Shengxin Cai

Spiro fused diterpene-indole alkaloids from a creek-bottom-derived Aspergillus terreus

Four metabolites, teraspiridoles A-D (2-5), formed from the merger of a diterpene and modified indole scaffold were obtained from an Aspergillus terreus isolate. The structures and absolute configurations of these natural products were established using NMR, mass spectrometry, Marfeys method, VCD, and ECD data. Teraspiridole B (3) exhibited weak inhibition of planaria regeneration/survival.

Polyketide glycosides from Bionectria ochroleuca inhibit Candida albicans biofilm formation.

One of the challenges presented by Candida infections is that many of the isolates encountered in the clinic produce biofilms, which can decrease these pathogens’ susceptibilities to standard-of-care antibiotic therapies. Inhibitors of fungal biofilm formation offer a potential solution to counteracting some of the problems associated with Candida infections. A screening campaign utilizing samples from our fungal extract library revealed that a Bionectria ochroleuca isolate cultured on Cheerios breakfast cereal produced metabolites that blocked the in vitro formation of Candida albicans biofilms. A scale-up culture of the fungus was undertaken using mycobags (also known as mushroom bags or spawn bags), which afforded four known [TMC-151s C–F (1–4)] and three new [bionectriols B–D (5–7)] polyketide glycosides. All seven metabolites exhibited potent biofilm inhibition against C. albicans SC5314, as well as exerted synergistic antifungal activities in combination with amphotericin B. In this report, we describe the structure determination of the new metabolites, as well as compare the secondary metabolome profiles of fungi grown in flasks and mycobags. These studies demonstrate that mycobags offer a useful alternative to flask-based cultures for the preparative production of fungal secondary metabolites.

Spatial Molecular Architecture of the Microbial Community of a Peltigera Lichen

Microbial communities have evolved over centuries to live symbiotically. The direct visualization of such communities at the chemical and functional level presents a challenge. Overcoming this challenge may allow one to visualize the spatial distributions of specific molecules involved in symbiosis and to define their functional roles in shaping the community structure. In this study, we examined the diversity of microbial genes and taxa and the presence of biosynthetic gene clusters by metagenomic sequencing and the compartmentalization of organic chemical components within a lichen using mass spectrometry. This approach allowed the identification of chemically distinct sections within this composite organism. Using our multipronged approach, various fungal natural products, not previously reported from lichens, were identified and two different fungal layers were visualized at the chemical level. ABSTRACT Microbes are commonly studied as individual species, but they exist as mixed assemblages in nature. At present, we know very little about the spatial organization of the molecules, including natural products that are produced within these microbial networks. Lichens represent a particularly specialized type of symbiotic microbial assemblage in which the component microorganisms exist together. These composite microbial assemblages are typically comprised of several types of microorganisms representing phylogenetically diverse life forms, including fungi, photosymbionts, bacteria, and other microbes. Here, we employed matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry to characterize the distributions of small molecules within a Peltigera lichen. In order to probe how small molecules are organized and localized within the microbial consortium, analytes were annotated and assigned to their respective producer microorganisms using mass spectrometry-based molecular networking and metagenome sequencing. The spatial analysis of the molecules not only reveals an ordered layering of molecules within the lichen but also supports the compartmentalization of unique functions attributed to various layers. These functions include chemical defense (e.g., antibiotics), light-harvesting functions associated with the cyanobacterial outer layer (e.g., chlorophyll), energy transfer (e.g., sugars) surrounding the sun-exposed cyanobacterial layer, and carbohydrates that may serve a structural or storage function and are observed with higher intensities in the non-sun-exposed areas (e.g., complex carbohydrates). IMPORTANCE Microbial communities have evolved over centuries to live symbiotically. The direct visualization of such communities at the chemical and functional level presents a challenge. Overcoming this challenge may allow one to visualize the spatial distributions of specific molecules involved in symbiosis and to define their functional roles in shaping the community structure. In this study, we examined the diversity of microbial genes and taxa and the presence of biosynthetic gene clusters by metagenomic sequencing and the compartmentalization of organic chemical components within a lichen using mass spectrometry. This approach allowed the identification of chemically distinct sections within this composite organism. Using our multipronged approach, various fungal natural products, not previously reported from lichens, were identified and two different fungal layers were visualized at the chemical level.

Bioactive sulfur-containing sulochrin dimers and other metabolites from an alternaria sp. isolate from a Hawaiian soil sample

Polluxochrin (1) and dioschrin (2), two new dimers of sulochrin linked by thioether bonds, were purified from an Alternaria sp. isolate obtained from a Hawaiian soil sample. The structures of the two metabolites were established by NMR, mass spectrometry data, and X-ray analysis. Metabolite 1 was determined to be susceptible to intramolecular cyclization under aqueous conditions, resulting in the generation of 2 as well as another dimeric compound, castochrin (3). An additional nine new metabolites were also obtained, including four new pyrenochaetic acid derivatives (8–11), one new asterric acid analogue (13), and four new secalonic acid analogues (14–17). Bioassay analysis of these compounds revealed 1–3 displayed antimicrobial and weak cytotoxic activities.

Identification of Compounds with Efficacy against Malaria Parasites from Common North American Plants

Some of the most valuable antimalarial compounds, including quinine and artemisinin, originated from plants. While these drugs have served important roles over many years for the treatment of malaria, drug resistance has become a widespread problem. Therefore, a critical need exists to identify new compounds that have efficacy against drug-resistant malaria strains. In the current study, extracts prepared from plants readily obtained from local sources were screened for activity against Plasmodium falciparum. Bioassay-guided fractionation was used to identify 18 compounds from five plant species. These compounds included eight lupane triterpenes (1-8), four kaempferol 3-O-rhamnosides (10-13), four kaempferol 3-O-glucosides (14-17), and the known compounds amentoflavone and knipholone. These compounds were tested for their efficacy against multi-drug-resistant malaria parasites and counterscreened against HeLa cells to measure their antimalarial selectivity. Most notably, one of the new lupane triterpenes (3) isolated from the supercritical extract of Buxus sempervirens, the common boxwood, showed activity against both drug-sensitive and -resistant malaria strains at a concentration that was 75-fold more selective for the drug-resistant malaria parasites as compared to HeLa cells. This study demonstrates that new antimalarial compounds with efficacy against drug-resistant strains can be identified from native and introduced plant species in the United States, which traditionally have received scant investigation compared to more heavily explored tropical and semitropical botanical resources from around the world.

Structure–Activity Relationships of New Natural Product-Based Diaryloxazoles with Selective Activity against Androgen Receptor-Positive Breast Cancer Cells

Targeted therapies for ER+/PR+ and HER2-amplified breast cancers have improved patient survival, but there are no therapies for triple negative breast cancers (TNBC) that lack expression of estrogen and progesterone receptors (ER/PR), or amplification or overexpression of HER2. Gene expression profiling of TNBC has identified molecular subtypes and representative cell lines. An extract of the Texas native plant Amyris texana was found to have selective activity against MDA-MB-453 cells, a model of the luminal androgen receptor (LAR) subtype of TNBC. Bioassay-guided fractionation identified two oxazole natural products with selective activity against this cell line. Conducted analog synthesis and structure-activity relationship studies provided analogs with more potent and selective activity against two LAR subtype cell line models, culminating in the discovery of compound 30 (CIDD-0067106). Lead compounds discovered have potent and selective antiproliferative activities, and mechanisms of action studies show they inhibit the activity of the mTORC1 pathway.

Texas Native Plants Yield Compounds with Cytotoxic Activities against Prostate Cancer Cells

There remains a critical need for more effective therapies for the treatment of late-stage and metastatic prostate cancers. Three Texas native plants yielded three new and three known compounds with antiproliferative and cytotoxic activities against prostate cancer cells with IC50 values in the range of 1.7-35.0 μM. A new sesquiterpene named espadalide (1), isolated from Gochnatia hypoleuca, had low micromolar potency and was highly effective in clonogenic assays. Two known bioactive germacranolides (2 and 3) were additionally isolated from G. hypoleuca. Dalea frutescens yielded two new isoprenylated chalcones, named sanjuanolide (4) and sanjoseolide (5), and the known sesquiterpenediol verbesindiol (6) was isolated from Verbesina virginica. Mechanistic studies showed that 1-4 caused G2/M accumulation and the formation of abnormal mitotic spindles. Tubulin polymerization assays revealed that 4 increased the initial rate of tubulin polymerization, but did not change total tubulin polymer levels, and 1-3 had no effects on tubulin polymerization. Despite its cytotoxic activity, compound 6 did not initiate changes in cell cycle distribution and has a mechanism of action different from the other compounds. This study demonstrates that new compounds with significant biological activities germane to unmet oncological needs can be isolated from Texas native plants.

Abstract P5-03-04: Identification of compounds from natural sources with selective activity against triple-negative breast cancer molecular subtypes

Triple negative breast cancers (TNBCs) lack expression of the estrogen and progesterone receptors (ER/PR) and do not have amplified HER2. While targeted therapies for ER+/PR+ and HER2-amplified breast cancers have greatly improved patient survival, there are no targeted therapies for TNBCs and no effective therapies to treat metastatic disease. There is a need to identify new therapeutic agents and molecular targets for treating TNBCs, but efforts have been limited by a lack of understanding of the subtypes of these heterogeneous diseases. However, gene expression profiling of TNBC patients recently identified 6 molecular subtypes of TNBC and representative cell lines, providing the first opportunity to identify subtype-specific leads for TNBC. We performed high-content screening to evaluate novel libraries of extracts from Texas plants and diverse fungal cultures for antiproliferative and/or cytotoxic activity in a panel of cell lines modeling five different TNBC molecular subtypes. The aim was to identify extracts with selective activity in a single cell line. We hypothesized that extracts found to have selective activity in one of these cell lines may target a protein or cellular process critical to the growth of that subtype. We identified 11 extracts with selective activity against cell lines representing four different TNBC molecular subtypes. From a fungal culture we identified a new compound called maximiscin, which was found to have selective cytotoxic efficacy against the MDA-MB-468 cell line of the basal-like 1 subtype. From a plant extract we isolated deguelin, which had selective activity in the MDA-MB-453 cell line, a model of the luminal androgen receptor (LAR) subtype. The molecular mechanisms of action of each compound were investigated in cell line models. Initial cell cycle studies using flow cytometry showed that maximiscin caused an accumulation of cells in G1 after 18h of treatment. Protein microarray studies indicated that maximiscin increased levels of phospho-p53, which was consistent with the observed G1 accumulation. Based on these findings, we hypothesized that maximiscin induces DNA damage and investigated the effects of maximiscin on the phosphorylation of several DNA damage response proteins. Maximiscin increased phosphorylation of Chk1, Chk2, p53 and H2A.X as soon as 2h after treatment, indicating an accumulation of DNA damage. Previous studies have shown that LAR TNBC cells are particularly sensitive to PI3K inhibitors in vitro compared to other TNBC subtypes. The effects of deguelin on PI3K-Akt-mTORC1 signaling were evaluated in both MDA-MB-453 and MDA-MB-231 cells. Phosphorylation of both ribosomal protein S6 and 4E-BP1 were dramatically reduced in MDA-MB-453 cells 2h after deguelin treatment. Interestingly this was not observed in MDA-MB-231 cells, suggesting inhibition of mTORC1 signaling may be involved in the selective activity of deguelin in MDA-MB-453 cells. Preliminary studies suggest deguelin may also decrease androgen receptor abundance in MDA-MB-453 cells, indicating multiple molecular mechanisms may be involved in its selective effects. These results demonstrate that compounds with selective activity against TNBC subtypes can be identified from nature. Citation Format: Robles AJ, Du L, Cai S, Risinger AL, Cichewicz RH, Mooberry SL. Identification of compounds from natural sources with selective activity against triple-negative breast cancer molecular subtypes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-04.

Abstract P5-04-18: Extracts derived from fungi and plants demonstrate specificity for subtyptes of triple negative breast cancer

New effective therapies are needed for patients with triple negative breast cancers (TNBC). The identification by Lehmann and Bauer 1 of distinct subtypes of TNBC and representative cell lines that are driven by different defects and signaling pathways provided the opportunity, for the first time, to screen for selective activities against these subtypes of TNBC. Using this knowledge, we initiated a screen of diverse natural product extract libraries with the goal of identifying extracts selective for subtypes of TNBC. The compounds with this selective activity will then be purified using bioassay-guided fractionation. Drugs derived from plants and fungi have provided some of the most important pharmaceuticals used today, including numerous anticancer agents. 2 Natural products occupy a biologically validated chemical space that does not overlap with compounds found in most synthetic chemical libraries. 3 Additionally, there are differences in chemical space between plant and fungal-derived compounds 4 and different compound classes are expected to be isolated from these two sources. A total of 1,953 extracts of fungi collected from diverse environments, including Great Lakes sediments and 2,200 plant extracts from tropical environments have been screened for selective cytotoxic activities against cell lines representing 5 subtypes of TNBC. These subtypes are the basal-like 1 and 2 (BL1, BL2), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR). The initial screening using one concentration, 2 µg/ml for fungal extracts and 20 µg/ml for plant extracts, identified many extracts with selective activity against the TNBC subtypes. Detailed dose response curves were then generated with these extracts in each of the TNBC cell lines. A total of 4 fungal extracts and 7 plant extracts with selective cytotoxic activities were identified with selectivity up to 100-fold for 3 of the extracts. Bioassay-guided fractionation is ongoing to identify the active constituents. These results demonstrate that natural product extracts can yield selective actions against TNBC subtypes. We expect that these plant and fungal extracts will yield compounds that target molecular drivers specific to the TNBC subtypes. It is our expectation that compounds with selective, targeted activities will continue to be isolated from these extract collections. 1. Lehman BD and Bauer JA et al. J Clin Invest: 121, 2750-2767, 2011. 2. Newman DJ and Cragg GM. J Nat Prod: 75, 311-335, 2012. 3. Feher M and Schmidt JM. J Chem Inf Comput Sci: 43, 218-227, 2003. 4. El-Elimat, T et al. ACS Med Chem Lett: 3, 645-649, 2012. Citation Format: Shaffer CV, Cai S, Perez A, Risinger AL, Du L, O9Keefe BR, Cichewicz RH, Mooberry SL. Extracts derived from fungi and plants demonstrate specificity for subtyptes of triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-18.

Abstract 5545: Deguelin selectively inhibits proliferation of luminal androgen receptor (LAR) triple-negative breast cancer cells

Triple negative breast cancers (TNBCs) lack expression of the estrogen and progesterone receptors (ER/PR) and HER2 amplification. These aggressive malignancies have higher mortality rates than other breast cancer subtypes. While targeted therapies for ER+/PR+ and HER2-amplified breast cancers have greatly improved survival for these patients, there are no approved targeted therapies for TNBCs. Development of targeted therapies for TNBC will likely provide a similar survival benefit over traditional cytotoxic therapy. The identification of molecular targets for TNBC has been limited without a better understanding of the subtypes of these heterogeneous diseases. Gene expression profiling recently identified 6 defined subtypes of TNBC and representative cell lines, providing the first opportunity to identify subtype-specific leads for TNBC. In this study, we used high-content phenotypic screening to evaluate novel libraries of plant and fungal extracts for antiproliferative and cytotoxic activity in a panel of cell lines modeling the different TNBC subtypes. The aim was to identify extracts with selective activity in a single cell line with the hypothesis that the selectivity resulted from a target relevant specifically to that subtype. A plant extract selectively inhibited proliferation of MDA-MB-453 cells, which represent the luminal androgen receptor (LAR) subtype. Bioassay-guided fractionation identified deguelin, which showed potent and highly selective activity in MDA-MB-453 cells compared to all the other TNBC cell lines, with 240 to 1000-fold higher potency. Further studies investigated the molecular mechanisms of action of degeulin in MDA-MB-453 cells. Based on the sensitivity of LAR TNBCs to both antiandrogens and Hsp90 inhibitors, as well as prior studies demonstrating Hsp90 inhibition by deguelin, we hypothesized that deguelin9s mechanism in MDA-MB-453 cells involves Hsp90 inhibition, resulting in AR destabilization and reduced proliferation. Immunoblotting of deguelin-treated MDA-MB-453 whole-cell lysates indicated that deguelin reduced cellular levels of AR, which are noticeable 8 h after treatment. Immunofluorescence microscopy studies also suggested reduced nuclear localization of AR 18 h after treatment. Previous studies showed that LAR TNBC cells are highly sensitive to PI3K inhibitors compared to other TNBC subtypes, and thus the effects of deguelin on PI3K-Akt signaling were evaluated in MDA-MB-453 cells. Immunoblotting indicated that deguelin reduced the relative phosphorylation of Akt at T308, but had no effect on phosphorylation of S473, suggesting inhibition of PI3K but not mTORC2. Ongoing studies are aimed at better characterizing the mechanisms of deguelin in MDA-MB-453 cells with the goal of identifying targets to develop better therapies for LAR TNBC patients. Citation Format: Andrew J. Robles, Shengxin Cai, Robert H. Cichewicz, Susan L. Mooberry. Deguelin selectively inhibits proliferation of luminal androgen receptor (LAR) triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5545. doi:10.1158/1538-7445.AM2015-5545