Natural-Product-Inspired Compounds as Countermeasures against the Liver Carcinogen Aflatoxin B1.

Aflatoxin B1 (AfB1) ranks among the most potent liver carcinogens known, and the accidental or intentional exposure of humans and livestock to this toxin remains a serious global threat. One protective measure that had been proposed is employing small-molecule therapeutics capable of mitigating the toxicity of AfB1; however, to date, these efforts have had little clinical success. To identify molecular scaffolds that reduce the toxicity of AfB1, we developed a cell-based high-throughput high-content imaging assay that enabled our team to test natural products (pure compounds, fractions, and extracts) for protection of monolayers and spheroids composed of HepG2 liver cells against AfB1. The spheroid assay showed notable potential for further development, as it afforded greater sensitivity of HepG2 cells to AfB1, which is believed to better mimic the in vivo response of hepatocytes to the toxin. One of the most bioactive compounds to arise from this investigation was alternariol-9-methyl ether (1, purified from an Alternaria sp. isolate), which inspired the synthesis and testing of several structurally related molecules. Based on these findings, it is proposed that several types of natural and synthetic polyarene molecules that have undergone oxidative functionalization (e.g., compounds containing 3-methoxyphenol moieties) are promising starting points for the development of new agents that protect against AfB1 toxicity.

Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling.

Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated.

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides.

Mass-spectrometry-based metabolomics and molecular phylogeny data were used to identify a metabolically prolific strain of Tolypocladium that was obtained from a deep-water Great Lakes sediment sample. An investigation of the isolate's secondary metabolome resulted in the purification of a 22-mer peptaibol, gichigamin A (1). This peptidic natural product exhibited an amino acid sequence including several ß-alanines that occurred in a repeating ααß motif, causing the compound to adopt a unique right-handed 311 helical structure. The unusual secondary structure of 1 was confirmed by spectroscopic approaches including solution NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. Artificial and cell-based membrane permeability assays provided evidence that the unusual combination of structural features in gichigamins conferred on them an ability to penetrate the outer membranes of mammalian cells. Compound 1 exhibited potent in vitro cytotoxicity (GI50 0.55 ± 0.04 µM) and in vivo antitumor effects in a MIA PaCa-2 xenograft mouse model. While the primary mechanism of cytotoxicity for 1 was consistent with ion leakage, we found that it was also able to directly depolarize mitochondria. Semisynthetic modification of 1 provided several analogs, including a C-terminus-linked coumarin derivative (22) that exhibited appreciably increased potency (GI50 5.4 ± 0.1 nM), but lacked ion leakage capabilities associated with a majority of naturally occurring peptaibols such as alamethicin. Compound 22 was found to enter intact cells and induced cell death in a process that was preceded by mitochondrial depolarization.

Osteoclast Differentiation is Downregulated by Select Polyphenolic Fractions from Dried Plum via Suppression of MAPKs and Nfatc1 in Mouse C57BL/6 Primary Bone Marrow Cells.

Background: Clinical and preclinical studies have shown that dietary supplementation with dried plum improves bone health. These osteoprotective effects are a result, in part, of the antiresorptive properties of the fruit, which appear to be mediated by its polyphenolic compounds. Objective: This study was designed to determine if certain fractions of the polyphenolic compounds in dried plums are responsible for the antiresorptive effects and whether they alter mitogen-activated protein kinase (MAPK) and calcium signaling, which are essential to osteoclast differentiation and activity, under normal and inflammatory conditions. Methods: Six polyphenolic fractions were derived from the total polyphenolic extract of dried plum based on solubility. Initial screening, with the use of the Raw 264.7 monocyte and macrophage cell line, showed that 3 fractions had the most marked capacity to downregulate osteoclast differentiation. This response was confirmed in 2 of the fractions by using primary bone marrow-derived cultures and in all subsequent experiments to determine how osteoclast differentiation and function were altered with a focus on these 2 fractions in primary cultures. Data were analyzed by using ANOVA followed by post hoc analyses. Results: Both of the polyphenol fractions decreased osteoclast differentiation and activity coincident with downregulating nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (Nfatc1), which is required for osteoclast differentiation. Calcium signaling, essential for the auto-amplification of Nfatc1, was suppressed by the polyphenolic fractions under normal conditions as indicated by suppressed mRNA expression of costimulatory receptors osteoclast-associated receptor (Oscar), signaling regulatory protein ß1 (Sirpb1), and triggering receptor expressed on myeloid cells 2 (Trem2). In contrast, in the presence of tumor necrosis factor α (TNF-α), only Sirpb1 was downregulated. In addition to calcium signaling, phosphorylation of extracellular signal-regulated kinase (Erk) and p38 MAPK, involved in the expression and activation of Nfatc1, was also suppressed by the polyphenolic fractions. Conclusion: These results show that certain types of polyphenolic compounds from dried plum downregulate calcium and MAPK signaling, resulting in suppression of Nfatc1 expression, which ultimately decreases osteoclast formation and activity.

A potent HDAC inhibitor, 1-alaninechlamydocin, from a Tolypocladium sp. induces G2/M cell cycle arrest and apoptosis in MIA PaCa-2 cells.

The cyclic tetrapeptide 1-alaninechlamydocin was purified from a Great Lakes-derived fungal isolate identified as a Tolypocladium sp. Although the planar structure was previously described, a detailed analysis of its spectroscopic data and biological activity are reported here for the first time. Its absolute configuration was determined using a combination of spectroscopic ((1)H-(1)H ROESY, ECD, and X-ray diffraction) and chemical (Marfey's analysis) methods. 1-Alaninechlamydocin showed potent antiproliferative/cytotoxic activities in a human pancreatic cancer cell line (MIA PaCa-2) at low-nanomolar concentrations (GI50 5.3 nM, TGI 8.8 nM, LC50 22 nM). Further analysis revealed that 1-alaninechlamydocin induced G2/M cell cycle arrest and apoptosis. Similar to other cyclic epoxytetrapeptides, the inhibitory effects of 1-alaninechlamydocin are proposed to be produced primarily via inhibition of histone deacetylase (HDAC) activity.

Cytotoxic dimeric epipolythiodiketopiperazines from the ascomycetous fungus Preussia typharum.

Two new dimeric epipolythiodiketopiperazines, preussiadins A (1) and B (2), together with two known diastereomers, leptosins C (6) and A (7), were obtained from the mycelia of a Preussia typharum isolate. The structures of the new compounds were established by spectroscopic methods, and the absolute configurations of 1 and 2 were assigned by chemical transformations and comparisons of quantum chemical ECD and VCD calculations to experimental data. Compound 1 exhibited potent cytotoxic activity in the NCI-60 cell line panel with an average LC50 value of 251 nM. Further studies demonstrated that 1 circumvents P-glycoprotein-mediated drug resistance, yet had no significant antitumor activity in a xenograft UACC-62 melanoma model.

Crowdsourcing natural products discovery to access uncharted dimensions of fungal metabolite diversity.

A fundamental component for success in drug discovery is the ability to assemble and screen compounds that encompass a broad swath of biologically relevant chemical-diversity space. Achieving this goal in a natural-products-based setting requires access to a wide range of biologically diverse specimens. For this reason, we introduced a crowdsourcing program in which citizen scientists furnish soil samples from which new microbial isolates are procured. Illustrating the strength of this approach, we obtained a unique fungal metabolite, maximiscin, from a crowdsourced Alaskan soil sample. Maximiscin, which exhibits a putative combination of polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and shikimate pathway components, was identified as an inhibitor of UACC-62 melanoma cells (LC50=0.93 µM). The metabolite also exhibited efficacy in a xenograft mouse model. These results underscore the value of building cooperative relationships between research teams and citizen scientists to enrich drug discovery efforts.

Production of cytotoxic glidobactins/luminmycins by Photorhabdus asymbiotica in liquid media and live crickets.

Photorhabdus asymbiotica engages in a two-part life cycle that requires adaptation to both symbiotic and pathogenic phases. The genome of P. asymbiotica contains several gene clusters, which are predicted to be involved in the biosynthesis of unique secondary metabolites that are hypothesized to enhance the bacterium's pathogenic capabilities. However, recent reports on Photorhabdus secondary metabolite production have indicated that many of its genes are silent under laboratory culture conditions. Using a circumscribed panel of media and alternative fermentation conditions, we have successfully achieved the production of a series of new and known glidobactin/luminmycin derivatives from P. asymbiotica including glidobactin A (1), luminmycin A (2), and luminmycin D (3). These compounds were also obtained upon infection of live crickets with the bacterium. Luminmycin D showed cytotoxicity against human pancreatic cells (IC50 of 0.11 µM), as well as proteasome inhibition (IC50 of 0.38 µM).

Secondary metabolites produced by fungi derived from a microbial mat encountered in an iron-rich natural spring.

A collection of fungal isolates was obtained from a complex microbial mat, which occupied an iron-rich freshwater spring that feeds into Clear Creek, Golden, Colorado, USA. Two of the fungal isolates, a Glomeromycete (possible Entrophospora sp.) and a Dothideomycete (possible Phaeosphaeria sp.), were investigated for bioactive secondary metabolites. In total, six new compounds consisting of clearanols A-E (5, 6, 10-12) and disulochrin (7) were purified and their structures were determined. Disulochrin exhibited modest antibacterial activity against methicillin-resistant Staphylococcus aureus, whereas clearanol C showed weak inhibitory activity against Candida albicans biofilm formation.

Association of a functional variant downstream of TNFAIP3 with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE, MIM152700) is an autoimmune disease characterized by self-reactive antibodies resulting in systemic inflammation and organ failure. TNFAIP3, encoding the ubiquitin-modifying enzyme A20, is an established susceptibility locus for SLE. By fine mapping and genomic re-sequencing in ethnically diverse populations, we fully characterized the TNFAIP3 risk haplotype and identified a TT>A polymorphic dinucleotide (deletion T followed by a T to A transversion) associated with SLE in subjects of European (P = 1.58 × 10(-8), odds ratio = 1.70) and Korean (P = 8.33 × 10(-10), odds ratio = 2.54) ancestry. This variant, located in a region of high conservation and regulatory potential, bound a nuclear protein complex composed of NF-κB subunits with reduced avidity. Further, compared with the non-risk haplotype, the haplotype carrying this variant resulted in reduced TNFAIP3 mRNA and A20 protein expression. These results establish this TT>A variant as the most likely functional polymorphism responsible for the association between TNFAIP3 and SLE.

A novel, species-specific class of uncompetitive inhibitors of gamma-glutamyl transpeptidase.

Expression of gamma-glutamyl transpeptidase (GGT) in tumors contributes to resistance to radiation and chemotherapy. GGT is inhibited by glutamine analogues that compete with the substrate for the gamma-glutamyl binding site. However, the glutamine analogues that have been evaluated in clinical trials are too toxic for use in humans. We have used high throughput screening to evaluate small molecules for their ability to inhibit GGT and have identified a novel class of inhibitors that are not glutamine analogues. These compounds are uncompetitive inhibitors, binding the gamma-glutamyl enzyme complex. OU749, the lead compound, has an intrinsic K(i) of 17.6 microm. It is a competitive inhibitor of the acceptor glycyl-glycine, which indicates that OU749 occupies the acceptor site while binding to the gamma-glutamyl substrate complex. OU749 is more than 150-fold less toxic than the GGT inhibitor acivicin toward dividing cells. Inhibition of GGT by OU749 is species-specific, inhibiting GGT isolated from human kidney with 7-10-fold greater potency than GGT isolated from rat or mouse kidney. OU749 does not inhibit GGT from pig cells. Human GGT expressed in mouse fibroblasts is inhibited by OU749 similarly to GGT from human cells, which indicates that the species specificity is determined by differences in the primary structure of the protein rather than species-specific, post-translational modifications. These studies have identified a novel class of inhibitors of GGT, providing the basis for further development of a new group of therapeutics that inhibit GGT by a mechanism distinct from the toxic glutamine analogues.

Role of glutathione S-transferase Pi in cisplatin-induced nephrotoxicity.

One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyl transpeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes (GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have approximately 10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum-glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences.