Metabolic Engineering-Based Rapid Characterization of a Sesquiterpene Cyclase and the Skeletons of Fusariumdiene and Fusagramineol from <i>Fusarium graminearum</i>

The potential power of sesquiterpene synthase FgJ03939 from <i>Fusarium graminearum</i> was fully exploited in a farnesyl diphosphate-overexpressing <i>Saccharomyces cerevisiae</i> chassis to produce the novel sesquiterpenes fusariumdiene (<b>1</b>), <i>epi</i>-fusagramineol (<b>2</b>), and fusagramineol (<b>3</b>) with 5/7 bicyclic and 5/6/3 tricyclic ring systems, respectively, as well as five known sesquiterpenes (<b>4</b>–<b>8</b>). The structure of the unusual skeletons was characterized, and an absolute configuration was proposed. A mechanism for the biosynthesis of <b>1</b>–<b>8</b> was also proposed

Cheminformatic Insight into the Differences between Terrestrial and Marine Originated Natural Products

This is a new golden age for drug discovery based on natural products derived from both marine and terrestrial sources. Herein, a straightforward but important question is “what are the major structural differences between marine natural products (MNPs) and terrestrial natural products (TNPs)?” To answer this question, we analyzed the important physicochemical properties, structural features, and drug-likeness of the two types of natural products and discussed their differences from the perspective of evolution. In general, MNPs have lower solubility and are often larger than TNPs. On average, particularly from the perspective of unique fragments and scaffolds, MNPs usually possess more long chains and large rings, especially 8- to 10-membered rings. MNPs also have more nitrogen atoms and halogens, notably bromines, and fewer oxygen atoms, suggesting that MNPs may be synthesized by more diverse biosynthetic pathways than TNPs. Analysis of the frequently occurring Murcko frameworks in MNPs and TNPS also reveals a striking difference between MNPs and TNPs. The scaffolds of the former tend to be longer and often contain ester bonds connected to 10-membered rings, while the scaffolds of the latter tend to be shorter and often bear more stable ring systems and bond types. Besides, the prediction from the naïve Bayesian drug-likeness classification model suggests that most compounds in MNPs and TNPs are drug-like, although MNPs are slightly more drug-like than TNPs. We believe that MNPs and TNPs with novel drug-like scaffolds have great potential to be drug leads or drug candidates in drug discovery campaigns

Exploring the Biologically Relevant Chemical Space for Drug Discovery

Both recent studies and our calculation suggest that the physicochemical properties of launched drugs changed continuously over the past decades. Besides shifting of commonly used properties, the average biological relevance (BR) and similarity to natural products (NPs) of launched drugs decreased, reflecting the fact that current drug discovery deviated away from NPs. To change the current situation characterized by high investment but low productivity in drug discovery, efforts should be made to improve the BR of the screening library and hunt drugs more effectively in the biologically relevant chemical space. Additionally, a multiple dimensional molecular descriptor, named the biologically relevant spectrum (BRS) was proposed for quantitative structure–activity relationships (QSAR) study or screening library preparation. Prediction models for 43 biological activity categories were developed with BRS and support vector machine (SVM). In most cases, the overall prediction accuracies were around 95% and the Matthew’s correlation coefficients (MCC) were over 0.8. Thirty-seven out of 48 drug-activity associations were successfully predicted for drugs that launched from 2006 to 2012, which were not included in the training data set. A web-server named BioRel (http://ibi.hzau.edu.cn/biorel) was developed to provide services including BR, BRS calculation, activity class, and pharmacokinetic property prediction

Infection of Vero E6 cells by bat SARSr-CoV WIV1, Rs4874, WIV1-Rs4231S and WIV1-Rs7327S.

<p>(A) The successful infection was confirmed by immunofluorescent antibody staining using rabbit antibody against the SARSr-CoV Rp3 nucleocapsid protein. The columns (from left to right) show staining of nuclei (blue), virus replication (red), and both nuclei and virus replication (merged double-stain images). (B) The growth curves in Vero E6 cells with a MOI of 1.0 and 0.01.</p

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus - Fig 8

<p><b>Analysis of receptor usage by immunofluorescence assay (A) and real-time PCR (B).</b> Virus infectivity of Rs4874, WIV1-Rs4231S and WIV1-Rs7327S was determined in HeLa cells with and without the expression of human ACE2. ACE2 expression was detected with goat anti-human ACE2 antibody followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG. Virus replication was detected with rabbit antibody against the SARSr-CoV Rp3 nucleocapsid protein followed by cyanine 3 (Cy3)-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI (49,6-diamidino-2-phenylindole).The columns (from left to right) show staining of nuclei (blue), ACE2 expression (green), virus replication (red) and the merged triple-stained images, respectively.</p

Detection of potential recombination events by similarity plot and boot scan analysis.

<p>(A) Full-length genome sequence of SARSr-CoV WIV16 was used as query sequence and WIV1, Rs4231 and Rs4081 as reference sequences. (B) Full-length genome sequence of SARS-CoV SZ3 was used as query sequence and SARSr-CoV WIV16, Rf4092 and Rs4081 as reference sequences. All analyses were performed with a Kimura model, a window size of 1500 base pairs, and a step size of 150 base pairs. The gene map of query genome sequences are used to position breakpoints.</p

Functional characterization of diverse ORF8 and ORF8a proteins of bat SARSr-CoVs.

<p>(A) The ORF8 proteins of SARS-CoV and bat SARSr-CoVs induces the ATF6-dependent transcriptional activity. HeLa cells were transiently transfected with the pcAGGS expression plasmids of the ORF8 of SARS-CoV GZ02, bat SARSr-CoV Rf1, WIV1 and Rf4092 and the reporter plasmid 5×ATF6-GL3 for 40h. Control cells were co-transfected with the reporter plasmid and the empty pCAGGS vector for 24h, and treated with or without TM (2μg/ml) for an additional 16h. The cell lysates were harvested for dual luciferase assay and data are shown as the average values from triplicate wells. (B) The ORF8a proteins of SARS-CoV and bat SARSr-CoV triggered apoptosis. 293T cells were transfected with the expression plasmids of the ORF8a of SARS-CoV Tor2 and bat SARSr-CoV Rs4084 and a pcAGGS vector control for 24h. Apoptosis was analyzed by flow cytometry after annexin V staining and the percentage of apoptotic cells were calculated. Data are shown as the average values from triplicate cells. Error bars indicate SDs. * <i>P</i><0.05.</p

Similarity plot based on the full-length genome sequence of civet SARS CoV SZ3.

<p>Full-length genome sequences of all SARSr-CoV detected in bats from the cave investigated in this study were used as reference sequences. The analysis was performed with the Kimura model, a window size of 1500 base pairs and a step size of 150 base pairs.</p

Amino acid sequence comparison of the S1 subunit (corresponding to aa 1–660 of the spike protein of SARS-CoV).

<p>The receptor-binding domain (aa 318–510) of SARS-CoV and the homologous region of bat SARSr-CoVs are indicated by the red box. The key aa residues involved in the interaction with human ACE2 are numbered on top of the aligned sequences. SARS-CoV GZ02, BJ01 and Tor2 were isolated from patients in the early, middle and late phase, respectively, of the SARS outbreak in 2003. SARS-CoV SZ3 was identified from civets in 2003. SARSr-CoV Rs 672 and YN2013 were identified from <i>R</i>. <i>sinicus</i> collected in Guizhou and Yunnan Province, respectively. SARSr-CoV Rf1 and JL2012 were identified from <i>R</i>. <i>ferrumequinum</i> collected in Hubei and Jilin Province, respectively. WIV1, WIV16, RsSHC014, Rs4081, Rs4084, Rs4231, Rs4237, Rs4247, Rs7327 and Rs4874 were identified from <i>R</i>.<i>sinicus</i>, and Rf4092 from <i>R</i>. <i>ferrumequinum</i> in the cave surveyed in this study.</p

Alignment of nucleotide sequences of ORF8 or ORF8a/8b.

<p>The start codons and stop codons of ORF8, 8a and 8b are marked with black boxes and the forward and reverse arrows, respectively. The deletion responsible for the split ORF8a and 8b in human SARS-CoV BJ01, Tor2 and bat SARSr-CoV Rs4084 is marked with red boxes. See the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006698#ppat.1006698.g003" target="_blank">Fig 3</a> for the origin of various sequences used in this alignment.</p