Chitin Synthase Inhibitor Research Articles

Design, synthesis, and biological evaluation of novel spiro[pyrrolidine-2,3′-quinolin]-2′-one derivatives as potential chitin synthase inhibitors and antifungal agents

A series of novel spiro-quinolinone derivatives were designed and synthesized and their structures were confirmed by spectroscopic methods. The enzymatic experiments showed that all the seventeen synthesized compounds had inhibition potency against chitin synthase, among them five compounds had excellent inhibition potency that equal to that of polyoxin B. The Kinetic parameters of enzymatic assays indicated that these compounds were non-competitive inhibitors of chitin synthase. The antimicrobial experiments displayed that the synthesized compounds had selectively and broad-spectrum antifungal activity in vitro Among them, two compounds had stronger antifungal activity against C. albicans than that of fluconazole meanwhile five others compounds showed antifungal activity against C. albicans being equal to that of fluconazole. Moreover, there are four or five compounds that possessed antifungal activities against C. neoformans, A. fumigatus and A. flavus as high as fluconazole had, respectively. The sorbitol protection assay and evaluation of antifungal activity against micafungin-resistant strain further verified that these compounds possessed antifungal activity through inhibiting the synthesis of chitin of cell wall. The evaluation of antifungal activity against others drug-resistant fungi variants showed these designed compounds had significant antifungal activity against these tested variants. The combination use experiments exhibited that the synthesized compounds had synergistic effects or additive effects with current used drugs in clinic. These results demonstrated that these synthesized compounds were chitin synthase inhibitors and had selective and broad-spectra antifungal activities.

Intra-hemocoel injection of pseurotin A from Metarhizium anisopliae, induces dose-dependent reversible paralysis in the Greater Wax Moth (Galleria mellonella)

Species from the Metarhizium genus are the causal agents of the green muscardine disease of insects. These fungi have been successfully employed for the biological control of pests over decades. Besides the biocontrol applications, recent efforts for genome sequencing of species in this genus have revealed a great diversity of biosynthetic gene clusters potentially associated with secondary metabolite synthesis. Amongst such molecules are the pseurotins, compounds with several activities, as chitin synthase inhibitors, and immunoglobulin E suppressors. Here, we report, for the first time, the isolation of pseurotin A from the culture broth of M. anisopliae, as well as the characterization of the effects of this compound over the model-arthropod Galleria mellonella. Pseurotin A displayed dose-dependent reversible paralysis effects when injected into the larvae hemocoel. However, the posterior challenge of the treated insects with M. anisopliae conidia did not lead to increased mortality, suggesting that pseurotin A treatment did not increase larvae susceptibility to the green muscardine disease. Although apparent insecticidal effects were not observed for pseurotin A, the paralysis effect observed can be important in M. anisopliae infection development.

Efficacy of nikkomycin Z in murine CNS coccidioidomycosis: modelling sustained-release dosing.

Meningitis is the most feared coccidioidomycosis complication. Nikkomycin Z (nikZ) is a chitin synthase inhibitor. A concern is short half-life, necessitating multiple dose/day regimens. We simulated extended release, providing nikZ in drinking water. Extended release would enhance convenience, and adherence, for patients. Coccidioides posadasii was injected intracerebrally into mice. Twelve day treatments began on Day 3. Fluconazole was given 100 mg/kg once daily (gavage); designed doses of nikZ 30, 100 or 300 mg/kg/day in drinking water. On Day 30 post-treatment, survivors were euthanized, brain cfu quantitated and cfu in other organs assessed. nikZ was stable in drinking water. Survival was 11%, 50%, 70%, 90% and 100% in untreated controls, fluconazole and nikZ 30, 100 and 300 mg/kg/day, respectively ; nikZ 300 mg/kg/day was superior (P ≤ 0.01) to fluconazole. Brains were sterilized in 0%, 20%, 86%, 89% and 80% of mice, respectively; nikZ 100 or 300 mg/kg/day was superior (P ≤ 0.01) to fluconazole. Clearance of infection in other organs was similar. All decreased drinking after infection, causing nikZ mice to ingest less than the desired dose in early therapy; despite this, they recovered sufficiently to resume pre-infection drinking and designed drug intakes. Thus, when sickest, even less than the designed dose was sufficient to enable recovery. This efficacy supports the development of sustained-release nikZ. Decreased intake wouldn't be a factor in humans, receiving drug via extended-release pill or continuous IV infusion. In prior studies (twice daily nikZ) of murine coccidioidal meningitis, results were inferior, suggesting sustained release may provide both convenience and superior outcomes.

Nikkomycin Z against Disseminated Coccidioidomycosis in a Murine Model of Sustained-Release Dosing.

Nikkomycin Z (nikZ) is a chitin synthase inhibitor. Efficacy against Coccidioides has been demonstrated in animal models of pulmonary or brain infection. Its short half-life in mice and in humans would necessitate divided daily dosing. We assayed nikZ efficacy in disseminated coccidioidomycosis (in a reduction of CFU design) and whether sustained release might be useful. Mice were challenged intravenously with low or high arthroconidial inocula. Fluconazole, clinically the most commonly used anticoccidioidal drug, was compared (gavage) at high dose to a dose range of nikZ administered intraperitoneally or, to mimic sustained release, administered continuously in drinking water. Therapy was given for 5 days. In vitro, both fluconazole and nikZ inhibited the isolate studied; nikZ was fungicidal. Oral nikZ therapy gave similar results to intraperitoneal nikZ and sterilized infection in most animals after low-inoculum challenge. In both challenges, oral nikZ produced greater reduction of CFU in organs (lung, liver, and spleen) than fluconazole. Oral nikZ doses of ≥200 mg/kg of body weight/day were particularly effective in all organs and were well tolerated. This efficacy occurred even though, after severe challenge, mice had reduced water intake, resulting in ingesting less than the desired dose, particularly initially after infection. This study shows, for the first time, efficacy of nikZ against disseminated coccidioidomycosis. Efficacy was shown after challenges producing different levels of severity of disease. This study also suggests the likely benefits of developing an extended release formulation supplying continuous systemic concentrations of nikZ.

AOP Report: Inhibition of Chitin Synthase 1 Leading to Increased Mortality in Arthropods.

AOP Report: Inhibition of Chitin Synthase 1 Leading to Increased Mortality in Arthropods.

Design, synthesis and biological evaluation of novel diazaspirodecanone derivatives containing piperidine-4-carboxamide as chitin synthase inhibitors and antifungal agents

A series of novel 2-oxo-(1-oxo-2,8-diazaspiro[4.5]decane-8-yl)ethylpiperidine carboxamide derivatives were designed, synthesized and characterized by 1H NMR, 13C NMR and HRMS spectroscopy. All eighteen newly prepared compounds were evaluated for their inhibition against chitin synthase (CHS) and antifungal activities in vitro. The enzyme assay revealed that compound 5h showed excellent inhibitory activity against CHS with IC50 value of 0.10 mM, and the compounds 5b, 5d and 5q showed good inhibition against chitin synthase with IC50 values of 0.13 mM, 0.18 mM and 0.15 mM, respectively, while IC50 value of ployoxin B was 0.08 mM. Meanwhile, the others of these compounds exhibited moderate inhibition potency against chitin synthase. The antifungal assay showed compound 5h had excellent antifungal activity compared with the control drugs fluconazole and polyoxin B against these tested strains including C. albicans, A. fumigatus, C. neoformans and A. flavus. Its excellent antifungal activity was consistent with its excellent chitin synthase inhibition. Compound 5k and 5l against C. albicans were comparable with fluconazole, and they showed strong antifungal potency against A. flavus with MIC values of 0.07 mmol/L and 0.13 mmol/L respectively. Compound 5m had similar MIC value against A. fumigatus to fluconazole. The phenomenon that compounds 5b, 5d and 5q that showed good enzymatic inhibition didn’t exert good antifungal activity, while compounds 5k, 5l and 5m that showed moderate chitin synthase inhibition exhibited excellent antifungal activity was discussed. Furthermore, the trial of drug combination showed that compounds had synergistic effects or additive effects with fluconazole against tested fungi which also verified that these designed compounds targeted different targets from that of fluconazole. Additionally, the antibacterial trial showed that all synthesized compounds had little potency against tested bacteria strains. These results indicated that the designed compounds were potential chitin synthase inhibitors and had selectively antifungal activities.

In silico Binding Profile Analysis and In vitro Investigation on Chitin Synthase Substrate and Inhibitors from Maize Stem Borer, Chilo partellus.

Insect growth and metamorphosis are strictly dependent on the structural changes that occur in chitin containing tissues and organs. Chitin synthase catalyzes chitin polymerization by β-(1, 4) glycosidic linkage of N-acetyl-D-glucosamine (GlcNAc) monomers; the major component of insect cuticles. Targeting this enzyme could be a promising strategy to control insect pests while avoiding adverse effects on coexisting populations. Nikkomycin Z and polyoxins are commercially available fungal inhibitors known to bind to the nucleotide-binding sites of insects and fungal chitin synthase. But the binding mode of chitin synthase has not been explored to date as its structure is not available yet. To understand the structural features of the Chilo partellus chitin synthase enzyme (CpCHS), the three-dimensional (3D) structure of the CpCHS catalytic domain was modeled using ROBETTA webserver. The obtained model was used to investigate the binding mode of its substrate, uridine diphosphate-N-acetyl-D-glucosamine (UDP-GlcNAc), and inhibitors (nikkomycin Z and polyoxins) by molecular docking approach using Schrödinger Suite-Maestro v9.2. The docked complexes were further investigated for their interaction stability by performing molecular dynamics (MD) simulations using GROMACS v5.1.2. Our study highlighted the significance of various interactions made by CHS residues present in the Walker-B loop and donor-binding motifs with the substrate (UDP-GlcNAc), and GEDR motif with an acceptor (GlcNAc). Also, the interactions of the QRRRW motif while forming chitin polymer were explored. We observed that the inhibitors exhibited good binding affinity with these motifs, indicated by their docking and binding affinity scores. In vitro analysis suggested that nikkomycin Z showed higher inhibition of chitin synthase activity at a concentration of 2.5 μg.L-1. Our study provided insights into the crucial interactions of chitin synthase while designing inhibitors against insect pests.

Screening and Application of Chitin Synthase Inhibitors

Chitin is an important part of the fungal cell wall, but is not found in plants and mammals, so chitin synthase (CHS) can be a green fungicide target. In this paper, 35 maleimide compounds were designed and synthesized as CHS inhibitors. All the screened compounds showed different degrees of CHS inhibitory activity and antifungal activity in vitro. In particular, the half–inhibitory concentration (IC50) value of compound 20 on CHS was 0.12 mM, and the inhibitory effect was better than that of the control polyoxin B (IC50 = 0.19 mM). At the same time, this compound also showed good antifungal activity and has further development value.

Design, synthesis and biological evaluation of novel 3,4-dihydro-2(1H)-quinolinone derivatives as potential chitin synthase inhibitors and antifungal agents

A series of 3,4-dihydro-2(1H)-quinolinone derivatives contained butenediamide fragment were designed and synthesized. Their inhibition potency against chitin synthase and antimicrobial activities were screened in vitro. The enzymatic assays showed that all the synthesized compounds had inhibition potency against chitin synthase at concentration of 300 μg/mL. Compound 2d displayed excellent potency with inhibition percentage (IP) value of 82.3%, while IP value of the control polyoxin B was 87.5%. Compounds 2b, 2e and 2s whose IP values were above 70% showed good inhibition potency against chitin synthase. Moreover, the IC50 value of 2b was comparable with that of polyoxin B (0.09 mM). The Ki of compound 2b was 0.12 mM and the result from Lineweaver-Burk plot showed that 2b was non-competitive inhibitor to bind chitin synthase. The antifungal experiment showed that these compounds had excellent antifungal activity against fungal strains, especially for candida albicans. The antifungal activities against C .albicans of compounds 2b, 2d, 2e and 2l were comparable with that of fluconazole and were superior to that of polyoxin B. Meanwhile, the other compounds against C. albicans showed better antifungal activity (MIC 2 μg/mL) than polyoxin B except for compound 2n (MIC 4 μg/mL). The trial of drug combination use showed that these synthesized compounds had synergistic effects with fluconazole and polyoxin B. It indicated that these compounds were not competing with polyoxin B to bind with chitin synthase, which was also consistence with the result of enzymatic assays. The antibacterial experiment showed that these compounds had no activity against selected strains including three Gram-positive and three Gram-negative bacteria. These results showed that the designed compounds were chitin synthase inhibitors and had selective antifungal activity.

Metabolic mechanisms of resistance to spirodiclofen and spiromesifen in Iranian populations of Panonychus ulmi

The European red mite, Panonychus ulmi (Koch), is one of the major pests of apple trees worldwide. Cyclic keto-enol compounds such as spirodiclofen and spiromesifen are frequently used to control phytophagous spider mites in agricultural crops, including P. ulmi on apple trees. Spider mites, however, can rapidly develop resistance against acaricides and, in this study, multiple P. ulmi populations from apple orchards in Iran were monitored for spirodiclofen and spiromesifen resistance. The Urmia and Shahin Dej population showed the highest spirodiclofen resistance ratio (more than 150-fold) compared to the susceptible Ahar population. Toxicity bioassays also revealed the presence of moderate cross-resistance between spiromesifen and spirodiclofen, but not towards the chitin synthase inhibitor etoxazole. As a first step towards elucidating spirodiclofen resistance mechanisms, the role of detoxification enzymes (cytochrome P450 monooxygenases, carboxyl/choline esterases and glutathione S-transferases) was investigated by in vivo synergism and in vitro enzyme assays. PBO pretreatment synergized spirodiclofen toxicity in the populations of Urmia and Shahin Dej to a higher extent than in the susceptible Ahar population. Furthermore, enzyme activity measurements showed relatively higher activity of detoxifying enzymes in the resistant populations. In conclusion, increased detoxification is most likely underlying spirodiclofen resistance and results in limited cross-resistance to spiromesifen.

Potent Chitin Synthase Inhibitors from Plants

Background:Chitin is the main component of fungal, protozoan and helminth cell wall. They help to maintain the structural and functional characteristics of these organisms. The chitin wall is dynamic and is repaired, rearranged and synthesized as the cells develop. Active synthesis can be noticed during cytokinesis, laying of primary septum, maintenance of lateral cell wall integrity and hyphal tip growth. Chitin synthesis involves coordinated action of two enzymes namely, chitin synthase (that lays new cell wall) and chitinase (that removes the older ones). Since chitin synthase is conserved in different eukaryotic microorganisms that can be a ‘soft target’ for inhibition with small molecules. When chitin synthase is inhibited, it leads to the loss of viability of cells owing to the self- disruption of the cell wall by existing chitinase.Methods:In the described study, small molecules from plant sources were screened for their ability to interfere with hyphal tip growth, by employing Hyphal Tip Burst assay (HTB). Aspergillus niger was used as the model organism. The specific role of these small molecules in interfering with chitin synthesis was established with an in-vitro method. The enzyme required was isolated from Aspergillus niger and its activity was deduced through a novel method involving non-radioactively labelled substrate. The activity of the potential lead molecules were also checked against Candida albicans and Caenorhabditis elegans. The latter was adopted as a surrogate for the pathogenic helminths as it shares similarity with regard to cell wall structure and biochemistry. Moreover, it is widely studied and the methodologies are well established.Results:Out of the 11 compounds and extracts screened, 8 were found to be prospective. They were also found to be effective against Candida albicans and Caenorhabditis elegans.Conclusion:Purified Methyl Ethyl Ketone (MEK) Fraction1 (F1) of Coconut (Cocos nucifera) Shell Extract (COSE) was found to be more effective against Candida albicans with an IC50 value of 3.04 μg/mL and on L4 stage of Caenorhabditis elegans with an IC50 of 77.8 μg/mL.

Design, synthesis and biological evaluation of novel diazaspiro[4.5]decan-1-one derivatives as potential chitin synthase inhibitors and antifungal agents

A series of 2,8-diazaspiro[4.5]decan-1-one derivatives were designed, synthesized and screened for their inhibition activities against chitin synthase (CHS) and antimicrobial activities in vitro. The biological assays revealed that compounds 4a, 4e, 4h, 4j, 4o, 4q and 4r exhibited moderated to excellent potency against CHS with IC50 values ranging from 0.12 to 0.29 mM. Compounds 4e, 4j with IC50 value of 0.13 mM, 0.12 mM respectively, showed excellent inhibition potency among these compounds, which were similar to that of polyoxin B whose IC50 value was 0.08 mM. Meanwhile, the screening of the antifungal activity showed that compounds 4j and 4r had the same potency of inhibiting the growth of A. fumigatus with MIC value of 0.08 mmol/L. Compound 4d displayed excellent activity against C. albicans (ATCC 90023) with MIC value of 0.04 mmol/L, which was superior to fluconazole (0.104 mmol/L) and polyoxin B (0.129 mmol/L). The result of antibacterial assay showed that these compounds had little potency against those selected bacteria strains including three Gram-positive bacteria and three Gram-negative bacteria. Furthermore, the combination use of 4c-fluconazole, 4i-fluconazole, 4j-fluconazole, and 4o-fluconazole against C. albicans,A. fumigatus and A. flavus showed additive or synergistic effects. These results indicated that the designed compounds serve as potential chitin synthase inhibitors and have selectively antifungal activities.

Design, synthesis and biological evaluation of novel 5-(piperazin-1-yl)quinolin-2(1H)-one derivatives as potential chitin synthase inhibitors and antifungal agents

A series of 5-(4-substituted piperazin-1-yl)quinolin-2(1H)-one derivatives (4a-4w) has been designed as chitin synthase inhibitors and antifungal agents. The designed compounds were obtained by an environmentally benign route in four steps starting from 5-amino-3,4-dihydroquinolin-2(1H)-one which was offered by an easily achieved synthetic method. The synthesized compounds were tested for their inhibition potency against chitin synthase. Compounds 4a and 4c exhibited excellent inhibitory activity with IC50 values of 0.10 mM and 0.15 mM, respectively, which is better than that of Polyoxin B whose IC50 value is 0.18 mM. Compounds 4h, 4i, 4j, 4k and 4n exerted moderate inhibition potency with IC50 values of 0.38, 0.36, 0.47, 0.47 and 0.37 mM, respectively. These synthesized compounds were also evaluated for their in vitro antifungal activity against Candida albicans, Crytococcus neoformans, and Aspergillus flavus. Compounds 4a, 4i and 4j exhibited the most potent antifungal activity against C. albicans with MIC of 32 μg/mL, which were similar to that of Polyoxin B. The results of antibacterial activity against selected strains showed that the designed compounds have little potency against bacteria and indicated that these compounds were chitin synthase inhibitors and have selectively antifungal activity.

Diversity and evolution of chitin synthases in oomycetes (Straminipila: Oomycota)

The oomycetes are filamentous eukaryotic microorganisms, distinct from true fungi, many of which act as crop or fish pathogens that cause devastating losses in agriculture and aquaculture. Chitin is present in all true fungi, but it occurs in only small amounts in some Saprolegniomycetes and it is absent in Peronosporomycetes. However, the growth of several oomycetes is severely impacted by competitive chitin synthase (CHS) inhibitors. Here, we shed light on the diversity, evolution and function of oomycete CHS proteins. We show by phylogenetic analysis of 93 putative CHSs from 48 highly diverse oomycetes, including the early diverging Eurychasma dicksonii, that all available oomycete genomes contain at least one putative CHS gene. All gene products contain conserved CHS motifs essential for enzymatic activity and form two Peronosporomycete-specific and six Saprolegniale-specific clades. Proteins of all clades, except one, contain an N-terminal microtubule interacting and trafficking (MIT) domain as predicted by protein domain databases or manual analysis, which is supported by homology modelling and comparison of conserved structural features from sequence logos. We identified at least three groups of CHSs conserved among all oomycete lineages and used phylogenetic reconciliation analysis to infer the dynamic evolution of CHSs in oomycetes. The evolutionary aspects of CHS diversity in modern-day oomycetes are discussed. In addition, we observed hyphal tip rupture in Phytophthora infestans upon treatment with the CHS inhibitor nikkomycin Z. Combining data on phylogeny, gene expression, and response to CHS inhibitors, we propose the association of different CHS clades with certain developmental stages.

Design, synthesis and biological evaluation of novel quinazoline-2,4-diones conjugated with different amino acids as potential chitin synthase inhibitors

A series of (2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl) acetamido) acids) (6 a-m), (7) has been designed to inhibit the action of fungus chitin synthase enzyme (CHS). The synthesis of the designed compounds was carried out in four steps starting from the reaction between 1-methylquinazoline-2,4(1H,3H)-dione and ethyl chloroacetate to yield the ethyl acetate derivative. This ester was hydrolyzed to the corresponding carboxylic acid derivative that was then utilized to couple several amino acids getting the final designed compounds. The synthesized compounds were tested for their inhibition against CHS. Compound 7 showed the highest potency among others with minimum inhibitory concentration (IC50) of 0.166 mmol/L, while polyoxin B (the positive control) had IC50 of 0.17 mmol/L. The synthesized compounds were also evaluated for their in vitro antifungal activity using Aspergillus fumigates, Aspergillus flavus, Crytococcus neoformans and Candida albicans. Unfortunately, the 14 synthesized compounds showed lower in vitro activity compared to the used active controls. However, compound 6m and fluconazole have synergistic effect on Aspergillus flavus; Compounds 7 and fluconazole have synergistic effects on Aspergillus fumigates.

LYS12 LysM receptor decelerates Phytophthora palmivora disease progression in Lotus japonicus.

Phytophthora palmivora is a devastating oomycete plant pathogen. We found that P. palmivora induces disease in Lotus japonicus and used this interaction to identify cellular and molecular events in response to this oomycete, which has a broad host range. Transcript quantification revealed that Lys12 was highly and rapidly induced during P. palmivora infection. Mutants of Lys12 displayed accelerated disease progression, earlier plant death and a lower level of defence gene expression than the wild type, while the defence program after chitin, laminarin, oligogalacturonide or flg22 treatment and the root symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhiza were similar to the wild type. On the microbial side, we found that P. palmivora encodes an active chitin synthase-like protein, and mycelial growth is impaired after treatment with a chitin-synthase inhibitor. However, wheat germ agglutinin-detectable N-acetyl-glucosamine (GlcNAc) epitopes were not identified when the oomycete was grown invitro or while infecting the roots. This indicates that conventional GlcNAc-mers are unlikely to be produced and/or accumulate in P. palmivora cell walls and that LYS12 might perceive an unknown carbohydrate. The impact of Lys12 on progression of root rot disease, together with the finding that similar genes are present in other P. palmivora hosts, suggests that LYS12 might mediate a common early response to this pathogen.

The Gastrointestinal Tract Is a Major Source of Echinocandin Drug Resistance in a Murine Model of Candida glabrata Colonization and Systemic Dissemination.

ABSTRACTCandida species are a part of the human microbiome and can cause systemic infection upon immune suppression. Candida glabrata infections are increasing and have greater rates of antifungal resistance than other species. Here, we present a C. glabrata gastrointestinal (GI) colonization model to explore whether colonized yeast exposed to caspofungin, an echinocandin antifungal, develop characteristic resistance mutations and, upon immunosuppression, breakthrough causing systemic infection. Daily therapeutic dosing (5 mg/kg of body weight) of caspofungin resulted in no reduction in fecal burdens, organ breakthrough rates similar to control groups, and resistance rates (0 to 10%) similar to those reported clinically. Treatment with 20 mg/kg caspofungin initially reduced burdens, but a rebound following 5 to 9 days of treatment was accompanied by high levels of resistance (FKS1/FKS2 mutants). Although breakthrough rates decreased in this group, the same FKS mutants were recovered from organs. In an attempt to negate drug tolerance that is critical for resistance development, we cotreated mice with daily caspofungin and the chitin synthase inhibitor nikkomycin Z. The largest reduction (3 log) in GI burdens was obtained within 3 to 5 days of 20 mg/kg caspofungin plus nikkomycin treatment. Yet, echinocandin resistance, characterized by a novel Fks1-L630R substitution, was identified following 5 to 7 days of treatment. Therapeutic caspofungin plus nikkomycin treatment left GI burdens unchanged but significantly reduced organ breakthrough rates (20%; P < 0.05). Single-dose pharmacokinetics demonstrated low levels of drug penetration into the GI lumen posttreatment with caspofungin. Overall, we show that C. glabrata echinocandin resistance can arise within the GI tract and that resistant mutants can readily disseminate upon immunosuppression.

20 微生物の生産する新規キチン合成酵素阻害剤Guanofosfocin類の単離と構造決定(口頭発表の部)

20 微生物の生産する新規キチン合成酵素阻害剤Guanofosfocin類の単離と構造決定(口頭発表の部)

Design and synthesis of chitin synthase inhibitors as potent fungicides

Chitin is a structural component of fungal cell walls but is absent in vertebrates, mammals, and humans. Chitin synthase is thus an attractive molecular target for developing fungicides. Based on the structure of its donor substrate, UDP-N-acetyl-glucosamine, as well as the modelled structure of the bacterial chitin synthase NodC, we designed a novel scaffold which was then further optimized into a series of chitin synthase inhibitors. The most potent inhibitor, compound 13, exhibited high chitin synthase inhibitory activity with an IC50 value of 64.5μmol/L. All of the inhibitors exhibited antifungal activities against the growth of agriculturally-destructive fungi, Fusarium graminearum, Botrytis cinerea, and Colletotrichum lagenarium. This work presents a new scaffold which can be used for the development of novel fungicides.

The Gastrointestinal Tract Is a Major Source of Echinocandin Drug Resistance in a Candida glabrata Colonization Mouse Model

Background Gastrointestinal (GI) Candida commensals may be a major source of invasive candidiasis and a hidden reservoir of antifungal resistance. Candida glabrata resistance rates have increased greater than those of other species. Here, we present a C. glabrata GI colonization model to explore how antifungal drugs affect resistance acquisition and systemic breakthrough infections.Methods Immunocompetent mice were treated with antibiotics to clear native GI bacteria and then inoculated via oral gavage with C. glabrata. Fecal samples were collected throughout the study to assess fungal GI colonization. Daily administration of caspofungin (CSF; 5 or 20 mg/kg i.p.), chitin synthase inhibitor nikkomycin Z (Nz; 100 mg/kg oral), or saline was initiated on day 3 post inoculation. CSF-resistant colony frequencies were determined through selection of fecal samples on CSF-supplemented media, and FKS mutations were identified using the newly developed molecular beacon diagnostic assays. Dexamethasone was administered to induce immunosuppression. Upon completion of the experiment, blood, and organs were harvested and yeast burden levels determined.ResultsDaily therapeutic dosing (5 mg/kg) of CSF resulted in no reduction in fecal burdens, little resistance (0–10%), and organ breakthrough rates similar to control groups. Treatment with high dose (20 mg/kg) CSF caused a 2.5-log decrease in average burden, yet high levels (10/10 mice) of resistance (fks1/2 mutants) were observed following 5–9 days of treatment. Although breakthrough rates decreased in this group, yeast recovered from organs contained fks mutations. The largest reduction (3 log) in GI burdens was obtained within 3–5 days of high dose CSF plus Nz (100 mg/kg; oral) treatment. However, echinocandin resistance was again observed from all mice (10/10) following 5–7 days of treatment. Treatment with the therapeutic dose plus Nz left GI burdens unchanged, but did significantly reduce organ breakthrough rates (20%; P < 0.05).Conclusion We have developed a C. glabrata GI colonization and dissemination model. Systemic breakthrough depends on both gut C. glabrata population composition and serum/tissue drug level.Disclosures D. Perlin, Merck: Grant Investigator and Scientific Advisor, Research supportPfizer: Grant Investigator, Research support. Astellas: Consultant and Grant Investigator, Research support and Speaker honorarium. F2G: Consultant, Consulting fee