Mycobacterium tuberculosis Decaprenylphosphoryl-ß-d-ribose Oxidase Inhibitors: Expeditious Reconstruction of Suboptimal Hits into a Series with Potent in Vivo Activity.

Decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) is an essential enzyme in Mycobacterium tuberculosis and has recently been studied as a potential drug target, with inhibitors progressing to clinical studies. Here we describe the identification of a novel series of morpholino-pyrimidine DprE1 inhibitors. These were derived from a phenotypic high-throughput screening (HTS) hit with suboptimal physicochemical properties. Optimization strategies included scaffold-hopping, synthesis, and evaluation of fragments of the lead compounds and property-focused optimization. The resulting optimized compounds had much improved physicochemical properties and maintained enzyme and cellular potency. These molecules demonstrated potent efficacy in an in vivo tuberculosis murine infection model.

Identification of Off-Patent Compounds That Present Antifungal Activity Against the Emerging Fungal Pathogen Candida auris.

Candida auris is an emerging fungal pathogen of great concern among the scientific community because it is causing an increasing number of hospital outbreaks of difficult management worldwide. In addition, isolates from this species frequently present reduced susceptibility to azole and echinocandin drugs. For this reason, it is necessary to develop new antifungal strategies to better control the disease caused by this yeast. In this work, we screened drugs from the Prestwick chemical library, which contains 1,280 off-patent compounds that are already approved by the Food and Drug Administration, with the aim of identifying molecules with antifungal activity against C. auris. In an initial screening, we looked for drugs that inhibited the growth of three different C. auris strains and found 27 of them which it did so. Ten active compounds were selected to test the susceptibility profile by using the EUCAST protocol. Antifungal activity was confirmed for seven drugs with MICs ranging from 0.5 to 64 mg/L. Some of these drugs were also tested in combination with voriconazole and anidulafungin at sub-inhibitory concentrations. Our results suggest synergistic interactions between suloctidil and voriconazole with fractional inhibitory concentration index (FICI) values of 0.11 to 0.5 and between ebselen and anidulafungin (FICI, 0.12 to 0.44). Our findings indicate that drug repurposing could be a viable alternative to managing infections by C. auris.

Insight into the Significance of Aspergillus fumigatus cyp51A Polymorphisms.

Triazole antifungal compounds are the first treatment choice for invasive aspergillosis. However, in the last decade the rate of azole resistance among Aspergillus fumigatus strains has increased notoriously. The main resistance mechanisms are well defined and mostly related to point mutations of the azole target, 14-α sterol demethylase (cyp51A), with or without tandem repeat integrations in the cyp51A promoter. Furthermore, different combinations of five Cyp51A mutations (F46Y, M172V, N248T, D255E, and E427K) have been reported worldwide in about 10% of all A. fumigatus isolates tested. The azole susceptibility profile of these strains shows elevated azole MICs, although on the basis of the azole susceptibility breakpoints, these strains are not considered azole resistant. The purpose of the study was to determine whether these cyp51A polymorphisms (single nucleotide polymorphisms [SNPs]) are responsible for the azole susceptibility profile and whether they are reflected in a poorer azole treatment response in vivo that could compromise patient treatment and outcome. A mutant with a cyp51A deletion was generated and became fully susceptible to all azoles tested. Also, three cyp51A gene constructions with different combinations of SNPs were generated and reintroduced into an azole-susceptible wild-type (WT) strain (the ΔakuBKU80 strain). The alternative model host Galleria mellonella was used to compare the virulence and voriconazole response of G. mellonella larvae infected with A. fumigatus strains with WT cyp51A or cyp51A with SNPs. All strains were pathogenic in G. mellonella larvae, although they did not respond similarly to voriconazole therapeutic doses. Finally, the full genomes of these strains were sequenced and analyzed in comparison with those of A. fumigatus WT strains, revealing that they belong to different strain clusters or lineages.

Could the determination of Aspergillus fumigatus mating type have prognostic value in invasive aspergillosis?

A clear link between mating type and virulence has been demonstrated for some fungal pathogens, but not for Aspergillus fumigatus as of yet. An association between mating type and invasiveness has recently been established. The mating type proportion (MAT1-1:MAT1-2) of 213 A. fumigatus strains was determined (48.5%:51.5%) and results were in agreement with previous studies. However, these percentages changed when the strain collection was divided into azole-susceptible and -resistant strains. The 163 susceptible strains kept these proportions, but among the 50 azole-resistant strains 60.0% MAT1-1 and 40% MAT1-2 were found. Moreover, looking at the clinical outcome associated to 27 azole-resistant strains, we found that MAT1-1 was linked to a high mortality rate (64%), whereas the rate associated to MAT1-2 genotype was markedly lower (15%). The pathogenicity linked to the Mat type was tested in a Galleria mellonella model of infection, showing that MAT1-1 strains were consistently more pathogenic than MAT1-2, independently of their susceptibility phenotype. This data would suggest that A. fumigatus mating type determination at the time of diagnosis could have a prognostic value in invasive aspergillosis.

A New Aspergillus fumigatus Typing Method Based on Hypervariable Tandem Repeats Located within Exons of Surface Protein Coding Genes (TRESP).

Aspergillus fumigatus is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For A. fumigatus genotyping, the short tandem repeat method (STRAf) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. A. fumigatus isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated A. fumigatus strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson's index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for A. fumigatus has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks.

Hitting the caspofungin salvage pathway of human-pathogenic fungi with the novel lasso peptide humidimycin (MDN-0010).

Fungal infections have increased dramatically in the last 2 decades, and fighting infectious diseases requires innovative approaches such as the combination of two drugs acting on different targets or even targeting a salvage pathway of one of the drugs. The fungal cell wall biosynthesis is inhibited by the clinically used antifungal drug caspofungin. This antifungal activity has been found to be potentiated by humidimycin, a new natural product identified from the screening of a collection of 20,000 microbial extracts, which has no major effect when used alone. An analysis of transcriptomes and selected Aspergillus fumigatus mutants indicated that humidimycin affects the high osmolarity glycerol response pathway. By combining humidimycin and caspofungin, a strong increase in caspofungin efficacy was achieved, demonstrating that targeting different signaling pathways provides an excellent basis to develop novel anti-infective strategies.

Cyclic colisporifungin and linear cavinafungins, antifungal lipopeptides isolated from Colispora cavincola.

Colisporifungin (1), a cyclic depsilipopeptide structurally related to the aselacins, and cavinafungins A and B, two linear peptides, were isolated from liquid culture broths of the hitherto unstudied fungus Colispora cavincola using a Candida albicans whole-cell assay as well as a bioassay to detect compounds potentiating the antifungal activity of caspofungin. The structural elucidation, including the absolute configuration of the new molecules, was accomplished using a combination of spectroscopic and chemical techniques, including 1D and 2D NMR, HRMS, and Marfey's analysis. The cyclic peptide colisporifungin displayed a strong potentiation of the growth inhibitory effect of caspofungin against Aspergillus fumigatus and, to a lesser extent, against Candida albicans. The linear peptides displayed broad-spectrum antifungal activities inhibiting growth of Candida species (MIC values 0.5-4 µg/mL) as well as A. fumigatus with a prominent inhibition of 8 µg/mL.

Assessing the effects of adsorptive polymeric resin additions on fungal secondary metabolite chemical diversity.

Adsorptive polymeric resins have been occasionally described to enhance the production of specific secondary metabolites (SMs) of interest. Methods that induce the expression of new chemical entities in fungal fermentations may lead to the discovery of new bioactive molecules and should be addressed as possible tools for the creation of new microbial chemical libraries for drug lead discovery. Herein, we apply both biological activity and chemical evaluations to assess the use of adsorptive resins as tools for the differential expression of SMs in fungal strain sets. Data automation approaches were applied to ultra high performance liquid chromatography analysis of extracts to evaluate the general influence in generating new chemical entities or in changing the production of specific SMs by fungi grown in the presence of resins and different base media.

Novel antifungal agents: a patent review (2011 - present).

INTRODUCTION: Invasive fungal infections (IFI) have increased significantly over the past decades. The mortality rate of IFI is alarming, and early and accurate diagnosis is difficult. Most used antifungal drugs are not completely effective due to the development of increasing resistance and undesirable side effects which limit their use. In this scenario, new effective broad spectrum and safer antifungal drugs are urgently needed. AREAS COVERED: This review summarizes the latest advances in the discovery of new antifungal compounds through the patents granted from 2011 to August 2013. In the 26 patents reviewed here, either derivatives of existing antifungal drugs or novel structures are included. New imidazoles, fluconazole analogs and adducts of azoles with 2,6-di-tert-butyl-4-methylphenol are described. The review also includes chitinases, ß-1,3-D-glucan and chitin synthases inhibitors and novel structures. EXPERT OPINION: In the patents reviewed here, progress has been made to accomplish at least one of the necessary requirements for the development of novel antifungal agents, such as broad spectrum of activity, more favorable pharmacokinetic profile, good bioavailability and low adverse effects. However, in vivo activity, mechanisms of action, drug-drug interactions and other aspects that make a compound a good antifungal agent need further development.

Antimicrobial activity of heterotrophic bacterial communities from the marine sponge Erylus discophorus (Astrophorida, Geodiidae).

Heterotrophic bacteria associated with two specimens of the marine sponge Erylus discophorus were screened for their capacity to produce bioactive compounds against a panel of human pathogens (Staphylococcus aureus wild type and methicillin-resistant S. aureus (MRSA), Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumanii, Candida albicans and Aspergillus fumigatus), fish pathogen (Aliivibrio fischeri) and environmentally relevant bacteria (Vibrio harveyi). The sponges were collected in Berlengas Islands, Portugal. Of the 212 isolated heterotrophic bacteria belonging to Alpha- and Gammaproteobacteria, Actinobacteria and Firmicutes, 31% produced antimicrobial metabolites. Bioactivity was found against both Gram positive and Gram negative and clinically and environmentally relevant target microorganisms. Bioactivity was found mainly against B. subtilis and some bioactivity against S. aureus MRSA, V. harveyi and A. fisheri. No antifungal activity was detected. The three most bioactive genera were Pseudovibrio (47.0%), Vibrio (22.7%) and Bacillus (7.6%). Other less bioactive genera were Labrenzia, Acinetobacter, Microbulbifer, Pseudomonas, Gordonia, Microbacterium, Micrococcus and Mycobacterium, Paenibacillus and Staphylococcus. The search of polyketide I synthases (PKS-I) and nonribosomal peptide synthetases (NRPSs) genes in 59 of the bioactive bacteria suggested the presence of PKS-I in 12 strains, NRPS in 3 strains and both genes in 3 strains. Our results show the potential of the bacterial community associated with Erylus discophorus sponges as producers of bioactive compounds.

Anti-fungal and anti-bacterial activities of ethanol extracts of selected traditional Chinese medicinal herbs.

OBJECTIVE: To evaluate in vitro antimicrobial activities of selected 58 ethno-medicinal plant extracts with a view to assess their therapeutic potential. METHODS: A total of 58 traditional Chinese medicinal plants were carefully selected based on the literature review and their traditional use. The antimicrobial activities of ethanol extracts of these medicinal plants were tested against fungi (Aspergillus fumigatus), yeast (Candida albicans), gram-negative (Acinetobacter baumannii and Pseudomonas aeruginosa) and gram-positive bacteria (Staphylococcus aureus). The activities were tested at three different concentrations of 1.00, 0.10 and 0.01 mg/mL. The data was analysed using Gene data Screener program. RESULTS: The measured antimicrobial activities indicated that out of the 58 plant extracts, 15 extracts showed anti-fungal activity and 23 extracts exhibited anti-bacterial activity. Eight plant extracts have exhibited both anti-bacterial and anti-fungal activities. For instance, Eucommia ulmoides, Polygonum cuspidatum, Poria cocos and Uncaria rhyncophylla showed activity against both bacterial and fungal strains, indicating their broad spectrum of activity. CONCLUSIONS: The results revealed that the ethanol extracts of 30 plants out of the selected 58 possess significant antimicrobial activities. It is interesting to note that the findings from the current study are consistent with the traditional use. A clear correlation has also been found between the antimicrobial activity and the flavonoid content of the plant extracts which is in agreement with the literature. Hence, the results presented here can be used to guide the selection of potential plant species for the isolation and structure elucidation of novel antimicrobial compounds in order to establish the structure-activity relationship. This in turn is expected to lead the way to the discovery of novel antimicrobial agents for therapeutic use.

Balibalosides, an original family of glucosylated sesterterpenes produced by the Mediterranean sponge Oscarella balibaloi.

The chemical investigation of the recently described Mediterranean Homoscleromorpha sponge Oscarella balibaloi revealed an original family of five closely related glucosylated sesterterpenes 1-4, named balibalosides. Their structure elucidation was mainly inferred from NMR and HRMS data analyses. Balibalosides differ by the pattern of acetyl substitutions on the three sugar residues linked to the same aglycone sesterterpenoid core. From a biosynthetic perspective, these compounds may represent intermediates in the pathways leading to more complex sesterterpenes frequently found in Dictyoceratida, a sponge Order belonging to Demospongiae, a clade which is phylogenetically distinct from the Homoscleromorpha. While steroid and triterpenoid saponins were already well known from marine sponges, balibalosides are the first examples of glycosilated sesterterpenes.

A yeast-based in vivo bioassay to screen for class I phosphatidylinositol 3-kinase specific inhibitors.

The phosphatidylinositol 3-kinase (PI3K) pathway couples receptor-mediated signaling to essential cellular functions by generating the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate. This pathway is implicated in multiple aspects of oncogenesis. A low-cost bioassay that readily measures PI3K inhibition in vivo would serve as a valuable tool for research in this field. Using heterologous expression, we have previously reconstituted the PI3K pathway in the model organism Saccharomyces cerevisiae. On the basis of the fact that the overproduction of PI3K is toxic in yeast, we tested the ability of commercial PI3K inhibitors to rescue cell growth. All compounds tested counteracted the PI3K-induced toxicity. Among them, 15e and PI-103 were the most active. Strategies to raise the intracellular drug concentration, specifically the use of 0.003% sodium dodecyl sulfate and the elimination of the Snq2 detoxification pump, optimized the bioassay by enhancing its sensitivity. The humanized yeast-based assay was then tested on a pilot scale for high-throughput screening (HTS) purposes using a collection of natural products of microbial origin. From 9600 extracts tested, 0.6% led to a recovery of yeast growth reproducibly, selectively, and in a dose-dependent manner. Cumulatively, we show that the developed PI3K inhibition bioassay is robust and applicable to large-scale HTS.

A new approach to drug discovery: high-throughput screening of microbial natural extracts against Aspergillus fumigatus using resazurin.

Natural products are an inexhaustible source for drug discovery. However, the validation and selection of primary screening assays are vital to guarantee a selection of extracts or molecules with relevant pharmacological action and worthy of following up. The assay must be rapid, simple, easy to implement, and produce quick results and preferably at a low cost. In this work, we developed and validated a colorimetric microtiter assay using the resazurin viability dye. The parameters of the resazurin method for high-throughput screening (HTS) using natural extracts against Aspergillus fumigatus were optimized and set up. The extracts plus RPMI-1640 modified medium containing the spores and 0.002% resazurin were added per well. The fluorescence was read after 24 to 30 h of incubation. The resazurin proved to be as suitable as Alamar Blue for determining the minimal inhibitory concentration of different antifungals against A. fumigatus and effective to analyze fungicidal and fungistatic compounds. An HTS of 12 000 microbial extracts was carried out against two A. fumigatus strains, and 2.7% of the extracts displayed antifungal activity. Our group has been the first to use this methodology for screening a collection of natural extracts to identify compounds with antifungal activity against the medically important human pathogen A. fumigatus.