Cloning, functional characterization and screening of potential inhibitors for Chilo partellus chitin synthase A using in silico, in vitro and in vivo approaches.

Chitin synthase (CHS) is one of the crucial enzymes that play an essential role in chitin synthesis during the molting process, and it is considered to be the specific target to control insect pests. Currently, there are no potent inhibitors available in the market, which specifically target this enzyme. Pyrimidine nucleoside peptide, nikkomycin Z, binds to nucleotide-binding sites of fungal and insect CHS. But, their mode of action is still fragmentary due to the lack of a 3Dstructure of CHS. Chilo partellus is a severe pest insect of major food crops such as maize and sorghum, in an attempt to target integument expressed cuticular CpCHS. The CpChsA cDNA was cloned, and subsequently, their developmental and tissue-specific expression was studied. The 3D structure of the CHS catalytic domain was modeled, after which natural compounds were screened using a virtual screening workflow and resulted in the identification of five hit molecules. Molecular dynamics simulations were performed to investigate the dynamics and interactions of hits with CpCHS. The obtained results revealed that the compounds kasugamycin, rutin and robinin could act as potent inhibitors of CpCHS. All three molecules were observed to significantly reduce the chitin production as validated using in vitro and in vivo studies. Thus, this study aims to provide a set of novel inhibitor molecules against CpCHS for controlling the pest population. Communicated by Ramaswamy H. Sarma.

Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents.

A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC50 of 0.10 mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC50 of 0.18 mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4 µg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents.

Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies.

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall ß-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca(2+)-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening.

Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC50 values of 1.8 and 10µM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100µM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties.

Novel small molecules for the treatment of infections caused by Candida albicans: a patent review (2002-2010).

INTRODUCTION: The fungal pathogen Candida albicans is one of the leading causes of infections affecting immunodeficient individuals, including those HIV-infected and patients undergoing cancer therapy. Emerging problems in terms of therapeutic efficacy and drug resistance have highlighted the need to consider new therapeutic approaches, based on the exploitation of virulence factors as alternatives to conventional drug targets. AREAS COVERED: Advances in the development of anti-Candida drugs are examined in this review, as reflected by the patent literature since 2002 along with selected peer-reviewed publications. Taking into account a total of 26 patents, the discussion encompasses several therapeutic approaches, including azoles as ergosterol biosynthesis inhibitors, glucan and chitin synthase inhibitors, and secreted aspartyl protease inhibitors. EXPERT OPINION: New analogs of existing drugs are being developed as broad spectrum antifungals to improve efficacy and circumvent drug resistance. Also, candidate drugs targeting new virulence factors are promising to overcome limitations due to poor efficacy and the rising of drug resistance observed for several available drugs. Efforts for the discovery and development of antifungal agents should be equivalent to other therapeutic areas, and advances in the generation of therapeutic agents with fungus-specific mechanisms of action are of highest priority.

Chitin synthase 2 inhibitory activity of O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one, isolated from Chamaecyparis pisifera.

In the course of search for potent chitin synthase inhibitors from plant extracts, the chitin synthase 2 inhibitors, O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one which have diterpene skeleton, were isolated from the leaves of Chamaecyparis pisifera. These compounds inhibited chitin synthase 2 of Saccharomyces cerevisiae with the IC50 values of 5.8 and 226.4 microM, respectively. Especially, O-methyl pisiferic acid showed 15.3-fold stronger inhibitory activity than polyoxin D (IC50=88.6 microM), a well-known chitin synthase inhibitor. These compounds exhibited weaker inhibitory activities against chitin synthase 1 than chitin synthase 2, whereas it showed no inhibitory activity for chitin synthase 3. The compound exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate (Ki=5 microM). These results indicated that O-methyl pisiferic acid is a specific inhibitor of chitin synthase 2. The compound also inhibited chitin synthase 1 of Candida albicans, which represents analogues to chitin synthase 2 of S. cerevisiae, with an IC50 of 75.6 microM, which represents 1.8-fold weaker activity than that of polyoxin D. Although O-methyl pisiferic acid has been reported for antibacterial and insecticidal activities, the present study is the first report on its inhibitory activity against chitin synthase 2.

Inhibition of chitin synthase 2 and antifungal activity of lignans from the stem bark of Lindera erythrocarpa.

Potent chitin synthase 2 inhibitors, methyllinderone (1), linderone (2) and kanakugiol (3) were isolated from the stem bark of L. erythrocarpa Makino (Lauraceae). These compounds inhibited chitin synthase 2 with IC(50) values of 23.3, 21.4 and 23.8 microg/mL, respectively. Methyllinderone (1) and linderone (2) exhibited no inhibitory activities for chitin synthases 1 and 3 from S. cerevisiae, and chitin synthase 1 from Candida albicans up to the concentration of 280 microg/mL, while kanakugiol (3) exhibited very weak activity against chitin synthase 1 of C. albicans with an IC(50) of 160 microg/mL. All of the compounds showed moderate to weak antifungal activities against various pathogenic fungi (MIC: 8 - >128 microg/mL) including Cryptococcus neoformans, Aspergillus fumigatus, and Colletotrichum lagenarium. The results indicate that these compounds are specific inhibitors of chitin synthase 2 and can potentially serve as antifungal agents.

Inhibition of chitin synthases and antifungal activities by 2'-benzoyloxycinnamaldehyde from Pleuropterus ciliinervis and its derivatives.

In the course of search for potent chitin synthase inhibitors from natural resources, a novel chitin synthases inhibitor, 2'-benzoyloxycinnamaldehyde (2'-BCA) (I), was isolated from the aerial parts of Pleuropterus ciliinervis NAKAI. 2'-BCA inhibited chitin synthase 1 and 2 of Saccharomyces cerevisiae with the IC50s of 54.9 and 70.8 microg/ml, respectively, whereas it exhibited no inhibitory activity for chitin synthase 3 up to 280 microg/ml. Its derivatives, 2'-chloro- (V) and 2(-bromo-cinnamaldehyde (VI), each showed 1.9 and 2.7-fold stronger inhibitory activities than 2'-BCA, with the IC50s of 37.2 and 26.6 microg/ml, respectively. Especially, the IC50 of compound VI against chitin synthase 2 represented 1.7-fold more potent inhibitory activity than polyoxin D, a well-known chitin synthase inhibitor. Furthermore, compounds V and VI showed potent antifungal activities against various fungi including human pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC = 16 microg/ml). Although the chemical synthesis of this compound has been reported, the present study is the first report to describe the isolation of 2'-BCA from natural resources and chitin synthases inhibitory activities of its derivatives. These results suggested that 2'-BCA and its derivatives can potentially serve as useful lead compounds for development of antifungal agents.

Obovatols, new chitin synthase 2 inhibitors of Saccharomyces cerevisiae from Magnolia obovata.

In the course of the search for inhibitors of ScCHS2 from natural sources, we have isolated a new type of chitin synthase 2 inhibitor, obovatol, which has a biphenol skeleton, from Magnolia obovata. Obovatol inhibited chitin synthase 2 activity of Saccharomyces cerevisiae with an IC(50) of 38 microM. Its derivative, tetrahydroobovatol, inhibited chitin synthase 2 activity under the same conditions with an IC(50) of 59 microM. These compounds exhibited no inhibitory activity for ScCHS3, and showed less inhibitory activity for chitin synthase 1 than for chitin synthase 2 (IC(50) > 1 mM). These results indicated that obovatol and tetrahydroobovatol are specific inhibitors of ScCHS2. They also inhibited CaCHS1, which is structurally and functionally analogous to ScCHS2, with similar IC(50)s to ScCHS2 (IC(50) 28 and 51 microM, respectively). The compounds exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate [inhibition constant (K(i)) 21.8 microM for obovatol and 23.1 microM for tetrahydroobovatol]. Furthermore, they showed antifungal activities against various pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC 7.8 mg/L). The results indicate that obovatol and tetrahydroobovatol can potentially serve as antifungal agents.

Inhibitory activity for chitin synthase II from Saccharomyces cerevisiae by tannins and related compounds.

In the course of search for potent inhibitors of chitin synthase II from natural resources, seven tannins and related compounds were isolated from the aerial part of Euphorbia pekinensis and identified as gallic acid (1), methyl gallate (2), 3-O-galloyl-(-)-shikimic acid (3), corilagin (4), geraniin (5), quercetin-3-O-(2"-O-galloyl)-beta-D-glucoside (6), and kaempferol-3-O-(2"-O-galloyl)-beta-D-glucoside (7). These and nine related compounds, (-)-quinic acid (8), (-)-shikimic acid (9), ellagic acid (10), kaempferol (11), quercetin (12), quercitrin (13), rutin (14), quercetin-3-O-(2"-O-galloyl)-beta-D-rutinoside (15) and 1,3,4,6-tetra-O-galloyl-beta-D-glucose (16), were evaluated for the inhibitory activity against chitin synthase II and III. They inhibited chitin synthase II with IC(50) values of 18-206 microM, except for two organic acids, (-)-quinic acid (8) and (-)-shikimic acid (9). Among them, 3-O-galloyl-(-)-shikimic acid (3) was the most potent inhibitor against chitin synthase II of Saccharomyces cerevisiae with an IC(50) value of 18 microM. The inhibition appears to be selective for chitin synthase II, as they did not appreciably inhibit chitin synthase III.

Extremophilic orgainisms as and unexplored source fo antifungal compounds.

Extracts of the biomasses and fermentation broths of 217 extremophilic microorganisms isolated from a number of locales were screened for antifungal activity using whole-cell and mechanism-based in vitro assays. Importantly, eleven broth extracts had activity against several Candida species and Aspergillus fumigatus in whole-cell in vitro assays. One broth specifically inhibited (1,3)beta-glucan synthase activity and four specifically inhibited ketol-isomerase activity, suggesting a mode of action of the antifungal compound(s) present in these extracts. The extract from one thermophile, a novel species of Pseudomonas, was fractionated, an active compound purified and its structure determined. The compound was identified as pyochelin, a previously identified iron-binding compound with heretofore undescribed antifungal activity. To our knowledge, this is the first report demonstrating that extremophiles synthesize compounds that have antifungal activity.

Chitin synthase II inhibitory activity of ursolic acid, isolated from Crataegus pinnatifida.

Two triterpenoid compounds, ursolic acid and uvaol, were isolated from Crataegus pinnatifida Bunge leaves. Ursolic acid inhibits chitin synthase II from S. cerevisiae with an IC50 value of 0.84 microgram/ml and the inhibition appears to be selective for chitin synthase II, whereas uvaol has no inhibitory activity up to 280 micrograms/ml. Oleanolic acid, alpha-hederin hydrate, and betulic acid inhibited the chitin synthase II activity under the same conditions with an IC50 of 5.6, 64.3, and 98.7 micrograms/ml, respectively.

Inhibition of chitin synthase II by catechins from stem bark of Taxus cuspidata.

Two flavonoids, (+/-)-catechin and (-)-epicatechin, were isolated from the stem bark of Taxus cuspidata by monitoring chitin synthase II inhibitory activity. The compounds inhibit chitin synthase II with an IC50 of 15 and 29 micrograms/ml, respectively and appear to be selective for chitin synthase II. They did not inhibit chitin synthase III.

Design of anticandidal agents: synthesis and biological properties of analogues of polyoxin L.

Six analogues of polyoxin L were synthesized from uridine. All of these analogues inhibited chitin synthetase from Candida albicans. Derivatization of the amine terminus of the polyoxin analogues resulted in loss of activity, and analogues containing aromatic amino acid residues were the most efficient inhibitors of chitin synthetase. The concentration of tryptophanyl uracil polyoxin C, 8, which caused 50% inhibition of chitin synthetase activity, was 1.6 X 10(-6) M. This was virtually identical with the activity found for polyoxin D. None of the inhibitors effectively competed with the entry of (Met)3 into C. albicans. All of the analogues caused severe morphological distortions of the yeast in culture, and a number of analogues killed C. albicans at millimolar concentrations. The results suggest that chitin synthetase inhibitors may have potential as anticandidal drugs.

Metabolic products of microorganisms. 181. Chitin synthase from fungi, a test model for substances with insecticidal properties.

Chitin synthase from Coprinus cinereus (Schaeff. ex Fr.) S. F. Gray (= C. lagopus sensu Buller) was used as a model for chitin synthase from insects. The effect of dimilin (difluorobenzuron), captan (trichloromethylsulfonyl fungicide), kitazin P (organophosphorus ester fungicide) and parathion (organophosphorus insecticide) on the fungal enzyme was compared with the effect of nikkomycin (nucleosidepeptide antibiotic).