Discovery of New Tricyclic Oxime Sampangine Derivatives as Potent Antifungal Agents for the Treatment of Cryptococcosis and Candidiasis.

Cryptococcus neoformans (C. neoformans) and Candida albicans (C. albicans) are classified as the critical priority groups among the pathogenic fungi, highlighting the urgent need for developing more effective antifungal therapies. On the basis of antifungal natural product sampangine, herein, a series of tricyclic oxime and oxime ether derivatives were designed. Among them, compound WZ-2 showed excellent inhibitory activity against C. neoformans (MIC80 = 0.016 µg/mL) and synergized with fluconazole to treat resistant C. albicans (FICI = 0.078). Interestingly, compound WZ-2 effectively inhibited virulence factors (e.g., capsule, biofilm, and yeast-to-hypha morphological transition), suggesting the potential to overcome drug resistance. In a mouse model of cryptococcal meningitis, compound WZ-2 (5 mg/kg) effectively reduced the brain C. neoformans H99 burden. Furthermore, compound WZ-2 alone and its combination with fluconazole also significantly reduced the kidney burden of the drug-resistant strain (0304103) and sensitive strain (SC5314) of C. albicans.

Discovery of Pyrazolone Carbothioamide Derivatives as Inhibitors of the Pdr1-KIX Interaction for Combinational Treatment of Azole-Resistant Candidiasis.

Candida glabrata has emerged as an important opportunistic pathogen of invasive candidiasis due to increasing drug resistance. Targeting Pdr1-KIX interactions with small molecules represents a potential strategy for treating drug-resistant candidiasis. However, effective Pdr1-KIX inhibitors are rather limited, hindering the validation of target druggability. Here, new Pdr1-KIX inhibitors were designed and assayed. Particularly, compound B8 possessed a new chemical scaffold and exhibited potent KIX binding affinity, leading to enhanced synergistic efficacy with fluconazole to treat resistant C. glabrata infection (FICI = 0.28). Compound B8 acted by inhibiting the efflux pump and down-regulating resistance-associated genes through blocking the Pdr1-KIX interaction. Compound B8 exhibited excellent in vitro and in vivo antifungal potency in combination with fluconazole against azole-resistant C. glabrata. It also had direct antifungal effect to treat C. glabrata infection, suggesting new mechanisms of action independent of Pdr1-KIX inhibition. Therefore, compound B8 represents a promising lead compound for antifungal drug development.

Controlling antifungal activity with light: Optical regulation of fungal ergosterol biosynthetic pathway with photo-responsive CYP51 inhibitors.

Invasive fungal infections (IFIs) have been associated with high mortality, highlighting the urgent need for developing novel antifungal strategies. Herein the first light-responsive antifungal agents were designed by optical control of fungal ergosterol biosynthesis pathway with photocaged triazole lanosterol 14α-demethylase (CYP51) inhibitors. The photocaged triazoles completely shielded the CYP51 inhibition. The content of ergosterol in fungi before photoactivation and after photoactivation was 4.4% and 83.7%, respectively. Importantly, the shielded antifungal activity (MIC80 ≥ 64 µg/mL) could be efficiently recovered (MIC80 = 0.5-8 µg/mL) by light irradiation. The new chemical tools enable optical control of fungal growth arrest, morphological conversion and biofilm formation. The ability for high-precision antifungal treatment was validated by in vivo models. The light-activated compound A1 was comparable to fluconazole in prolonging survival in Galleria mellonella larvae with a median survival of 14 days and reducing fungal burden in the mouse skin infection model. Overall, this study paves the way for precise regulation of antifungal therapy with improved efficacy and safety.

Target- and prodrug-based design for fungal diseases and cancer-associated fungal infections.

Invasive fungal infections (IFIs) are emerging as a serious threat to public health and are associated with high incidence and mortality. IFIs also represent a frequent complication in patients with cancer who are undergoing chemotherapy. However, effective and safe antifungal agents remain limited, and the development of severe drug resistance further undermines the efficacy of antifungal therapy. Therefore, there is an urgent need for novel antifungal agents to treat life-threatening fungal diseases, especially those with new mode of action, favorable pharmacokinetic profiles, and anti-resistance activity. In this review, we summarize new antifungal targets and target-based inhibitor design, with a focus on their antifungal activity, selectivity, and mechanism. We also illustrate the prodrug design strategy used to improve the physicochemical and pharmacokinetic profiles of antifungal agents. Dual-targeting antifungal agents offer a new strategy for the treatment of resistant infections and cancer-associated fungal infections.

Discovery of BRD4-HDAC Dual Inhibitors with Improved Fungal Selectivity and Potent Synergistic Antifungal Activity against Fluconazole-Resistant Candida albicans.

Over the past several decades, invasive fungal infections, especially candidiasis, have caused dramatic morbidity and mortality due to ineffective antifungal drugs and severe drug resistance. Herein, new BRD4-histone deacetylase (HDAC) inhibitors were designed to restore the susceptibility of Candida albicans (C. albicans) to fluconazole (FLC). Interestingly, several compounds showed excellent selectivity against fungal HDACs. In particular, compound B2 showed excellent synergistic effect with FLC against resistant C. albicans (FICI = 0.063) with high selectivity against fungal HDACs (SI = 1653) and low cytotoxicity. Compound B2 effectively synergized with FLC and prevented biofilm formation and morphological transition in resistant C. albicans, potentiating the antifungal activity of FLC in vivo and significantly reducing kidney fungal loads. Thus, this drug combination is promising in the treatment of resistant C. albicans infections.

Discovery of a new chemical scaffold for the treatment of superbug Candida auris infections.

Candida auris has emerged as a serious threat of public health and caused global epidemic due to multi-drug resistance, remarkable transmissibility and high mortality. To tackle the challenging super fungus, novel benzoanilide antifungal agents were discovered by an integrated strategy of phenotypic screen, hit optimization, antifungal assays and mechanism exploration. The most promising compound A1 showed potent in vitro and in vivo efficacy against Candida auris infection. Mechanism investigation revealed that compound A1 blocked the biosynthesis of virulence factors and fungal cell walls through the inhibition of glycosylphosphatidylinositol (GPI) and GPI-anchored proteins. Thus, compound A1 represents a promising lead compound to combat drug-resistant candidiasis.

Small molecules for combating multidrug-resistant superbug Candida auris infections.

Candida auris is emerging as a major global threat to human health. C. auris infections are associated with high mortality due to intrinsic multi-drug resistance. Currently, therapeutic options for the treatment of C. auris infections are rather limited. We aim to provide a comprehensive review of current strategies, drug candidates, and lead compounds in the discovery and development of novel therapeutic agents against C. auris. The drug resistance profiles and mechanisms are briefly summarized. The structures and activities of clinical candidates, drug combinations, antifungal chemosensitizers, repositioned drugs, new targets, and new types of compounds will be illustrated in detail, and perspectives for guiding future research will be provided. We hope that this review will be helpful to prompting the drug development process to combat this fungal pathogen.

Jumonji Histone Demethylase Inhibitor JIB-04 as a Broad-Spectrum Antifungal Agent.

Invasive fungal infections are emerging as a global public health problem. The lack of effective antifungal drugs is the bottleneck of clinical antifungal treatment. To identify novel antifungal agents with new mechanisms of action, JIB-04, a Jumonji histone demethylase inhibitor, was identified to possess broad-spectrum antifungal activity by a cell-based screen. Particularly, JIB-04 effectively inhibited Jumonji demethylase activity and ergosterol biosynthesis of Cryptococcus neoformans cells, leading to in vitro and in vivo anti-Cryptococcus activity. It also significantly inhibited the virulence factors of C. neoformans including biofilm, melanin, capsule, and surface hydrophobicity. Thus, JIB-04 was validated as a potent antifungal agent for the treatment of cryptococcal meningitis and Jumonji histone demethylase was preliminarily identified as a potential target for the development of novel antifungal therapeutics.

Role of Azole Drugs in Promoting Fungal Cell Autophagy Revealed by an NIR Fluorescence-Based Theranostic Probe.

Autophagy, a widespread degradation system in eukaryotes, plays an important role in maintaining the homeostasis of the cellular environment and the recycling of substances. Optical probes for the tracking of autophagy can be used as an effective tool not only to visualize the autophagy process but also to study autophagy-targeted drugs. Various molecule probes for autophagy of cancer cells emerge but are very limited for that of fungal cells, resulting in the lack of research on antifungal drugs targeting autophagy. To address this issue, we report an azole NIR fluorescence-based theranostic probe AF-1 with antifungal activity that is sensitive to autophagy-associated pH. The unique design of this probe lies in the introduction of both the pH-sensitive fluorophore with a detection range matching the pH range of the autophagy process and the conserved core structural fragment of azole drugs, providing a strategy to investigate the relationship between antifungal drug action and autophagy. As such, AF-1 exhibited excellent spectral properties and was found to target and induce the autophagy of the fungal cell membrane while maintaining moderate antifungal activity. Of note, using this theranostic probe as both a dye and drug, the autophagy process of fungi was visualized in a ratiometric manner, revealing the role of azole antifungal drugs in promoting autophagy to induce fungal cell apoptosis.

Discovery of Novel Sertraline Derivatives as Potent Anti-Cryptococcus Agents.

Treatment of life-threatening cryptococcal meningitis (CM) is highly challenging due to the limited efficacy of the available antifungal drugs. Antidepressant sertraline (SER) has been proposed to be a potential antifungal agent for CM. However, clinical studies indicated that SER failed to achieve the expected therapeutic effects. Herein, novel SER derivatives were designed by scaffold hopping, and they showed improved anticryptococcal activity both in vitro and in vivo. In particular, compound D16 was identified as a promising anti-CM agent with a new antifungal mode of action. It acted by blocking the biosynthesis of ergosterol through the inhibition of Δ5,6-desaturase. This study provides a new target and a drug-like candidate for CM treatment.

Discovery of Piperidol Derivatives for Combinational Treatment of Azole-Resistant Candidiasis.

Effective strategies are needed to deal with invasive fungal infections caused by drug-resistant fungi. Previously, we designed a series of antifungal benzocyclane derivatives based on the drug repurposing of haloperidol. Herein, further structural optimization and antifungal mechanism studies were performed, leading to the discovery of new piperidol derivative B2 with improved synergistic antifungal potency, selectivity, and water solubility. In particular, the combination of compound B2 and fluconazole showed potent in vitro and in vivo antifungal activity against azole-resistant Candida albicans. Compound B2 inhibited important virulence factors by regulating virulence-associated genes and improved the efficacy of fluconazole by down-regulating the CYP51-coding gene and efflux pump gene. Taken together, the piperidol derivative B2 represents a promising lead compound for the combinational treatment of azole-resistant candidiasis.

[Status and further development of regulation for pesticide registration for Chinese medicinal materials].

Nowadays, the cultivation of traditional Chinese medicinal materials is faced with the problems of pesticides illegal uses and pesticides residues. In the early period of eco-agriculture of Chinese materia medica, the uses of pesticides in special time is still inevitable. However, the registration of pesticides for Chinese medicinal materials now is far from enough to meet demand. So it is necessary to publish policies to expand the pesticides registration for Chinese medicinal materials. We collected and analyzed the data of pesticide registration for Chinese medicinal materials, the results showed that till March 2018, there were 10 kinds of Chinese medicinal materials, 91 pesticide products and 46 kinds of pesticide ingredients registered. Based on the current status of the pesticide management in China and the experience of the registration pesticide for minor crops in countries around the world, we point out that the pesticide registration for Chinese medicinal materials should be carried out in the form of minor crops and some specific methods are also provided in this paper.

Bioaccessibility and risk assessment of heavy metals, and analysis of arsenic speciation in Cordyceps sinensis.

BACKGROUND: Cordyceps sinensis (C. sinensis) is a famous and precious Traditional Chinese Medicine (TCM), while frequent reports of heavy metals, especially arsenic, exceeding standards in C. sinensis in recent years have raised concerns of its safety. Therefore, it is urgent for a research on heavy metals (Cu, Pb, As, Cd, Hg) in C. sinensis, of its bioaccessibility, dietary exposure estimation, arsenic speciation analysis and health risks assessment to human body. METHODS: Three 30 g batches of mixed wild growth C. sinensis samples were collected from Qinghai Province and each batch were divided into three parts: the whole C. sinensis, the stroma and the caterpillar body. The in vitro gastrointestinal method was used to evaluate the bioaccessibility of the heavy metals in the samples. The arsenic speciation analysis in the in vitro gastrointestinal solutions and dilute nitric acid extracted solutions were conducted using high performance liquid chromatography-inductively coupled plasma mass (HPLC-ICP-MS) method. Finally, the target hazard quotient (THQ) developed by the US EPA (1989) was used to assess the health risks of heavy metals in C. sinensis. RESULTS: The contents of Cu, Pb, Cd and Hg in the stroma were higher than those in the caterpillar body. In contrast, As was mainly found in the caterpillar body. In the whole C. sinensis, the average bio-accessibilities of Cu, Pb, As, Hg and Cd were 41.29, 40.11, 64.46, 18.91, and 81.14%, respectively. While in the caterpillar body, the corresponding bio-accessibilities values were 48.26, 42.92, 66.15, 12.86, 87.07%, respectively, and were 38.30, 30.53, 30.18, 7.46, and 82.30%, respectively in the stroma part. Different arsenic speciations of arsenite [As(III)], arsenate [As(V)] and trace amounts of methylarsonic acid [MMA] were detected. Of the total As, 8.69% was in inorganic form, which was also the major form of dissolved As. Among the extracted inorganic species, the concentrations of As(III) and As(V) were 0.56 ± 0.16 and 0.29 ± 0.06 mg kg-1, respectively. In the gastrointestinal solutions, only As(III) and As(V) could be detected; the sum content of the two species was 2.00-2.73%. The bioaccessibility target hazard quotient (BTHQ) values for Cu, Pb, As, Cd and Hg in C. sinensis were 0.0041, 0.0040, 0.5334, 0.0020 and 0.0005, respectively, all less than 1. CONCLUSION: None of the five heavy metals in C. sinensis can be 100% absorbed by human body. The content of arsenic in C. sinensis is high, but the strong toxic inorganic arsenic accounted for only 8.69%. The heavy metals in C. sinensis presented no obvious risks to human health in a reasonable taking way.

Elucidation of Characteristic Sulfur-Fumigated Markers and Chemical Transformation Mechanism for Quality Control of Achyranthes bidentate Blume Using Metabolome and Sulfur Dioxide Residue Analysis.

Achyranthes bidentata Blume (AB) is a health food and a sulfur-free herbal medicine that is one of the most heavily sulfur-fumigated herbs in the marketplace. In this work, a comprehensive approach using ultra-performance liquid chromatography coupled with quadrupole time-of-flight-MS (UPLC-Q-TOF-MS) and multivariate statistical analysis was developed to identify characteristic sulfur-fumigation markers, elucidate chemical transformation mechanisms and characterize the degree of sulfur-fumigation of AB. Non-fumigated and sulfur-fumigated AB samples were compared by UPLC-Q-TOF-MS/MS analysis. Three triterpene saponins (Betavulgarosides II-IV) and two amides (Feruloyl-4-O-methyldopamine and Moupinamide) were identified as characteristic markers, which were positively correlated with two active AB components, namely oleanic acid and ferulic acid, respectively. Moreover, the extent of the sulfur-fumigation under different weight ratios of sulfur to herbal materials (1:20, 1:40, and 1:80) was analyzed based on chemical transformations and sulfur dioxide residues. Further verification showed that the ratio of 1:40 within 1 h was reasonable and efficient for herb quality preservation and assurance. This study provides a reliable sulfur-fumigation protocol for the quality control of AB and other herbs.

[Assessment of soil fertility for cultivation of Chinese herbal medicines].

The soil fertility has great impacts on the yielding of Chinese medicinal materials, as well as the generation of major components. The practices showed that soil fertility has been decayed with the growth of cultivation years,which is leading to adverse effect on quality and quantity of Chinese medicinal materials. However, there was a lack of domestic unified standard for assessment of soil fertility of Chinese medicinal material cultivation, which has seriously limited the soil management and quality control. In this text, we reviewed the progress on research of soil fertility evaluation, built the soil fertility evaluation index including soil texture,soil bulk density,soil organic matter,soil acidity and alkalinity,soil available nitrogen,soil available phosphorus, soil available potassium,soil animals and soil microorganisms. It would provide the direction and thought for standard-settin on soil fertility evaluation of Chinese medicinal material cultivation.

[Sulfur dioxide limit standard and residues in Chinese medicinal materials].

The traditional sulfur fumigation processing method has been widely used in the initial processing and storage of traditional Chinese medicinal materials due to its economy, efficiency, convenience, high operability and effect on mold and insect prevention. However, excessive sulfur fumigation of traditional Chinese medicinal materials would lead to the changes in chemical compositions, and even endanger human health. This study showed that traditional Chinese medicinal materials were sulfur fumigated directly after being harvested for quick drying, or fumigated after being weted in the storage process for preventing mold and insects. We found that the sulfur dioxide limits for traditional Chinese medicinal materials were stricter than those for foods. Based on the existing limit standards, we obtained the data of sulfur dioxide residues for 35 types of traditional Chinese medicinal materials in a total of 862 batches. According to the limit standard in the Chinese Pharmacopoeia (150， 400 mg·kg⁻¹), the average over-standard rate of sulfur dioxide was as high as 52.43%, but it was reduced to 29.47% if calculated based on the limit for vegetable additive standard (500 mg·kg⁻¹). Sulfur fumigation issue shall be considered correctly: sulfur dioxide is a type of low toxic substance and less dangerous than aflatoxin and other highly toxic substances, and a small amount of residue would not increase the toxicity of traditional Chinese medicinal materials. However, sulfur fumigation might change the content of chemical substances and affect the quality of traditional Chinese medicinal materials. Furthermore, the exposure hazards of toxic substances are comprehensively correlated with exposure cycle, exposure frequency, and application method. In conclusion, it is suggested to strengthen the studies on the limit standard of traditional Chinese medicinal materials, formulate practical and feasible limit standard for sulfur dioxide residues in traditional Chinese medicinal materials that are consistent with the medication characteristics of traditional Chinese medicinal materials and can guarantee people's demand for safe medication.

[Quantitative analysis and stability of p-hydroxybenzyl hydrogen sulfite in sulfur-fumigated Gastrodiae Rhizoma].

Studies on the characteristic chemical markers of sulfur fumigation have become an effective auxiliary way for quality control of traditional Chinese medicine. This study established a quantitative analysis method for the determination of p-hydroxybenzyl hydrogen sulfite (p-HS) in Gastrodiae Rhizoma (GR) based on UPLC-MS/MS. Then, 37 batches of GR were screened, and the results showed that 27 batches of them were sulfur-fumigated. Especially, the GR samples in Yunnan producing areas were sulfur-fumigated more seriously. Based on the stability of p-HS after different storage time and heat treatment methods, it was found that the content of p-HS was stable and reliable in the storage period of 8 months and under normal heat treatment (50, 60, 70, 80 °C) conditions. In conclusion, this study shows a high sensitivity, good selectivity and good stability of p-HS, which can provide reference for the quality control and sulfur fumigation screening of GR.

[Changes of chemical compositions and sulfur dioxide residues of Gastrodiae Rhizoma with different storage times].

To study the effect of different storage time on the chemical compositions and sulfur dioxide residues of sulfur-fumigated Gastrodiae Rhizoma (GR), and provide scientific basis for solving the quality and safety issues of sulfur-fumigated traditional Chinese medicinal materials. GR, sulfur-fumigated GR and its medicinal slices were stored under the same conditions, and then 8 active ingredients and sulfur dioxide residues were measured respectively. The results showed that the content of gastrodins in sulfur-fumigated GR and its medicinal slices was significantly lower than that in the non-fumigated GR. Moreover, the content of sulfur dioxide residue in sulfur-fumigated GR was significantly higher than that in its medicinal slices. That is to say, sulfur fumigation degree had significantly higher effect on GR quality as compared with its medicinal slices. During the whole storage time (8 months), the content of the eight chemical components in GR was not changed greatly in general. However, after the storage for 4 months, the content of 8 components and sulfur dioxide residues in all of GR samples were significantly changed. In particular, the content of sulfur dioxide residue in GR medicinal materials decreased up to 50% or more.

[Quality changes in Gastrodia Rhizoma of different origins and forms before and after sulfur fumigation].

As Gastrodiae Rhizoma (GR) is one of the herbs more seriously affected by sulfur fumigation, so its quality has been always of a great concern. In this paper, GR samples collected from eight main producing areas and in three forms were fumigated with sulfur and quantitatively and qualitatively analyzed based on UPLC-Q-TOF-MS/MS. The results showed that the contents of gastrodin, parishin, parishin B and parishin C were decreased, while the content of parishin E was increased after sulfur fumigation treatment. Besides, a new sulfur marker named p-hydroxybenzyl hydrogen sulfite was produced in sulfur-fumigated GR samples. As compared with producing origins, forms had a greater impact on the quality of GR, especially in Hongtianma and Wutianma. Besides, the contents of gastrodins and parishins in Hongtianma from Jilin were lowest as compared with those in other producing areas. This might be correlated with planting patterns and environmental factors. In conclusion, sulfur fumigation has a more obvious impact on the quality of GR than origins and forms, which is attributed to the generation of new sulfur fumigated markers.

[Study on distribution of five heavy metal elements in different parts of Cordyceps sinensis by microwave digestion ICP-MS].

The contents of five heavy metals (Cu, Pb, As, Cd, Hg) in 17 batches of Cordyceps sinensis were determined by microwave digestion-ICP-MS, and their distribution in C. sinensis were analyzed. The results showed that the contents of Cu, Pb, Cd and Hg in all batches were in accordance with the international standards of Chinese Medicine-Chinese Herbal Medicine Heavy Metal Limit, with their contents in the stroma higher than that in the caterpillar body, and the excess rate of As, which mainly concentrated in the caterpillar body part of C. sinensis, was 88.24%, as the content of As in the caterpillar body was 7 to 12 fold of that in the stroma. In this study, the distribution of five heavy metals in C. sinensis was clarified, and the existing problems of arsenic limit of heavy metal in C. sinensis were analyzed, and some suggestions were put forward. It is hoped that the reference standard can be provided for the limited standard of arsenic in C. sinensis.