulent cataract making clear the clinical heterogeneity of congenital cataract. This mutation has been associated with the phenotype of congenital cataract but also is considered an SNP in the NCBI data base. Our data and previous report suggest that p.R48H could be a disease-causing mutation and not an SNP.     

Modelling family 2 cystatins and their interaction with papain

Cystatins are extensively studied cysteine protease inhibitors, found in wide range of organisms with highly conserved structural folds. S-type of cystatins is well known for their abundance in saliva, high selectivity and poorer activity towards host cysteine proteases in comparison to their immediate ancestor cystatin C. Despite more than 90% sequence similarity, the members of this group show highly dissimilar binding affinity towards papain. Cystatin M/E is a potent inhibitor of legumain and papain like cysteine proteases and recognized for its involvement in skin barrier formation and potential role as a tumor suppressor gene. However, the structures of these proteins and their complexes with papain or legumain are still unknown. In the present study, we have employed computational methods to get insight into the interactions between papain and cystatins. Three-dimensional structures of the cystatins are generated by homology modelling, refined with molecular dynamics simulation, validated through numerous web servers and finally complexed with papain using ZDOCK algorithm in Discovery Studio. A high degree of shape complementarity is observed within the complexes, stabilized by numerous hydrogen bonds (HB) and hydrophobic interactions. Using interaction energy, HB and solvent accessible surface area analyses, we have identified a series of key residues that may be involved in papain–cystatin interaction. Differential approaches of cystatins towards papain are also noticed which are possibly responsible for diverse inhibitory activity within the group. These findings will improve our understanding of fundamental inhibitory mechanisms of cystatin and provide clues for further research.     

Heterogeneous Packing in the Folding/Unfolding Intermediate State of Bitter Gourd Trypsin Inhibitor

The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2–3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.     

Differential effect of long-term drug selection with doxorubicin and vorinostat on neuroblastoma cells with cancer stem cell characteristics

Numerous studies have confirmed that cancer stem cells (CSCs) are more resistant to chemotherapy; however, there is a paucity of data exploring the effect of long-term drug treatment on the CSC sub-population. The purpose of this study was to investigate whether long-term doxorubicin treatment could expand the neuroblastoma cells with CSC characteristics and histone acetylation could affect stemness gene expression during the development of drug resistance. Using n-myc amplified SK-N-Be(2)C and non-n-myc amplified SK-N-SH human neuroblastoma cells, our laboratory generated doxorubicin-resistant cell lines in parallel over 1 year; one cell line intermittently treated with the histone deacetylase inhibitor (HDACi) vorinostat and the other without exposure to HDACi. Cells'' sensitivity to chemotherapeutic drugs, the ability to form tumorspheres, and capacity for in vitro invasion were examined. Cell-surface markers and side populations (SPs) were analyzed using flow cytometry. Differentially expressed stemness genes were identified through whole genome analysis and confirmed with real-time PCR. Our results indicated that vorinostat increased the sensitivity of only SK-N-Be(2)C-resistant cells to chemotherapy, made cells lose the ability to form tumorspheres, and reduced in vitro invasion and the SP percentage. CD133 was not enriched in doxorubicin-resistant or vorinostat-treated doxorubicin-resistant cells. Nine stemness-linked genes (ABCB1, ABCC4, LMO2, SOX2, ERCC5, S100A10, IGFBP3, TCF3, and VIM) were downregulated in vorinostat-treated doxorubicin-resistant SK-N-Be(2)C cells relative to doxorubicin-resistant cells. A sub-population of cells with CSC characteristics is enriched during prolonged drug selection of n-myc amplified SK-N-Be(2)C neuroblastoma cells. Vorinostat treatment affects the reversal of drug resistance in SK-N-Be(2)C cells and may be associated with downregulation of stemness gene expression. This work may be valuable for clinicians to design treatment protocols specific for different neuroblastoma patients.