Sterol 24-C-methyltransferase: An enzymatic target for the disruption of ergosterol biosynthesis and homeostasis in Cryptococcus neoformans

Growth of Cryptococcus neoformans was inhibited by nine nitrogen and sulfur-containing sterols with a heteroatom positioned at C3, C7, C24, C25 or C32 in the lanostane frame. Analysis of the sterol composition of control and treated cells by GC-MS and ¹H NMR has proven that the C-methylation reaction catalyzed by the sterol 24-C-methyltransferase (24-SMT) is the crucial first step in a kinetically favored pathway that fails to include obtusifoliol or zymosterol as intermediates. Cultures fed [methyl-²H₃]methionine led to two deuterium atoms into each of the newly biosynthesized sterols forming a route lanosterol, eburicol (24(28)-methylene-24,25-dihydrolanosterol), 32-noreburicol and ergost-7-enol to ergosterol. Examination of the substrate specificity of a soluble 24-SMT from C. neoformans showed lanosterol to be the optimal acceptor molecule. Incubation with the test compounds generated induced amounts of lanosterol, eburicol or 32-noreburicol concurrent with a decrease of ergosterol. Among them 24(R,S),25-epiminolanosterol (inhibitor of 24-SMT) showed the most potent in vitro antifungal activity comparable to those of itraconazole (inhibitor of the 14-demethylase). Taken together, these data indicate that treatment with substrate-based inhibitors of 24-SMT, a catalyst not found in humans, can disrupt ergosterol homeostasis involved with fungal growth and therefore these compounds can provide leads for rational drug design of opportunistic pathogens.     

α-Bisabolol inhibits <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Af239 growth via affecting microsomal ∆<Superscript>24</Superscript>-sterol methyltransferase as a crucial enzyme in ergosterol biosynthesis pathway

Finding new compounds with antifungal properties is an important task due to the side effects of common antifungal drugs and emerging antifungal resistance in fungal strains. ?²⁴-sterol methyltransferase (24-SMT) is a crucial enzyme that plays important roles in fungal ergosterol biosynthesis pathway and is not found in humans. In the present study, the effects of α-bisabolol on Aspergillus fumigatus Af239 growth and ergosterol synthesis on the base of 24-SMT enzyme activity were studied; in addition, the expression of erg6, the gene encoded 24-SMT, was considered. To our knowledge, this is the first report demonstrating that α-bisabolol inhibits A. fumigatus growth specifically via suppressing fungal 24-SMT. Since this enzyme is a specific fungal enzyme not reported to exist in mammalian cells, α-bisabolol may serve as a lead compound in the development of new antifungal drugs. Fungi were cultured in presence of serial concentrations of α-bisabolol (0.281–9 mM) for 3 days at 35?°C. Mycelia dry weight was determined as an index of fungal growth and ergosterol content was assessed. Microsomal 24-SMT activity was assayed in presence of α-bisabolol as an inhibitor, lanosterol as a substrate and [methyl-H³] AdoMet (S-Adenosyl methionin). In addition, the expression of erg 6 gene (24-SMT encoding gene) was determined after treatments with various concentrations of α-bisabolol. Our results demonstrated that α-bisabolol strongly inhibited A. fumigatus growth (35.53–77.17%) and ergosterol synthesis (26.31–73.77%) dose-dependently and suppressed the expression of erg 6 gene by 76.14% at the highest concentration of 9 mM. α-bisabolol inhibited the activity of 24-SMT by 99% at the concentration of 5 mM. Taken together, these results provides an evidence for the first time that α-bisabolol inhibits A. fumigatus Af239 growth via affecting microsomal ?²⁴-sterol methyltransferase as a crucial enzyme in ergosterol biosynthetic pathway.     

The structure of a Burkholderia pseudomallei immunophilin-inhibitor complex reveals new approaches to antimicrobial development

TbSMT [Trypanosoma brucei 24-SMT (sterol C-24-methyltransferase)] synthesizes an unconventional 24-alkyl sterol product set consisting of Δ24(25)-, Δ24(28)- and Δ25(27)-olefins. The C-methylation reaction requires Si(β)-face C-24-methyl addition coupled to reversible migration of positive charge from C-24 to C-25. The hydride shifts responsible for charge migration in formation of multiple ergostane olefin isomers catalysed by TbSMT were examined by incubation of a series of sterol acceptors paired with AdoMet (S-adenosyl-L-methionine). Results obtained with zymosterol compared with the corresponding 24-2H and 27-13C derivatives revealed isotopic-sensitive branching in the hydride transfer reaction on the path to form a 24-methyl-Δ24(25)-olefin product (kinetic isotope effect, kH/kD=1.20), and stereospecific CH3→CH2 elimination at the C28 branch and C27 cis-terminal methyl to form Δ24(28) and Δ25(27) products respectively. Cholesta-5,7,22,24-tetraenol converted into ergosta-5,7,22,24(28)-tetraenol and 24β-hydroxy ergosta-5,7,23-trienol (new compound), whereas ergosta-5,24-dienol converted into 24-dimethyl ergosta-5,25(27)-dienol and cholesta-5,7,24-trienol converted into ergosta-5,7,25(27)trienol, ergosta-5,7,24(28)-trienol, ergosta-5,7,24-trienol and 24 dimethyl ergosta-5,7,25(27)-trienol. We made use of our prior research and molecular modelling of 24-SMT to identify contact amino acids that might affect catalysis. Conserved tyrosine residues at positions 66, 177 and 208 in TbSMT were replaced with phenylalanine residues. The substitutions generated variable loss of activity during the course of the first C-1-transfer reaction, which differs from the corresponding Erg6p mutants that afforded a gain in C-2-transfer activity. The results show that differences exist among 24-SMTs in control of C-1- and C-2-transfer activities by interactions of intermediate and aromatic residues in the activated complex and provide an opportunity for rational drug design of a parasite enzyme not synthesized by the human host.     

Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design

Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C₂₈- and C₂₉-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC₅₀ values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding ∼K_i 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.     

Homology modeling of lanosterol 14alpha-demethylase of Candida albicans and Aspergillus fumigatus and insights into the enzyme-substrate Interactions

The crystal structure of 14alpha-sterol demethylase from Mycobacterium tuberculosis (MT_14DM) provides a good template for modeling the three dimensional structure of lanosterol 14alpha-demethylase, which is the target of azole antifungal agents. Homologous 3D models of lanosterol 14alpha-demethylase from Candida albicans (CA_14DM) and Aspergillus fumigatus (AF_14DM) were built on the basis of the crystal coordinates of MT_14DM in complex with 4-phenylimidazole and fluconazole. The reliability of the two models was assessed by Ramachandran plots, Profile-3D analysis, and by analyzing the consistency of the two models with the experimental data on the P450(14DM). The overall structures of the resulting CA_14DM model and AF_14DM model are similar to those of the template structures. The two models remain the core structure characteristic for cytochrome P450s and most of the insertions and deletions expose the molecular surface. The structurally and functionally important residues such as the heme binding residues, the residues lining the substrate access channel, and residues in active site were identified from the model. To explore the binding mode of the substrate with the two models, 24(28)-methylene-24,25-dihydrolanosterol was docked into the active site of the two models and hydrophobic interaction and hydrogen-bonding were found to play an important role in substrate recognition and orientation. These results provided a basis for experiments to probe structure-function relationships in the P450(14DM). Although CA_14DM and AF_14DM shared similar core structural character, the active site of the two models were quite different, thus allowing the rational design of specific inhibitors to the target enzyme and the discovery of novel antifungal agents with broad spectrum.     

Naphtho[2,3-b][1,4]-thiazine-5,10-diones and 3-substituted-1,4-dioxo-1,4-dihydronaphthalen-2-yl-thioalkanoate derivatives: synthesis and biological evaluation as potential antibacterial and antifungal agents

A series of 3-substituted-1,4-dioxo-1,4-dihydronaphthalen-2-yl-thio-alkanoate derivatives 3-21 and naphtho[2,3-b][1,4]-thiazine-5,10-diones 24 were synthesized and evaluated for their antibacterial and antifungal activities. The structure-activity relationships of these compounds were studied and the results show that the compound 24a exhibited better antibacterial activity than Gentamycin in vitro against Staphylococcus aureus. In addition 24a also imparted marked antifungal activity in vitro against Cryptococcus neoformans, Sporothrix schenckii, and Trichophyton mentagraphytes when compared with Fluconazole. Compounds 15, 18, 19, and 21 also exhibited significant antibacterial activity in vitro against S. aureus.     

Antifungal and antimycobacterial activity of 1-(3,5-diaryl-4,5-dihydro-1H-pyrazol-4-yl)-1H-imidazole derivatives

1-(3,5-Diaryl-4,5-dihydro-1H-pyrazol-4-yl)-1H-imidazole derivatives were synthesized and tested for their in vitro antifungal and antimycobacterial activities. These imidazole derivatives showed an excellent antifungal activity against a clinical strain of Candida albicans and an interesting antitubercular activity against Mycobacterium tuberculosis H(37)Rv reference strain.     

Antifungal agents. Part 5: synthesis and antifungal activities of aminoguanidine derivatives of N-arylsulfonyl-3-acylindoles

In order to discover more promising antifungal agents, a series of aminoguanidine derivatives of N-arylsulfonyl-3-acylindoles (5a-r) were prepared and evaluated in vitro for their antifungal activities against seven phytopathogenic fungi. Especially compounds 5n and 5o exhibited more potent antifungal activities than or comparable to hymexazol, a commercially available agricultural fungicide at the concentration of 100 μg/mL. Preliminary structure-activity relationships study demonstrated that introduction of electron-donating substituents R(1) and R(2), and the proper length of substituent R(3) were usually very important for their antifungal activities.     

Synthesis and biological evaluation of novel N-acyl substituted quinolin-2(1H)-one derivatives as potential antimicrobial agents

A series of novel N-acyl substituted quinolin-2(1H)-one derivatives were synthesized and screened in vitro for their antibacterial and antifungal activities by disc diffusion method. All the compounds exhibited moderate to good antimicrobial activities, some of these compounds displayed comparable or better antibacterial or antifungal activities against some tested strains compared to the reference drugs Streptomycin and Fluconazole.     

Synthesis and antifungal activities of new fluconazole analogues with azaheterocycle moiety

A series of fluconazole analogues 5-20 incorporating azaindole and indole moieties were prepared using oxirane intermediates synthesized under microwave irradiation. All of the compounds were evaluated in vitro against two clinically important fungi, Candida albicans and Aspergillus fumigatus. Four derivatives 6, 13, 14 and 18 exerted high antifungal activity against C. albicans with MIC(80) values 3- to 28-fold lower than that of fluconazole.     

Synthesis and biological evaluation of vinyl ether-containing azole derivatives as inhibitors of Trichophyton rubrum

In an attempt to search for many target compounds with excellent activities, a series of vinyl ether-containing azole derivatives were designed, synthesized, and evaluated as antifungal agents. Results of preliminary antifungal tests against Trichophyton rubrum in vitro indicated that most of the synthesized compounds showed excellent activities. In comparison with fluconazole, itraconazole, voriconazole, omoconazole and amphotericin B, several compounds (such as 7d, 7g and 7h) exhibited more potent inhibitory activities, suggesting that they were promising leads for the development of novel antifungal agents.     

Synthesis and antifungal activities of phenylenedithioureas

A total of 20 new phenylenedithiourea derivatives was synthesized by reaction of phenylenediisothiocyanates with aromatic amines as aminobenzoic, aminosalicylic acid and their derivatives. Their chemical structures were confirmed by elemental analysis, IR spectrometry and 1H NMR. The compounds were screened for in vitro antifungal, antibacterial activities and some of them have strong antifungal activities comparable to the activity observed for ketoconazole.     

Side chain azasteroids and thiasteroids as sterol methyltransferase inhibitors in ergosterol biosynthesis

The synthesis of some novel azasteroids and thiasteroids based on a pregnan nucleus with a Δ7 double bond in two to five steps from the key aldehyde (3S,20S)-20-formylpregn-7-en-3-yl acetate has been disclosed herein. These compounds were evaluated as potential inhibitors of the enzyme Δ24-sterol methyltransferase (24-SMT), which is a key enzyme in the biosynthesis of ergosterol, and for their effects on the growth of the yeast Yarrowia lipolytica. Most of the side chain modified analogues were recognized as 24-SMT inhibitors, and in particular the 23-azasteroids 5f–5i and the 24-azasteroid 11 showed potent antifungal activity. The target enzyme could be identified unambiguously using an improved whole-cell assay combined with GC–MS analysis of the sterol pattern resulting upon incubation with the inhibitors.     

Preparation of transition-state analogues of sterol 24-methyl transferase as potential anti-parasitics

There is an urgent need for new drugs to treat leishmaniasis and Chagas disease. One important drug target in these organisms is sterol biosynthesis. In these organisms the main endogenous sterols are ergosta- and stigmata-like compounds in contrast to the situation in mammals, which have cholesterol as the sole sterol. In this paper we discuss the design, synthesis and evaluation of potential transition state analogues of the enzyme Delta24(25)-methyltransferase (24-SMT). This enzyme is essential for the biosynthesis of ergosterol, but not required for the biosynthesis of cholesterol. A series of compounds were successfully synthesised in which mimics of the S-adenosyl methionine co-factor were attached to the sterol nucleus. Compounds were evaluated against recombinant Leishmania major 24-SMT and the parasites L. donovani and Trypanosoma cruzi in vitro, causative organisms of leishmaniasis and Chagas disease, respectively. Some of the compounds showed inhibition of the recombinant Leishmania major 24-SMT and induced growth inhibition of the parasites. Some compounds also showed anti-parasitic activity against L. donovani and T. cruzi, but no inhibition of the enzyme. In addition, some of the compounds had anti-proliferative activity against the bloodstream forms of Trypanosoma brucei rhodesiense, which causes African trypanosomiasis.     

Synthesis, in vitro evaluation and molecular docking studies of new triazole derivatives as antifungal agents

On the basis of the active site of lanosterol 14α-demethylase from Candida albicans (CACYP51), a series of 1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,4-triazol-5(4H)-one derivatives were synthesized as fluconazole analogs. Results of the preliminary antifungal tests against eight human pathogenic fungi in vitro showed that these compounds exhibited activities to some extent, and some displayed excellent antifungal activities against C. albicans than reference drug fluconazole. Flexible molecular docking was used to analyze the structure-activity relationships (SARs) of the target compounds. The designed compounds interact with CACYP51 through hydrophobic, van der Waals and hydrogen-bonding interactions.     

Antifungal activities of N-arylbenzenesulfonamides against phytopathogens and control efficacy on wheat leaf rust and cabbage club root diseases

A set of N-arylbenzenesulfonamides with various substituents at the arylamine and benzenesulfonyl positions were prepared, and their antifungal properties were measured in vitro against such plant pathogenic fungi as Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, and Botrytis cinerea. Compounds 3, 4, 8, 9, 10, 14, 16, 18, 20, 21, 24 and 27 had antifungal activity over a broad spectrum of the phytopathogenic fungi tested, where 50% of inhibition (ED50) was in the range of 3-15 microg/ml. Based on the in vitro activity, six derivatives (3, 4, 10, 18, 21 and 27) were selected and tested further for their fungicidal efficacy in vivo. The fungicidal efficacy of 10, 21 and 27 had a disease control value of over 85% at 50 microg/ml against wheat leaf rust, while that of 4 was selective against cabbage club root disease.     

Synthesis of (1,4)-naphthoquinono-[3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives as antifungal, antibacterial, and anticancer agents

A series of (1,4)-naphthoquinono [3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives 2-7 were synthesized and evaluated for antifungal, antibacterial, and anticancer activities. The structure-activity relationship of these compounds was studied and the results show that the compound 2b exhibited in vitro antifungal activity against Candida albicans and Cryptococcus neoformans, and also possessed antibacterial profile against Klebsiella pneumoniae and Escherichia coli whereas 1c showed anticancer activity against Walker 256 Carcinosarcoma in rats.     

Synthesis,characterization and antifungal activity of quaternary derivatives of chitosan on Aspergillus flavus

Two series of new chitosan derivatives were synthesized by reaction of deacetylated chitosan (CH) with propyl (CH-Propyl) and pentyl (CH-Pentyl) trimethylammonium bromides to obtain derivatives with increasing degrees of substitution (DS). The derivatives were characterized by ¹H NMR and potentiometric titration techniques and their antifungal activities on the mycelial growth of Aspergillus flavus were investigated in vitro. The antifungal activities increase with DS and the more substituted derivatives of both series, CH-Propyl and CH-Pentyl, exhibited antifungal activities respectively three and six times higher than those obtained with commercial and deacetylated chitosan. The minimum inhibitory concentrations (MIC) were evaluated at 24, 48 and 72 h by varying the polymer concentration from 0.5 to 16 g/L and the results showed that the quaternary derivatives inhibited the fungus growth at polymer concentrations four times lower than that obtained with deacetylated chitosan (CH). The chitosans modified with pentyltrimethylammonium bromide exhibited higher activity and results are discussed taking into account the degree of substitution (DS).     

Synthesis and in vitro antifungal activities of new 3-substituted benzopyrone derivatives

A series of new benzopyrone compounds were designed and synthesized and their antifungal activities in vitro were evaluated. The results showed that the benzopyrone derivatives with short terminal alkyl chain exhibited potent antifungal activity, which represent a novel class of promising leads for the development of novel non-azole antifungal agents. Compound 5j is the most potent one with MIC(80) value 1.5 μg/mL against Trichophyton rubrum. Flexible molecular docking was used to analyze the structure-activity relationships (SARs) of the compounds. The designed compounds interact with CA-CYP51 through hydrophobic and van der Waals interactions.     

Synthesis and in vitro activity of 2-thiazolylhydrazone derivatives compared with the activity of clotrimazole against clinical isolates of Candida spp

In this paper, we report on the synthesis of a novel series of 2-thiazolylhydrazone derivatives and the influence of the substituents on the thiazole ring on antifungal activity. All synthesized compounds were screened for their in vitro activities against 22 clinical isolates of Candida spp., representing six different species, compared to clotrimazole as a reference compound. Some of the tested compounds were found to possess significant antifungal activity when compared to clotrimazole, in particular compound 14 which exhibited higher potency against most of the Candida spp. considered. The compounds that were most active as anti-Candida agents were also submitted to cytotoxic screening by the Trypan Blue dye exclusion assay and in general they were shown to induce low cytotoxic effects.