Discovery of Novel α,ß-Unsaturated Amide Derivatives as Candidate Antifungals to Overcome Fungal Resistance.

In our previous study, coumarin-containing CYP51 inhibitor A32 demonstrated potent antiresistance activity. However, compound A32 demonstrated unsatisfied metabolic stability, necessitating modifications to overcome these limitations. In this study, α,ß-unsaturated amides were used to replace the unstable coumarin ring, which increased metabolic stability by four times while maintaining antifungal activity, including activity against resistant strains. Subsequently, the sterol composition analysis and morphological observation experiments indicated that the target of these novel compounds is lanosterol 14α-demethylase (CYP51). Meanwhile, biofilm growth was inhibited and resistance genes (ERG11, CDR1, CDR2, and MDR1) expression was downregulated to find out how the antiresistance works. Importantly, compound C07 demonstrated the capacity to stimulate reactive oxygen species, thus displaying potent fungicidal activity. Moreover, C07 exhibited encouraging effectiveness in vivo following intraperitoneal administration. Additionally, the most potent compound C07 showed satisfactory pharmacokinetic properties and low toxicity. These α,ß-unsaturated amide derivatives, particularly C07, are potential candidates for treating azole-resistant candidiasis.

Developing Novel Coumarin-Containing Azoles Antifungal Agents by the Scaffold Merging Strategy for Treating Azole-Resistant Candidiasis.

The extensive use of antifungal drugs has resulted in severe drug resistance, making clinical treatment of fungal infections more difficult. Biofilm inhibitors can overcome drug resistance by inhibiting fungal biofilm formation. In this study, some coumarins with antibiofilm activity were merged into CYP51 inhibitors to produce novel molecules possessing potent antiresistance activity. As expected, most compounds exhibited excellent in vitro antifungal activity against pathogenic fungi, especially fluconazole-resistant candidiasis. Then, their mechanism was confirmed by sterol composition analysis and morphological observation. Biofilm inhibition and down-regulation of resistance-related genes were employed to confirm the compounds' antiresistance mechanisms. Significantly, compound A32 demonstrated fungicidal activity against fluconazole-resistant strain 904. Most importantly, compound A32 showed potent in vivo antifungal activity against pathogenic fungi and fluconazole-resistant strains. Preliminary pharmacokinetic and toxicity tests demonstrated that the compounds possessed favorable druggability. Taken together, compound A32 represents a promising lead to develop novel antifungal agents for treating azole-resistant candidiasis.

Design, synthesis and antifungal activities of novel triazole derivatives with selenium-containing hydrophobic side chains.

In this work, a series of novel 1,2,4-triazole derivatives with selenium-containing hydrophobic side chains were designed and synthesized based on the structure of lanosterol 14α-demethylase (CYP51). All compounds were characterized by HRMS, 1H NMR and 13C NMR. Then, their antifungal activities against eight human pathogenic fungi were evaluated in vitro by testing the minimal inhibitory concentrations. The results showed that nearly all tested compounds were found to be more potent against all tested fungal strains than control drug fluconazole. Further mechanism study demonstrated that the target compounds had fungal CYP51 inhibitory activity. Meanwhile, representative compounds revealed low cytotoxic effects toward mammalian cell lines. In addition, the docking results showed that the target compounds bound to Candida albicans CYP51 in a better pattern than fluconazole, especially in the narrow hydrophobic cleft. Overall, the novel 1,2,4-triazole derivatives with selenium-containing hydrophobic side chains can be further developed for the potential treatment of invasive fungal infections.

Discovery of novel selenium-containing azole derivatives as antifungal agents by exploiting the hydrophobic cleft of CYP51.

Herein, we report the design, synthesis and evaluation of a novel series of diselenide and selenide derivatives as potent antifungal agents by exploiting the hydrophobic cleft of CYP51. Among all synthesized compounds, the most potent compound B01 with low cytotoxic and hemolysis effect exhibited excellent activity against C.alb., C.gla., C.par. and C.kru., as well as selected fluconazole-resistant strains. Moreover, compound B01 could reduce the biofilm formation of the FCZ-resistant C.alb. Subsequently, metabolic stability assays using liver microsomes demonstrated that compound B01 showed good profiles of metabolic stability. With superior pharmacological profile, compound B01 was advanced into in vivo bioactivity evaluation. In a murine model of systemic C.alb. infection, compound B01 significantly reduced fungal load of kidneys. Furthermore, compound B01 revealed relatively low acute toxicity and subacute toxicity in mice. In addition, docking study performed into C.alb. CYP51, showed the binding mode between C.alb. CYP51 and compound B01. Collectively, diselenides compound B01 can be further developed for the potential treatment of invasive fungal infections.

Whole-genome resequencing of wild and cultivated cannabis reveals the genetic structure and adaptive selection of important traits.

BACKGROUND: Cannabis is an important industrial crop species whose fibre, seeds, flowers and leaves are widely used by humans. The study of cannabinoids extracted from plants has been popular research topic in recent years. China is one of the origins of cannabis and one of the few countries with wild cannabis plants. However, the genetic structure of Chinese cannabis and the degree of adaptive selection remain unclear. RESULTS: The main morphological characteristics of wild cannabis in China were assessed. Based on whole-genome resequencing SNPs, Chinese cannabis could be divided into five groups in terms of geographical source and ecotype: wild accessions growing in the northwestern region; wild accessions growing in the northeastern region; cultivated accessions grown for fibre in the northeastern region; cultivated accessions grown for seed in northwestern region, and cultivated accessions in southwestern region. We further identified genes related to flowering time, seed germination, seed size, embryogenesis, growth, and stress responses selected during the process of cannabis domestication. The expression of flowering-related genes under long-day (LD) and short-day (SD) conditions showed that Chinese cultivated cannabis is adapted to different photoperiods through the regulation of Flowering locus T-like (FT-like) expression. CONCLUSION: This study clarifies the genetic structure of Chinese cannabis and offers valuable genomic resources for cannabis breeding.

Discovery of novel aminosaccharide-based sulfonamide derivatives as potential carbonic anhydrase II inhibitors.

In this paper, a new class of novel sulfonamides incorporating aminosaccharide tails were designed and synthesized based on the sugar-tail approach. Then, all the novel compounds were evaluated for their inhibitory activities against three carbonic anhydrase (CA, EC 4.2.1.1) isoenzymes (hCA I, hCA II and hCA IX). Interestingly, effective inhibition of these three CA isoforms were observed, especially the glaucoma associated isoform hCA II. It is worth noting that these glycoconjugated sulfonamide derivatives also showed better CA inhibitory effects compared to the initial segment carzenide. Among them, compound 8d was the most effective inhibitor with IC50 of 60 nM against hCA II. Subsequent physicochemical properties studies showed that all compounds have good water solubility and neutral pH values in solutions. And these important physicochemical properties make target compounds acquire obvious advantages in the preparation of topical and nonirritating antiglaucoma drugs. Moreover, the target compounds showed lower corneal cytotoxicity than acetazolamide (AAZ) and good metabolic stability in vitro. In addition, molecular docking studies confirmed the interactions between aminosaccharide fragment and hydrophilic subpocket of hCA II active site were crucial for the enhanced CA inhibitory activity. Taken together, these results suggested 8d would be a promising lead compound for the development of topical antiglaucoma CAIs.

Discovery of 1,2,3-selenadiazole analogues as antifungal agents using a scaffold hopping approach.

With the increasing incidence of antifungal resistance, new antifungal agents having novel scaffolds hence are in an urgent need to combat infectious diseases caused by multidrug-resistant (MDR) pathogens. In this study, we reported the design, synthesis, and pharmacological evaluation of novel 1,2,3-selenadiazole analogues by scaffold hopping strategy. Preliminary results of antifungal activity demonstrated that the new class of compounds showed broad-spectrum fungistatic and fungicidal activity. Most importantly, these newly synthesized compounds can eliminate these azole-resistant fungi and inhibit the formation of C. albicans biofilm. In particular, compound S07 showed promising antifungal activity against five azole-resistant strains with MIC values ranging from 4 to 32 µg/mL. Then, further target identification and mechanistic studies indicated that representative compound S07 exert its inhibitory activity by inhibiting fungal lanosterol 14α-demethylase enzyme (CYP51). Interestingly, representative compounds showed low cytotoxicity on mammalian cell lines. In addition, the molecular docking studies elucidated the binding modes of these compounds toward CYP51. Altogether, these results suggest that compound S07 with novel skeleton is a promising CYP51 inhibitor for treatment of fungal infections.

Lead optimization generates selenium-containing miconazole CYP51 inhibitors with improved pharmacological profile for the treatment of fungal infections.

A series of selenium-containing miconazole derivatives were identified as potent antifungal drugs in our previous study. Representative compound A03 (MIC = 0.01 µg/mL against C.alb. 5314) proved efficacious in inhibiting the growth of fungal pathogens. However, further study showed lead compound A03 exhibited potential hemolysis, significant cytotoxic effect and unfavorable metabolic stability and was therefore modified to overcome these drawbacks. In this article, the further optimization of selenium-containing miconazole derivatives resulted in the discovery of similarly potent compound B17 (MIC = 0.02 µg/mL against C.alb. 5314), exhibiting a superior pharmacological profile with decreased rate of metabolism, cytotoxic effect and hemolysis. Furthermore, compound B17 showed fungicidal activity against Candida albicans and significant effects on the treatment of resistant Candida albicans infections. Meanwhile, compound B17 not only could reduce the ergosterol biosynthesis pathway by inhibiting CYP51, but also inhibited biofilm formation. More importantly, compound B17 also shows promising in vivo efficacy after intraperitoneal injection and the PK study of compound B17 was evaluated. In addition, molecular docking studies provide a model for the interaction between the compound B17 and the CYP51 protein. Overall, we believe that these selenium-containing miconazole compounds can be further developed for the potential treatment of fungal infections.

Discovery of novel artemisinin-sulfonamidehybrids as potential carbonicanhydraseIX inhibitors with improved antiproliferative activities.

A series of artemisinin-sulfonamide hybrids (1-16) have been designed and synthesized by using molecular hybridization approach and investigated for the inhibitory activity of four human (h) carbonic anhydrases (CAs, EC 4.2.1.1), hCA I, II, IX and XII. The results indicated most of the target compounds showed better CA IX and CA XII inhibitory activity than the starting segment sulfanilamide. Among all the compounds, compound 3 (IC50: 5 nM) showed the best CA IX inhibitory efficacy. The p-aminobenzenesulfonamide derivatives showed significant antiproliferative activities against MDA-MB-231 breast cancer cell line and HT-29 colon cancer cell line under hypoxic conditions where CA IX and CA XII are overexpressed and most of them showed no apparent cytotoxic effects toward MCF-10A normal mammary epithelial cell. Among these derivatives, compound 3 displayed the most potent antiproliferative activities (IC50: 0.65 µM) against HT-29 cell line under hypoxia and low cytotoxicity (IC50: 78.0 µM) toward normal cell line. Meanwhile, compound 3 was found to efficiently decrease the hypoxia-induced extracellular acidification in both cancer cells. Molecular docking studies of compounds 3, 4, 5 and 9 revealed the proper interactions between the hybrid molecules and the active site of CA IX. All the results proved the effectiveness of the hybridization approach to develop novel artemisinin-sulfonamide compounds targeting CA IX for cancer treatment.

Design, synthesis and biological evaluation of novel carbohydrate-based sulfonamide derivatives as antitumor agents.

A series of novel carbohydrate-based sulfonamides were designed and synthesized by the sugar-tail approach. The classical aromatic sulfonamide pharmacophore (ArSO2NH2) was directly linked to a hydrophilic sugar-tail moiety through a rigid 1, 2, 3-triazole linker by the click chemistry reaction. The inhibitory activity against three carbonic anhydrase (CA, EC 4.2.1.1) isozymes (hCA I, hCA II and hCA IX) of all new compounds so designed were investigated in vitro and efficient inhibition against all three CA isoforms, especially the tumor-associated hCA IX, were observed. These glycoconjugate sulfonamide derivatives displayed better inhibitory efficacy in comparison with the starting segments (SA and p-hydroxybenzene sulfonamide). In particular, compound 12g was found to be the most effective and rather selective inhibitor of hCA IX with inhibitory constant (IC50) value of 7 nM, being four times more potent than the clinical used agent acetazolamide (AAZ) (IC50 = 30 nM). Meanwhile, almost all compounds showed moderate antiproliferative activities against two cancer cell lines (HT-29 and MDA-MB-231) in both hypoxic and normoxic conditions while compound 12g also exhibited the most prominent antitumor activity. Furthermore, evident recovery (20-35% reduction of IC50 values) of cytotoxic efficiency of doxorubicin with the combination of compounds 12d, 12g and 22d as CAIs were detected on MDA-MB-231 cell line under hypoxic environment. In addition, docking studies revealed that the sugar-tail fragment of the target compounds participated in interactions with hydrophilic subpocket at the surface of hCA IX active site and supported the CA IX inhibitory activities of carbohydrate-based sulfonamide derivatives.

Design, synthesis, and biological evaluation of novel miconazole analogues containing selenium as potent antifungal agents.

Herein, based on the theory of bioisosterism, a series of novel miconazole analogues containing selenium were designed, synthesized and their inhibitory effects on thirteen strains of pathogenic fungi were evaluated. It is especially encouraging that all the novel target compounds displayed significant antifungal activities against all tested strains. Furthermore, all the target compounds showed excellent inhibitory effects on fluconazole-resistant fungi. Subsequently, preliminary mechanistic studies indicated that the representative compound A03 had a strong inhibitory effect on C.alb. CYP51. Moreover, the target compounds could prevent the formation of fungi biofilms. Further hemolysis test verified that potential compounds had higher safety than miconazole. In addition, molecular docking study provided the interaction modes between the target compounds and C.alb. CYP51. These results strongly suggested that some target compounds are promising as novel antifungal drugs.

Evaluation of reference genes for RT-qPCR analysis in wild and cultivated Cannabis.

RT-qPCR has been widely used for gene expression analysis in recent years. The accuracy of this technique largely depends on the selection of suitable reference genes. In order to facilitate gene expression analysis in wild and cultivated Cannabis, the expression stability of seven candidate reference genes (ACT2, 18S rRNA, GAPDH, UBQ, TUB, PP2A and EF1α) were assessed in leaves samples of different development stages and different organs of both wild and cultivated Cannabis in the present study. Their expression stabilities were evaluated through three software packages (GeNorm, Normfinder and Bestkeeper). Results showed that UBQ and EF1α were the highly ranked genes in different leaves samples, and PP2A was the most stable reference gene in different organs, while GAPDH was the least stable one. And the validation of the reference genes selected was further confirmed by the expression patterns of MDS and OLS.

Latitudinal Adaptation and Genetic Insights Into the Origins of Cannabis sativa L.

Cannabis is one of the most important industrial crops distributed worldwide. However, the phylogeographic structure and domestication knowledge of this crop remains poorly understood. In this study, sequence variations of five chloroplast DNA (cpDNA) regions were investigated to address these questions. For the 645 individuals from 52 Cannabis accessions sampled (25 wild populations and 27 domesticated populations or cultivars), three haplogroups (Haplogroup H, M, L) were identified and these lineages exhibited distinct high-middle-low latitudinal gradients distribution pattern. This pattern can most likely be explained as a consequence of climatic heterogeneity and geographical isolation. Therefore, we examined the correlations between genetic distances and geographical distances, and tested whether the climatic factors are correlated with the cpDNA haplogroup frequencies of populations. The "isolation-by-distance" models were detected for the phylogeographic structure, and the day-length was found to be the most important factor (among 20 BioClim factors) that influenced the population structures. Considering the distinctive phylogeographic structures and no reproductive isolation among members of these lineages, we recommend that Cannabis be recognized as a monotypic genus typified by Cannabis sativa L., containing three subspecies: subsp. sativa, subsp. Indica, and subsp. ruderalis. Within each haplogroup which possesses a relatively independent distribution region, the wild and domesticated populations shared the most common haplotypes, indicating that there are multiregional origins for the domesticated crop. Contrast to the prevalent Central-Asia-Origin hypothesis of C. saltiva, molecular evidence reveals for the first time that the low latitude haplogroup (Haplogroup L) is the earliest divergent lineage, implying that Cannabis is probably originated in low latitude region.

Design, Synthesis and Antifungal Activity of Novel Benzofuran-Triazole Hybrids.

A series of novel benzofuran-triazole hybrids was designed and synthesized by click chemistry, and their structures were characterized by HRMS, FTIR and NMR. The in vitro antifungal activity of target compounds was evaluated using the microdilution broth method against five strains of pathogenic fungi. The result indicated that the target compounds exhibited moderate to satisfactory activity. Furthermore, molecular docking was performed to investigate the binding affinities and interaction modes between the target compound and N-myristoyltransferase. Based on the results, preliminary structure activity relationships (SARs) were summarized to serve as a foundation for further investigation.