Microbiome metabolite quantification methods enabling insights into human health and disease.

Many of the health-associated impacts of the microbiome are mediated by its chemical activity, producing and modifying small molecules (metabolites). Thus, microbiome metabolite quantification has a central role in efforts to elucidate and measure microbiome function. In this review, we cover general considerations when designing experiments to quantify microbiome metabolites, including sample preparation, data acquisition and data processing, since these are critical to downstream data quality. We then discuss data analysis and experimental steps to demonstrate that a given metabolite feature is of microbial origin. We further discuss techniques used to quantify common microbial metabolites, including short-chain fatty acids (SCFA), secondary bile acids (BAs), tryptophan derivatives, N-acyl amides and trimethylamine N-oxide (TMAO). Lastly, we conclude with challenges and future directions for the field.

Iron(III) Binding Properties of PF1140, a Fungal N-Hydroxypyridone, and Activity against Mycoplasma genitalium.

Mycoplasma genitalium is a sexually transmitted bacterium associated with urogenital disease syndromes in the US and worldwide. The global rise in drug resistance in M. genitalium necessitates the development of novel drugs to treat this pathogen. To address this need, we have screened extracts from a library of fungal isolates assembled through the University of Oklahoma Citizen Science Soil Collection Program. Analysis of one of the bioactive extracts using bioassay-guided fractionation led to the purification of the compound PF1140 (1) along with a new and several other known pyridones. The N-hydroxy pyridones are generally regarded as siderophores with high binding affinity for iron(III) under physiological conditions. Results from UV-vis absorption spectroscopy-based titration experiments revealed that 1 complexes with Fe3+. As M. genitalium does not utilize iron, we propose that the PF1140-iron complex induces cytotoxicity by facilitating the cellular uptake of iron, which reacts with endogenous hydrogen peroxide to produce toxic hydroxyl radicals.

Inhibitor of Chromosome Segregation in Pseudomonas aeruginosa from Fungal Extracts.

Chromosome segregation is an essential cellular process that has the potential to yield numerous targets for drug development. This pathway is presently underutilized partially due to the difficulties in the development of robust reporter assays suitable for high throughput screening. In bacteria, chromosome segregation is mediated by two partially redundant systems, condensins and ParABS. Based on the synthetic lethality of the two systems, we developed an assay suitable for screening and then screened a library of fungal extracts for potential inhibitors of the ParABS pathway, as judged by their enhanced activity on condensin-deficient cells. We found such activity in extracts of Humicola sp. Fractionation of the extract led to the discovery of four new analogues of sterigmatocystin, one of which, 4-hydroxy-sterigmatocystin (4HS), displayed antibacterial activity. 4HS induced the phenotype typical for parAB mutants including defects in chromosome segregation and cell division. Specifically, bacteria exposed to 4HS produced anucleate cells and were impaired in the assembly of the FtsZ ring. Moreover, 4HS binds to purified ParB in a ParS-modulated manner and inhibits its ParS-dependent CTPase activity. The data describe a small molecule inhibitor of ParB and expand the known spectrum of activities of sterigmatocystin to include bacterial chromosome segregation.

Rationally Minimizing Natural Product Libraries Using Mass Spectrometry.

Natural product libraries are crucial to drug development, but large libraries drastically increase the time and cost during initial high throughput screens. Here, we developed a method that leverages liquid chromatography-tandem mass spectrometry spectral similarity to dramatically reduce library size, with minimal bioactive loss. This method offers a broadly applicable strategy for accelerated drug discovery with cost reductions, which enable implementation in resource-limited settings.

Yuanhuacine Is a Potent and Selective Inhibitor of the Basal-Like 2 Subtype of Triple Negative Breast Cancer with Immunogenic Potential.

The heterogeneity of triple negative breast cancer (TNBC) has led to efforts to further subtype this disease with the hope of identifying new molecular liabilities and drug targets. Furthermore, the finding that TNBC is the most inherently immunogenic type of breast cancer provides the potential for effective treatment with immune checkpoint inhibitors and immune adjuvants. Thus, we devised a dual screen to identify compounds from natural product extracts with TNBC subtype selectivity that also promote the expression of cytokines associated with antitumor immunity. These efforts led to the identification of yuanhuacine (1) as a potent and highly selective inhibitor of the basal-like 2 (BL2) subtype of TNBC that also promoted an antitumor associated cytokine signature in immune cells. The mechanism of action of yuanhuacine for both phenotypes depends on activation of protein kinase C (PKC), defining a novel target for the treatment of this clinical TNBC subtype. Yuanhuacine showed potent antitumor efficacy in animals bearing BL2 tumors further demonstrating that PKC could function as a potential pharmacological target for the treatment of the BL2 subtype of TNBC.