Physiological and transcriptomic analysis reveals the potential mechanism of Morinda officinalis How in response to freezing stress.

BACKGROUND: Morinda officinalis How (MO) is a vine shrub distributed in tropical and subtropical regions, known as one of the "Four Southern Herbal Medicines" in China. The unclear responsive mechanism by which MO adapt to freezing stress limits progress in molecular breeding for MO freezing tolerance. RESULTS: In this study, morphological, physiological and microstructure changes in MO exposed to -2℃ for 0 h, 3 h, 8 h and 24 h were comprehensively characterized. The results showed that freezing stress caused seedling dehydration, palisade cell and spongy mesophyll destruction. A significant increase in the content of proline, soluble protein and soluble sugars, as well as the activity of superoxide dismutase and peroxidase was observed. Subsequently, we analyzed the transcriptomic changes of MO leaves at different times under freezing treatment by RNA-seq. A total of 24,498 unigenes were annotated and 3252 unigenes were identified as differentially expressed genes (DEGs). Most of these DEGs were annotated in starch and sucrose metabolism, plant hormone signal transduction and MAPK signaling pathways. Family Enrichment analysis showed that the glucosyl/glucuronosyl transferases, oxidoreductase, chlorophyll a/b binding protein and calcium binding protein families were significantly enriched. We also characterized 7 types of transcription factors responding to freezing stress, among which the most abundant family was the MYBs, followed by the AP2/ERFs and NACs. Furthermore, 10 DEGs were selected for qRT-PCR analysis, which validated the reliability and accuracy of RNA-seq data. CONCLUSIONS: Our results provide an overall view of the dynamic changes in physiology and insight into the molecular regulation mechanisms of MO in response to freezing stress. This study will lay a foundation for freezing tolerance molecular breeding and improving the quality of MO.

Biocontrol fungi induced stem-base rot disease resistance of Morinda officinalis How revealed by transcriptome analysis.

Introduction: Morinda officinalis How (MO) is a Rubiaceae plant, and its medicinal part is dried root, which is one of the "Four Southern Medicines" in China. At present, the plant MO breed seedlings mainly by cutting methods. Long-term asexual propagation makes pathogenic fungi accumulate in MO, leading to stem-base rot, which is caused by Fusarium oxysporum (Fon). Methods: In this study, we used Trichoderma harzianum and Pestalotiopsis sp. as biocontrol fungi to investigate their antagonistic ability to Fon through in vitro antagonism and pot experiments, and combined with transcriptome sequencing to explore the mechanism of biocontrol. Results: The results showed that both Trichoderma harzianum and Pestalotiopsis sp. could inhibit the growth of Fon. In addition, Trichoderma harzianum and Pestalotiopsis sp. could also enhance the basic immunity to Fon by increasing the activities of defensive enzymes such as POD and SOD, chlorophyll content, soluble sugar content, and oligosaccharide content of MO. The mechanism of biological control of stem-base rot of MO was discussed by transcriptome technology. MO was treated with two treatments, root irrigation with biocontrol fungi or inoculation with Fon after root irrigation with biocontrol fungi. Transcriptome sequencing revealed that nearly 11,188 differentially expressed genes (DEGs) were involved in the process of inducing MO systemic resistance to Fon by biocontrol fungi. Meanwhile, Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, as well as transcription factor (TFs) prediction showed that there were significant differences in the expression levels of MO roots under different treatments. Also, the genes of the "MAPK signaling pathway" and "plant hormone signaling pathway" were analyzed, in which the ERFs gene of the ethylene signal transduction pathway participated in the metabolism of glycosyl compounds. It is speculated that the ethylene signal may participate in the immune response of the sugar signal to the infection of Fon. After qRT-PCR verification of 10 DEGs related to the ethylene signal transduction pathway, the expression trend is consistent with the results of transcriptome sequencing, which proves the reliability of transcriptome sequencing. Discussion: In conclusion, this study preliminarily identified the molecular mechanism of the biological control of MO stem-base rot and provided a scientific basis for further research on the prevention and control mechanism of MO stem-base rot.

Discovery of a Series of Potent, Selective, and Orally Bioavailable Nucleoside Inhibitors of CD73 That Demonstrates In Vivo Antitumor Activity.

CD73 (ecto-5'-nucleotidase) has emerged as an attractive target for cancer immunotherapy of many cancers. CD73 catalyzes the hydrolysis of adenosine monophosphate (AMP) into highly immunosuppressive adenosine that plays a critical role in tumor progression. Herein, we report our efforts in developing orally bioavailable and highly potent small-molecule CD73 inhibitors from the reported hit molecule 2 to lead molecule 20 and then finally to compound 49. Compound 49 was able to reverse AMP-mediated suppression of CD8+ T cells and completely inhibited CD73 activity in serum samples from various cancer patients. In preclinical in vivo studies, orally administered 49 showed a robust dose-dependent pharmacokinetic/pharmacodynamic (PK/PD) relationship that correlated with efficacy. Compound 49 also demonstrated the expected immune-mediated antitumor mechanism of action and was efficacious upon oral administration not only as a single agent but also in combination with either chemotherapeutics or checkpoint inhibitor in the mouse tumor model.

Structure-based rational design, synthesis and antifungal activity of oxime-containing azole derivatives.

In an attempt to find novel azole antifungal agents with improved activity and broader spectrum, computer modeling was used to design a series of new azoles with piperidin-4-one O-substituted oxime side chains. Molecular docking studies revealed that they formed hydrophobic and hydrogen-bonding interactions with lanosterol 14alpha-demethylase of Candida albicans (CACYP51). In vitro antifungal assay indicates that most of the synthesized compounds showed good activity against tested fungal pathogens. In comparison with fluconazole, itraconazole and voriconazole, several compounds (such as 10c, 10e, and 10i) show more potent antifungal activity and broader spectrum, suggesting that they are promising leads for the development of novel antifungal agents.

Phosphate ester derivatives of homocamptothecin: synthesis, solution stabilities and antitumor activities.

Homocamptothecins (hCPTs) represents a new promising class of topoisomerase I inhibitors with enhanced stability and superior antitumor activity. Some phosphodiesters and phosphotriesters homocamptothecin derivatives were designed and synthesized based on our previous synthetic route. The cytotoxicity in vitro on three cancer cell lines and antitumor activity in vivo, and inhibitory properties of topoisomerase I of these derivatives were evaluated. Among them compounds 24e and 24f exhibited higher cytotoxic activity than IRT and the former exhibited the best antitumor activity in vivo and solution stability both at pH 7.4 and pH 3.0.

Three-dimensional model of lanosterol 14 alpha-demethylase from Cryptococcus neoformans: active-site characterization and insights into azole binding.

Cryptococcus neoformans is one of the most important causes of life-threatening fungal infections in immunocompromised patients. Lanosterol 14 alpha-demethylase (CYP51) is the target of azole antifungal agents. This study describes, for the first time, the 3-dimensional model of CYP51 from Cryptococcus neoformans (CnCYP51). The model was further refined by energy minimization and molecular-dynamics simulations. The active site of CnCYP51 was well characterized by multiple-copy simultaneous-search calculations, and four functional regions important for rational drug design were identified. The mode of binding of the natural substrate and azole antifungal agents with CnCYP51 was identified by flexible molecular docking. A G484S substitution mechanism for azole resistance in CnCYP51, which might be important for the conformation of the heme environment, is suggested.

Discovery of highly potent novel antifungal azoles by structure-based rational design.

On the basis of the active site of lanosterol 14alpha-demethylase from Candida albicans (CACYP51), a series of new azoles were designed and synthesized. All the new azoles show excellent in vitro activity against most of the tested pathogenic fungi, which represent a class of promising leads for the development of novel antifungal agents. The MIC(80) value of compounds 8c, 8i and 8n against C. albicans is 0.001 microg/mL, indicating that these compounds are more potent than fluconazole, itraconazole and voriconazole. Flexible molecular docking was used to analyze the structure-activity relationships (SARs) of the compounds. The designed compounds interact with CACYP51 through hydrophobic, van der Waals and hydrogen-bonding interactions.

Design, synthesis, and antifungal activity of novel conformationally restricted triazole derivatives.

A series of new triazole derivatives were designed and synthesized on the basis of the active site of lanosterol 14alpha-demethylase from Candida albicans (CACYP51). 2-(2,4-Difluorophenyl)-3-(methyl-(3-phenoxyalkyl)amino)-1-(1H-1,2,4-triazol-1-yl)propan-2-ols show excellent in-vitro activity against most of the tested pathogenic fungi. The MIC(80) value of compound 8a against Candida albicans is 0.01 microM, which provides a good starting template for further structural optimization. The binding modes of the designed compounds were investigated by flexible molecular docking. The compounds interacted with CACYP51 through hydrophobic, van-der-Waals, and hydrogen-bonding interactions.

From antidiabetic to antifungal: discovery of highly potent triazole-thiazolidinedione hybrids as novel antifungal agents.

In an attempt to discover a new generation of triazole antifungal agents, a series of triazole-thiazolidinedione hybrids were designed and synthesized by molecular hybridization of the antifungal agent fluconazole and rosiglitazone (an antidiabetic). Most of the target compounds showed good to excellent inhibitory activity against a variety of clinically important fungal pathogens. In particular, compounds (Z)-5-(2,4-dichlorobenzylidene)-3-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)thiazolidine-2,4-dione) (15 c), (Z)-3-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-5-(furan-3-ylmethylene)thiazolidine-2,4-dione (15 j), and (Z)-3-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-5-(furan-3-ylmethylene)thiazolidine-2,4-dione (15 r) were highly active against Candida albicans, with MIC80 values in the range of 0.03-0.15 µM. Moreover, compounds 15 j and 15 r were found to be effective against four fluconazole-resistant clinical isolates; these two compounds are particularly promising antifungal leads for further optimization. Molecular docking studies revealed that the hydrogen bonding interactions between thiazolidinedione and CYP51 from C. albicans are important for antifungal activity. This study also demonstrates the effectiveness of molecular hybridization in antifungal drug discovery.

Design and synthesis of antifungal benzoheterocyclic derivatives by scaffold hopping.

The incidence of invasive fungal infections and associated mortality is increasing dramatically. Although azoles are first-line antifungal agents, cross-resistance and hepatic toxicity are their two major limitations. The discovery of novel non-azole lead compounds will be helpful to overcome these problems. On the basis of our previously reported benzopyran non-azole CYP51 inhibitor, scaffold hopping was used to design structurally diverse new compounds and expand the structure-activity relationships of the lead structure. Five kinds of scaffolds, namely benzimidazole, benzoxazole, benzothiazole, quinazolin-4-one and carboline, were chosen for synthesis. In vitro antifungal activity data and results from molecular docking revealed that the scaffold was important for the antifungal activity. Several compounds showed potent activity against both standard and clinically resistant fungal pathogens, suggesting that they can serve as a good starting point for the discovery of novel antifungal agents.

Design and synthesis of novel triazole antifungal derivatives by structure-based bioisosterism.

The incidence of life-threatening fungal infections is increasing dramatically. In an attempt to develop novel antifungal agents, our previously synthesized phenoxyalkylpiperazine triazole derivatives were used as lead structures for further optimization. By means of structure-based bioisosterism, triazolone was used as a new bioisostere of oxygen atom. This type of bioisosteric replacement can improve the water solubility without loss of hydrogen-bonding interaction with the target enzyme. A series of triazolone-containing triazoles were rationally designed and synthesized. As compared with fluconazole, several compounds showed higher antifungal activity with broader spectrum, suggesting their potential for further evaluations.

Structure-based design, synthesis, and antifungal activity of new triazole derivatives.

A series of new antifungal triazole derivatives with phenylacetamide side chain were rational designed and synthesized on the basis of the structural information of lanosterol 14-demethylase (CYP51). In vitro antifungal activity assay indicated that several compounds showed higher activity than fluconazole. Especially, compound 8h showed excellent inhibitory activity against Candida albicans and Cryptococcus neoformans (MIC=0.0156 µg/mL), suggesting that it is a promising lead for the development of novel antifungal agents. The binding mode of compound 8h was investigated by flexible molecular docking. It interacted with CACYP51 through hydrophobic and van der Waals interactions.

Trifluoromethyl-promoted homocamptothecins: synthesis and biological activity.

The homocamptothecin (hCPT) represents a new class of topoisomerase inhibitor which combines enhanced plasma stability and strong antitumor activity. Fluorine imparts desirable characteristics to drugs by modulating both the pharmacokinetics and pharmacodynamic properties of a drug. Therefore, in an attempt to improve the antitumor activity of homocamptothecins, seven new 7-trifluoromethylated homocamptothecin derivatives were prepared by proline-catalyzed Friedlander annulation. The antitumor activity in vitro and in vivo on cancer cell lines, and inhibitory properties of topoisomerase I-mediated DNA cleavage of compounds 6c and 8b were evaluated. Several of these trifluoromethylated hCPT derivatives (such as 6a, 6b and 6c) possessed higher in vitro antitumor activity than topotecan (TPT). Especially, the compound 6c showed effective in vivo antitumor activity comparable to that of TPT.

Improved model of lanosterol 14alpha-demethylase by ligand-supported homology modeling: validation by virtual screening and azole optimization.

Lanosterol 14alpha-demethylase (CYP51) is an important target for antifungal drugs. An improved three-dimensional model of CYP51 from Candida albicans (CACYP51) was constructed by ligand-supported homology modeling and molecular dynamics simulations. The accuracy of the constructed model was evaluated by its performance in a small-scale virtual screen. The results show that known CYP51 inhibitors were efficiently discriminated by the model, and it performed better than our previous CACYP51 model. The active site of CACYP51 was characterized by multiple copy simultaneous search (MCSS) calculations. On the basis of the MCSS results, a series of novel azoles were designed and synthesized, and they showed good in vitro antifungal activity with a broad spectrum. The MIC(80) value of four of these compounds against C. albicans is 0.001 microg mL(-1), indicating that they are promising leads for the discovery of novel antifungal agents.

Design, synthesis and antifungal activity of isosteric analogues of benzoheterocyclic N-myristoyltransferase inhibitors.

N-myristoyltransferase (NMT) has been a promising new target for the design of novel antifungal agents with new mode of action. A series of benzoxazole and indole derivatives were designed and synthesized as isosteric analogues of benzoheterocyclic NMT Inhibitors. In vitro antifungal assay indicated that the benzoxazole derivatives were far more potent than the indoles. Molecular docking studies revealed that the hydrogen bonding interaction between the benzoheterocyclic core and NMT might be essential in the orientation of the inhibitor to a proper position. The antifungal activity of benzoxazole derivative 8f was comparable or superior to that of fluconazole, which can serve as a good starting point for further studies of structural diversity of the benzoheterocyclic NMT inhibitors.

Synthesis and evaluation of 9-benzylideneamino derivatives of homocamptothecin as potent inhibitors of DNA topoisomerase I.

A series of novel 9-benzylideneamino derivatives of homocamptothecin were synthesized via Friedlaender cyclization from our obtained intermediate 5. All the compounds were evaluated for in vitro cytotoxicity against three cancer cell lines (A549, LOVO and MDA-MB-435). Most of these derivatives possessed potent growth inhibitory effect on all the tested cell lines and four compounds (6d, 6f, 6i, 6k) showed higher inhibitory activities with the IC50 values of 2.3 nM-9.8 nM against breast cancer cell than topotecan. As compared to CPT, compound 6f revealed higher topoisomerase I inhibitory activity.

New azoles with potent antifungal activity: design, synthesis and molecular docking.

In response to the urgent need for novel antifungal agents with improved activity and broader spectrum, computer modeling was used to rational design novel antifungal azoles. On the basis of the active site of lanosterol 14alpha-demethylase from Candida albicans (CACYP51), a series of new azoles with substituted-phenoxypropyl piperazine side chains were rational designed and synthesized. In vitro antifungal activity assay indicates that the new azoles show good activity against most of the tested pathogenic fungi. Interestingly, the designed compounds are also active against an azole-resistant clinical strain. Compared to fluconazole and itraconazole, several compounds (such as 12i, 12j and 12n) show higher antifungal activity and broader spectrum, which are promising leads for the development of novel antifungal agents.