Andrew J. Robles

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.

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.

Melampodium leucanthum, a Source of Cytotoxic Sesquiterpenes with Antimitotic Activities

A new tricyclic sesquiterpene, named meleucanthin (1), was isolated from an extract of the leaves and branches of Melampodium leucanthum, along with four known germacranolide sesquiterpene lactones, leucanthin-A (2), leucanthin-B (3), melampodin-A acetate (4), and 3α-hydroxyenhydrin (5). The chemical structure of 1 was elucidated by analysis of 1D and 2D NMR and mass spectrometric data. All compounds exhibited antiproliferative and cytotoxic efficacy against PC-3 and DU 145 prostate cancer cells, as well as HeLa cervical cancer cells, with IC50 values ranging from 0.18 to 9 μM. These compounds were effective in clonogenic assays and displayed high cellular persistence. They were also found to be capable of circumventing P-glycoprotein-mediated drug resistance. Mechanism of action studies showed that 4 caused an accumulation of cells in the G2/M phase of the cell cycle, and 2-5 caused the formation of abnormal mitotic spindles. These results suggest the cytotoxic effects of these germacranolides involve inhibition of mitotic spindle function, and it is likely that other mechanisms additionally contribute to cell death. These studies also demonstrate the possibility of isolating new, biologically active compounds from indigenous Texas plants.

Maximiscin Induces DNA Damage, Activates DNA Damage Response Pathways, and Has Selective Cytotoxic Activity against a Subtype of Triple-Negative Breast Cancer

Triple-negative breast cancers are highly aggressive, and patients with these types of tumors have poor long-term survival. These breast cancers do not express estrogen or progesterone receptors and do not have gene amplification of human epidermal growth factor receptor 2; therefore, they do not respond to available targeted therapies. The lack of targeted therapies for triple-negative breast cancers stems from their heterogeneous nature and lack of a clear definition of driver defects. Studies have recently identified triple-negative breast cancer molecular subtypes based on gene expression profiling and representative cell lines, allowing for the identification of subtype-specific drug leads and molecular targets. We previously reported the identification of a new fungal metabolite named maximiscin (1) identified through a crowdsourcing program. New results show that 1 has selective cytotoxic efficacy against basal-like 1 MDA-MB-468 cells compared to cell lines modeling other triple-negative breast cancer molecular subtypes. This compound also exhibited antitumor efficacy in a xenograft mouse model. The mechanisms of action of 1 in MDA-MB-468 cells were investigated to identify potential molecular targets and affected pathways. Compound 1 caused accumulation of cells in the G1 phase of the cell cycle, suggesting induction of DNA damage. Indeed, treatment with 1 caused DNA double-strand breaks with concomitant activation of the DNA damage response pathways, indicated by phosphorylation of p53, Chk1, and Chk2. Collectively, these results suggest basal-like triple-negative breast cancer may be inherently sensitive to DNA-damaging agents relative to other triple-negative breast cancer subtypes. These results also demonstrate the potential of our citizen crowdsourcing program to identify new lead molecules for treating the subtypes of triple-negative breast cancer.

Pharmacokinetic Analysis and in Vivo Antitumor Efficacy of Taccalonolides AF and AJ

The taccalonolides are microtubule stabilizers that covalently bind tubulin and circumvent clinically relevant forms of resistance to other drugs of this class. Efforts are under way to identify a taccalonolide with optimal properties for clinical development. The structurally similar taccalonolides AF and AJ have comparable microtubule-stabilizing activities in vitro, but taccalonolide AF has excellent in vivo antitumor efficacy when administered systemically, while taccalonolide AJ does not elicit this activity even at maximum tolerated dose. The hypothesis that pharmacokinetic differences underlie the differential efficacies of taccalonolides AF and AJ was tested. The effects of serum on their in vivo potency, metabolism by human liver microsomes and in vivo pharmacokinetic properties were evaluated. Taccalonolides AF and AJ were found to have elimination half-lives of 44 and 8.1 min, respectively. Furthermore, taccalonolide AJ was found to have excellent and highly persistent antitumor efficacy when administered directly to the tumor, suggesting that the lack of antitumor efficacy seen with systemic administration of AJ is likely due to its short half-life in vivo. These results help define why some, but not all, taccalonolides inhibit the growth of tumors at systemically tolerable doses and prompt studies to further improve their pharmacokinetic profile and antitumor efficacy.

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 5519: Investigation of the cellular mechanisms of action of maximiscin, a novel natural product with selective cytotoxic activity against a triple negative breast cancer molecular subtype

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Natural products have historically been an excellent resource for new pharmaceuticals, particularly anticancer agents. Fungi are a remarkable source of novel secondary metabolites that have never been evaluated for their therapeutic potential. However, a vast majority of fungal species found in diverse environments are inaccessible to research scientists. We recently isolated and identified the natural product maximiscin from a Tolypocladium sp. isolate obtained through a crowdsourcing initiative. This compound showed in vitro cytotoxic potency against multiple cancer cell lines in the NCI-60 in vitro screening panel and in vivo antitumor efficacy against a UACC-62 melanoma xenograft model. MDA-MB-468, a triple negative breast cancer (TNBC) cell line of the basal-like 1 (BL1) subtype, was found to be particularly sensitive to the cytotoxic effects of maximiscin compared to several other TNBC cell lines. This finding suggests maximiscin modulates a cellular signaling pathway that specifically drives the BL1 subtype of TNBC. In this study we investigated the cellular mechanisms of action of maximiscin using cell biological and biochemical approaches. The effects of maximiscin on cell cycle distribution were evaluated and maximiscin reduced the fraction of cells in the S and G2/M phases of the cell cycle, and began to induce apoptosis within 8 h. Intracellular signaling through multiple pathways was evaluated by immunoblotting, providing an indication of potential mechanisms of action. Maximiscin also circumvented P-glycoprotein-mediated multidrug resistance. These studies suggest maximiscin may have therapeutic potential for the treatment of BL1 TNBC, especially in cases where P-glycoprotein-mediated multidrug resistance has developed. Studies are ongoing to further characterize the cellular and antitumor effects of maximiscin in BL1 models. Citation Format: Andrew J. Robles, Lin Du, Jarrod B. King, Robert H. Cichewicz, Susan L. Mooberry. Investigation of the cellular mechanisms of action of maximiscin, a novel natural product with selective cytotoxic activity against a triple negative breast cancer molecular subtype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5519. doi:10.1158/1538-7445.AM2014-5519

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 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