Exploring Microbial Small-Molecule Chemistry Using 2D Nmr- And Lc/Esi-Ms-Based Comparative Metabolomics

Microbial biology is integrated with a seemingly endless number of pathways involving small organic molecules. The coordinated regulation of biosynthesis, export, and detection of metabolites confer fitness for the producing microbes in nearly every environment. Continued, rapid evolution has yielded small-molecule biosynthetic enzymes capable of catalyzing an array of reactions that produce chemical species of high structural complexity, diversity, and target specificity. The procurement rate of new chemical entities (NCEs) over the recent decades has decreased, largely due to the redundant discovery of previously characterized chemical species and an incomplete understanding of the regulation and biosynth etic steps involved in microbial metabolic pathways. Methodology to establish the full diversity of metabolites produced by a particular microbial species, its metabolome, and associated biosynthetic genes will greatly accelerate our ability to procure NCEs and explore their biological roles. Metabolomics requires dedicated tools to characterize the small molecule products and biosynthetic potential of an organism. NMR spectroscopy and mass spectrometry are powerful analytical tools for unambiguously characterizing the structures of organic molecules. Therefore, the development of these methods for exploring microbial metabolism should foster access to the yet to be described metabolites of bacterial and fungal small molecule chemistry. Described herein is the development of comparative metabolomics approaches, combining differential analysis by 2D NMR spectroscopy (DANS), LC/ESI-MS profiling, with specific gene overexpression, knock-out, or knock-down strategies to associate and characterize secondary metabolites with their corresponding biosynthetic genes or gene clusters. Applied to the fungus Aspergillus fumigatus's gli cluster, DANS has revealed nine novel gliZ-dependent diketopiperazines, and their identification provides insight into gliotoxin biosynthesis. In a project involving the bacterium Streptomyces clavuligerus's hlm gene cluster, comparative NMR and LC/ESI+-MS demonstrated that the organism largely relies on alkylation though dimerization and/or methylation to detoxify deleteriously reactive thiol hlm pathway intermediates. Finally, as an extension to the A. fumigatus study, DANS is demonstrated to be a powerful tool for systematically examining the products of orphan secondary metabolic gene clusters , supporting the discovery of seven novel alkaloids in Aspergillus flavus

Proton Exchange Reactions in Isotope Chemistry (II) Synthesis of Stable Isotope Labeled LCQ908

Proton exchange reaction, as part of our strategy for the synthesis of stable labeled compounds, was applied to the preparations of stable isotope labeled LCQ908. Suitable intermediate with alpha protons to carbonyl group was first subject to H-D exchange reaction; subsequent coupling of the carbonyl group with 13C2 labeled triethyl phosphonoacetate and hydrogenation converts the labile deuterium to stable ones. Incorporation of 13C2 in the molecule eliminates the presence of undesired M+0

Utilization of stable isotope labeling to facilitate the identification of polar metabolites of KAF156, an antimalarial agent

Identification of polar metabolites of drug candidates during development is often challenging. Several prominent polar metabolites of 2-amino-1-(2-(4-fluorophenyl)-3-((4-fluorophenyl)amino)-8,8-dimethyl- 5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone ([14C]KAF156), an antimalarial agent, were detected in rat urine from an absorption, distribution, metabolism, and excretion study but could not be characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) because of low ionization efficiency. In such instances, a strategy often chosen by investigators is to use a radiolabeled compound with high specific activity, having an isotopic mass ratio (i.e., [12C]/[14C]) and mass difference that serve as the basis for a mass filter using accurate mass spectrometry. Unfortunately, [14C]KAF156-1 was uniformly labeled (n = 1-6) with the mass ratio of ∼0.1. This ratio was insufficient to be useful as a mass filter despite the high specific activity (120 μCi/mg). At this stage in development, stable isotope labeled [13C6]KAF156-1 was available as the internal standard for the quantification of KAF156. We were thus able to design an oral dose as a mixture of [14C]KAF156-1 (specific activity 3.65 μCi/mg) and [13C6]KAF156-1 with a mass ratio of [12C]/[13C6] as 0.9 and the mass difference as 6.0202. By using this mass filter strategy, four polar metabolites were successfully identified in rat urine. Subsequently, using a similar dual labeling approach, [14C]KAF156-2 and [13C2]KAF156-2 were synthesized to allow the detection of any putative polar metabolites that may have lost labeling during biotransformations using the previous [14C]KAF156-1. Three polar metabolites were thereby identified and M43, a less polar metabolite, was proposed as the key intermediate metabolite leading to the formation of a total of seven polarmetabolites. Overall this dual labeling approach proved practical and valuable for the identification of polar metabolites by LC-MS/MS

RsmA Regulates <i>Aspergillus fumigatus</i> Gliotoxin Cluster Metabolites Including Cyclo(L-Phe-L-Ser), a Potential New Diagnostic Marker for Invasive Aspergillosis

<div><p>Dimeric basic leucine zipper (bZIP) proteins are conserved transcriptional enhancers found in all eukaryotes. A recently reported and novel function for bZIPs is association of these proteins with secondary metabolite production in filamentous fungi. In particular a Yap-like bZIP termed RsmA (<u>r</u>estorer of <u>s</u>econdary <u>m</u>etabolism A) was identified in <i>Aspergillus nidulans</i> that positively regulates the carcinogen sterigmatocystin. To assess for conserved function for RsmA, we examined a role of this protein in secondary metabolism in the pathogen <i>A. fumigatus.</i> RsmA was found to positively regulate gliotoxin where overexpression (OE) of <i>rsmA</i> led to 2–100 fold increases of twelve <i>gli</i> cluster metabolites in culture medium including the newly identified <i>gli</i> metabolite cyclo(L-Phe-L-Ser). Lungs from both wild type and <i>OErsmA</i> infected mice contained gliotoxin (2.3 fold higher in <i>OErsmA</i> treatment) as well as the gliotoxin precursor cyclo(L-Phe-L-Ser) (3.2 fold higher in <i>OErsmA</i> treatment). The data here presents a conserved role for RsmA in secondary metabolite cluster activation and suggests cyclo(L-Phe-L-Ser) may serve as an alternative marker for diagnosis of invasive aspergillosis.</p></div

Plasmids used in this study.

<p>Plasmids used in this study.</p

<i>OErsmA</i> mutants are resistant to menadione.

<p>For each strain, 10⁵ conidia in 5 µl were spotted on GMM plates with 20 µM, 30 µM and 40 µM of menadione, or on GMM only for a control. Each strain was replicated 5 times. The plates were incubated at 37°C for 48 h.</p

Average radial growth of <i>A. fumigatus</i> strains.

<p>10⁴ conidia of each strain were point inoculated on GMM and grown at 37°C for 4 days and at 25°C for 12 days (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062591#pone-0062591-g001" target="_blank">Figure 1</a>). Radial growth was measured at the end of each growth period. Means ± standard deviations are indicated for four replicates of each strain. Levels not connected by same letter are significantly different (<i>P</i><0.0001) according to ANOVA analysis.</p

Infected mouse lung extracts (IMLE) HPLC- single ion monitoring (SIM)MS ion chromatograms.

<p>HPLC-SIMMS analysis of crude mouse lung extracts corresponding to mice infected with wild type, or <i>OErsmA</i>. (<b>A</b>) Ion chromatogram showing compound <b>6</b> is approximately two to three times as abundant in the <i>OErsmA</i>-IMLE relative to WT-IMLE. (<b>B</b>) Similarly, gliotoxin is about two times as abundant in <i>OErsmA</i>-IMLE than WT-IMLE. Reference chromatograms (bottom panels) show diagnostic ions of cyclo(L-Phe-L-Ser) (<b>6</b>) and gliotoxin (<b>1</b>). Lung extract chromatograms are scaled to the peaks measured in the <i>OErsmA</i>-IMLE sample (bottom panels of standards are not to scale).</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p