Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors

Cytochrome P450 14α-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14α-methyl group from sterol precursors such as lanosterol, obtusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydrolanosterol. Inhibitors of CYP51 include triazole antifungal agents fluconazole and itraconazole, drugs used in treatment of topical and systemic mycoses. The 2.1- and 2.2-Å crystal structures reported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) are the first structures of an authentic P450 drug target. MTCYP51 exhibits the P450 fold with the exception of two striking differences—a bent I helix and an open conformation of BC loop—that define an active site-access channel running along the heme plane perpendicular to the direction observed for the substrate entry in P450BM3. Although a channel analogous to that in P450BM3 is evident also in MTCYP51, it is not open at the surface. The presence of two different channels, with one being open to the surface, suggests the possibility of conformationally regulated substrate-in/product-out openings in CYP51. Mapping mutations identified in Candida albicans azole-resistant isolates indicates that azole resistance in fungi develops in protein regions involved in orchestrating passage of CYP51 through different conformational stages along the catalytic cycle rather than in residues directly contacting fluconazole. These new structures provide a basis for rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids

The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

Candidacidal potential of licorice phenolic extract: emphasis on its mode of action

Medicinal plants bave gained a special attention in the last years, due to its renowned health benefits, such as antimicrobial effects [I]. In fact, several natural matrices bave been increasingly studied, namely for its antifungal activity against opportunistic fungi [2,3]. Candida species, although commensa! microorganisms, have caused severe organic dysfunctions to the host, once current antifungal agents have lost their recognized efficiency [2]. So, numerous studies have been carried out focusing the mechanisms of acquired drug-resistance by Candida species [

In vitro anti-Candida activity of Glycyrrhiza glabra L.

The severity and frequency of opportunistic fungal infections still growing, concomitantly to the increasing rates of antimicrobial drug’s resistance. Natural matrices have been used over years due to its multitude of health benefits, including antifungal potential. Thus, the present work aims to evaluate the anti-Candida potential of the phenolic extract and individual phenolic compounds of Glycyrrhiza glabra L. (licorice), by disc diffusion assay, followed by determination of the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for both planktonic cells and biofilms. Licorice extract evidenced inhibitory potential against the nineteen tested Candida strains, but no pronounced effect was observed by testing the most abundant individual phenolic compounds. Candida tropicalis strains were the most sensible, followed by Candida glabrata, Candida parapsilosis and, then, Candida albicans. Lower MIC and MFC values were achieved to C. glabrata and C. tropicalis, which confirms its susceptibility to licorice extract; however, for C. tropicalis strains a higher variability was observed. Anti-biofilm potential was also achieved, being most evident in some C. glabrata and C. tropicalis strains. In general, a twice concentration of the MIC was necessary for planktonic cells to obtain a similar potential to that one observed for biofilms. Thus, an upcoming approach for new antifungal agents, more effective and safer than the current ones, is stablished; notwithstanding, further studies are necessary in order to understand its mechanism of action, as also to assess kinetic parameters.

Exploration of the anticandidal mechanism of Cassia spectabilis in debilitating candidiasis

Candida albicans has become resistant to the commercially available, toxic, and expensive anti-Candida agents that are on the market. These factors force the search for new antifungal agents from natural resources. Cassia spectabilis had been traditionally employed by healers for many generations. The possible mechanisms of the C. spectabilis leaf extract were determined by potassium leakage study and the effect of the extract on the constituents of the cell wall and enzymes as well as the morphological changes on C. albicans cells were studied along with cytotoxicity assays. The cytotoxicity result indicated that the extract is nontoxic as was clearly substantiated by a half maximal inhibitory concentration (IC50) value of 59.10 μg/mL. The treated cells (C. spectabilis extract) demonstrated potassium leakage of 1039 parts per million (ppm) compared to Amphotericin B (AmpB)-treated cells with a released potassium value of 1115 ppm. The effects of the extract on the cell wall proteins illustrated that there were three major types of variations in the expression of treated cell wall proteins: the presence of new proteins, the absence of proteins, and the amount of expressed protein. The activities of two enzymes, α-glucosidase and proteinase, were determined to be significantly high, thereby not fully coinciding with the properties of the antifungal reaction triggered by C. spectabilis. The morphology of C. albicans cells treated with the C. spectabilis extract showed that the cells had abnormalities and were damaged or detached within the microcolonies. Our study verifies C. spectabilis leaf extract as an effective anti-C. albicans agent.

Green mitigation strategy for cultural heritage: bacterial potential for biocide production

Several biosurfactants with antagonistic activity are produced by a variety of microorganisms. Lipopeptides (LPPs) produced by some Bacillus strains, including surfactin, fengycin and iturin are synthesized nonribosomally by mega-peptide synthetase (NRPS) units and they are particularly relevant as antifungal agents. Characterisation, identification and evaluation of the potentials of several bacterial isolates were undertaken in order to establish the production of active lipopeptides against biodeteriogenic fungi from heritage assets. Analysis of the iturin operon revealed four open reading frames (ORFs) with the structural organisation of the peptide synthetases. Therefore, this work adopted a molecular procedure to access antifungal potential of LPP production by Bacillus strains in order to exploit the bioactive compounds synthesis as a green natural approach to be applied in biodegraded cultural heritage context. The results reveal that the bacterial strains with higher antifungal potential exhibit the same morphological and biochemical characteristics, belonging to the genera Bacillus. On the other hand, the higher iturinic genetic expression, for Bacillus sp. 3 and Bacillus sp. 4, is in accordance with the culture antifungal spectra. Accordingly, the adopted methodology combining antifungal screening and molecular data is represent a valuable tool for quick identification of iturin-producing strains, constituting an effective approach for confirming the selection of lipopeptides producer strains.

Synthesis of new 4-isopropylthiazole hydrazide analogs and some derived clubbed triazole, oxadiazole ring systems - A novel class of potential antibacterial, antifungal and antitubercular agents

In the present study a series of 4-isopropylthiazole-2-carbohydrazide analogs, derived clubbed oxadiazole-thiazole and triazole-thiazole derivatives have been synthesized and characterized by IR, H-1 NMR, C-13 NMR, elemental and mass spectral analyses. The synthesized compounds were evaluated for their preliminary in vitro antibacterial, antifungal and antitubercular activity against Mycobacterium tuberculosis H(37)Rv strain by broth dilution assay method. The synthesized compounds 7a, 7b, 7d and 4 showed an antitubercular efficacy considerably greater than that of the parent 4-isopropyl-1,3-thiazole-2-carbohydrazide 1, suggesting that the substituted 4-isopropylthiazole-2-carbohydrazide moiety plays an important role in enhancing the antitubercular properties of this class of compounds. Compounds 2c, 3, 4, 6d, 7a and 7b exhibited good or moderate antibacterial and antifungal activity. Compounds 4 and 7b showed appreciable cytotoxicity at a concentration of 250 mu M.

Design, synthesis of some new (2-aminothiazol-4-yl)methylester derivatives as possible antimicrobial and antitubercular agents

A series of (2-aminothiazol-4-yl)methylester (5a-t) derivatives were synthesized in good yields and characterized by H-1 NMR, C-13 NMR, mass spectral and elemental analyses. The crystal structure of 5a was evidenced by X-ray diffraction study. The compounds were evaluated for their preliminary in vitro antibacterial, antifungal activity and were screened for antitubercular activity against Mycobacterium tuberculosis H37Rv strain. The synthesized compounds displayed interesting antimicrobial activity. (C) 2012 Elsevier Masson SAS. All rights reserved.

Design and synthesis of positional isomers of 5 and 6-bromo-1-(phenyl)sulfonyl]-2-(4-nitrophenoxy)methyl]-1H-benzimida zoles as possible antimicrobial and antitubercular agents

In this Letter, we report the structure activity relationship (SAR) studies on series of positional isomers of 5(6)-bromo-1-(phenyl)sulfonyl]-2-(4-nitrophenoxy)methyl]-1H-benzim idazoles derivatives 7(a-j) and 8(a j) synthesized in good yields and characterized by H-1 NMR, C-13 NMR and mass spectral analyses. The crystal structure of 7a was evidenced by X-ray diffraction study. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus, (Gram-positive), Escherichia coil and Klebsiella pneumoniae (Gram-negative), antifungal activity against Candida albicans, Aspergillus flavus and Rhizopus sp. and antitubercular activity against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis, Mycobacterium fortuitum and MDR-TB strains. The synthesized compounds displayed interesting antimicrobial activity. The compounds 7b, 7e and 7h displayed significant activity against Mycobacterium tuberculosis H37Rv strain.

High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of approximate to 20,000 microbial extracts, 12 hits were identified with broadspectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Synthesis, Antifungal and Antitumor Activity of Novel (Z)-5-Hetarylmethylidene-1,3-thiazol-4-ones and (Z)-5-Ethylidene-1,3-thiazol-4-ones

New hetaryl- and alkylidenerhodanine derivatives 3a-d, 3e, and 4a-d were prepared from heterocyclic aldehydes 1a-d or acetaldehyde 1e. The treatment of several rhodanine derivatives 3a-d and 3e with piperidine or morpholine in THF under reflux, afforded (Z)-5-(hetarylmethylidene)-2-(piperidin-1-yl) thiazol-4(5H)-ones and 2-morpholinothiazol-4(5H)-ones 5a-d, 6a-d, and (Z)-5-ethylidene-2-morpholinothiazol-4(5H)-one (5e), respectively, in good yields. Structures of all compounds were determined by IR, 1D and 2D NMR and mass spectrometry. Several of these compounds were screened by the U.S. National Cancer Institute (NCI) to assess their antitumor activity against 60 different human tumor cell lines. Compound 3c showed high activity against HOP-92 (Non-Small Cell Lung Cancer), which was the most sensitive cell line, with GI(50) = 0.62 mu M and LC50 > 100 mu M from the in vitro assays. In vitro antifungal activity of these compounds was also determined against 10 fungal strains. Compound 3e showed activity against all fungal strains tested, but showed high activity against Saccharomyces cerevisiae (MIC 3.9 mu g/mL).

In Vitro Activity of the Antifungal Plant Defensin RsAFP2 against Candida Isolates and Its In Vivo Efficacy in Prophylactic Murine Models of Candidiasis

We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Composition and antifungal activity against Candida albicans, Candida parapsilosis, Candida krusei and Cryptococcus neoformans of essential oils from leaves of Piper and Peperomia species

This study was addressed to investigate the composition and antifungal activity of essential oils from leaves of Piperaceae species (Piper aduncum, Piper amalago, Piper cernuum, Piper diospyrifolium, Piper crassinervium, Piper gaudichaudianum, Piper solmsianum, Piper regnellii, Piper tuberculatum, Piper umbelata and Peperomia obtusifolia) against Candida albicans, C. parapsilosis, C. krusei and Cryptococcus neoformans. The essential oils from P. aduncum, P. gaudichaudianum and P. solmsianum showed the highest antifungal activity against Cryptococcus neoformans with the MIC of 62.5, 62.5 and 3.9 mg.mL-1, respectively. The oil from P. gaudichaudianum showed activity against C. krusei with MIC of 31.25 mg.mL-1.