Article pubs.acs.org/jnp
Investigation of the Physical and Bioactive Properties of Bromo- and Iodo-Containing Sponge-Derived Compounds Possessing an Oxyphenylethanamine Core Erin P. McCauley,† Hanh Lam,‡ Nicholas Lorig-Roach,† Justin Luu,‡ Cameron Lloyd,‡ Karen Tenney,† Halina Pietraszkiewicz,§ Cristina Diaz,⊥ Frederick A. Valeriote,§ Victoria Auerbuch,*,‡ and Phillip Crews*,† †
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, California 95064, United States § Department of Internal Medicine, Division of Hematology and Oncology, Henry Ford Hospital, Detroit, Michigan 48202, United States ⊥ Harbor Branch Oceanographic Institute, Florida Atlantic University, Fort Pierce, Florida 34946, United States ‡
S Supporting Information *
ABSTRACT: This research set out to identify compounds from marine sponges that can act as bacterial virulence blockers. Extracts from a total of 80 sponges collected from throughout Indonesia were screened in a high-throughput NF-κB-based screen that identifies compounds capable of inhibiting the bacterial type III secretion system (T3SS) in Yersinia pseudotuberculosis. An extract that was shown to inhibit T3SS-driven NFκB expression was obtained from an Iotrochota cf. iota sponge and was the source of seven new bromo- and iodo-containing compounds, all of which contain a 2-(4-oxyphenyl)ethan-1-amine core. Five were determined to be new compounds and named enisorines A−E (1−5). The remaining two were determined to be new hemibastadinol analogues named (+)-1-O-methylhemibastadinol 2 (6) and (+)-1-O-methylhemibastadinol 4 (7). All seven compounds inhibited T3SS-dependent YopE secretion and did not affect the growth or metabolic activity of Y. pseudotuberculosis. The most potent inhibitors of T3SS activity were enisorine C (3), enisorine E (5), and (+)-1-O-methylhemibastadinol 2 (6), all of which inhibited YopE secretion by >50% at 30 μM.
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Gram-negative pathogens that serves to inject bacterial effector proteins inside host cells to manipulate host defenses.5 Our campaign to identify new T3SS inhibitors was motivated by the recent shift in antibiotic research away from traditional bactericidal or bacteriostatic drugs to considering agents that function as virulence blockers.5,6 Traditional antibiotics affect both pathogenic and commensal bacteria alike, whereas virulence blockers target factors, such as the T3SS, rarely employed by nonpathogenic bacteria but are required by pathogens for full virulence. When it is nonfunctional most T3SS-expressing bacteria are rendered inactive and can be easily cleared by the host’s immune system.7 A panel of marine sponge extracts was assayed in a highthroughput screen (HTS) that utilizes the host immune response to measure T3SS function. The assay measures NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), a heterodimeric transcription factor that binds to NF-κB DNA elements to control transcription of genes involved in
t is now widely recognized that marine sponges are a source of immense chemical diversity including compounds that represent most of the major biosynthetic classes.1 Strikingly, the annual reviews of marine natural products continue to show that a significant number of new compounds surveyed are isolated from sponges.2−4 The 2017 marine natural products review revealed that 32% of the marine natural products reported over the past 50 years were obtained from the phylum Porifera; the next most productive phylum was Cnidaria, which accounted for only 16% of reported compounds.4 Furthermore, in the early 1980s the Crews lab at the University of California Santa Cruz (UCSC) began building a repository of sponges from Indo-Pacific areas considered to have immense biodiversity. At this juncture, the UCSC collection contains more than 3000 sponge samples that are continually being used to jumpstart bioassay campaigns focused on new screening paradigms. The results disclosed in this project were derived by interfacing extracts and compounds from our repository with bioassay tools developed in the Auerbuch Lab at UCSC to identify compounds that target the bacterial type III secretion system (T3SS), a virulence factor employed by dozens of © 2017 American Chemical Society and American Society of Pharmacognosy
Received: August 11, 2017 Published: November 16, 2017 3255
DOI: 10.1021/acs.jnatprod.7b00694 J. Nat. Prod. 2017, 80, 3255−3266
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Chart 1
Scheme 1. Pathway to Sponge-Derived Secondary Metabolites That Inhibit the T3SS in Yersinia pseudotuberculosis
inflammation and development in mammalian cells.8 The pathogen Yersinia pseudotuberculosis induces NF-κB activation in human HEK293 (human embryonic kidney) cells when the T3SS is functional.9 Therefore, by measuring NF-κB activation, it is possible to detect if the T3SS is operational.6,10 In order to identify T3SS inhibitors, a subset of sponges collected from throughout Indonesia were selected from our repository, extracted, and screened using HEK293 cells expressing an NF-κB-driven green fluorescence protein (GFP) reporter gene.10 This led to the identification of seven new bromoand iodo-tyrosine-derived alkaloids, all of which contained a 2(4-oxyphenyl)ethan-1-amine core. Five were determined to be new compounds and named enisorines A−E (1−5). The remaining two were determined to be new hemibastadinol analogues and termed (+)-1-O-methylhemibastadinol 2 (6) and (+)-1-O-methylhemibastadinol 4 (7). All of these compounds
were evaluated for their ability to inhibit the T3SS in Y. pseudotuberculosis, and their structures and bioactivities are outlined below.
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RESULTS AND DISCUSSION NF-κB-Based High-Throughput Screen of Sponge Extracts. A total of 80 sponges collected from various regions in Indonesia [Bali (2), Sangihe Islands (9), Togian Islands (44), North Sulawesi (12), and Halmahera (13)] were examined (Scheme 1).11 Their extracts were divided into eight fractions obtained from liquid−liquid extraction followed by preparative HPLC, providing a total of 640 fractions. To identify putative T3SS inhibitors, the fractions were tested in a screen utilizing NF-κB-GFP HEK293 cells inoculated with Y. pseudotuberculosis.10 These cells were incubated with each of the 640 sponge extract fractions or a DMSO control. If the 3256
DOI: 10.1021/acs.jnatprod.7b00694 J. Nat. Prod. 2017, 80, 3255−3266
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Figure 1. Zones of inhibition data for disk diffusion soft agar colony formation in human cancer cell lines. Cell lines: H-116 = colon adenocarcinoma; H-125 = lung adenocarcinoma; MCF-7 = breast adenocarcinoma; LNCaP = prostate adenocarcinoma; OVCAR-5 = ovarian carcinoma; U251N = glioma cells; PANC-1 = prostrate adenocarcinoma; MDA = breast adenocarcinoma; CEM = leukemia cells. A high priority score of 3 is given to any fraction that had a zone of inhibition of ≤3 mm in all cell lines (low cytotoxicity), a medium priority score of 2 is given to any fraction with a zone of inhibition of >3 mm but 3 mm but 50% at 30 μM. Less potent were enisorine A (1) and (+)-1-O-methylhemibastadinol 4 (7), which inhibited YopE secretion by >50% at 60 μM. Lastly, enisorines B (2) and D (4) were much less potent, as they inhibited YopE secretion by >50% at 120 μM. The overall structural features present in enisorines (1−5) and (+)-1-O-methylhemibastadinols (6 and 7) are distinct compared to those contained in known T3SS inhibitors including clioxanide (19)21 or salicylidene acylhydrazides INP0007 (20) and INP0010 (21).21,22 While there are common features among all of these compounds, such as the presence of two aryl rings (labeled as “A” and “B”), these 3261
DOI: 10.1021/acs.jnatprod.7b00694 J. Nat. Prod. 2017, 80, 3255−3266
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Figure 5. Effects of enisorines A−E (1−5) and (+)-1-O-methylhemibastadinols (6 and 7) on YopE secretion in Yersinia pseudotuberculosis. Wild-type Y. pseudotuberculosis was grown under T3SS-inducing conditions in increasing concentrations of compounds 1−7. Secretion of T3SS cargo was assessed by precipitation of secreted proteins and visualization with Coomassie blue. YopE band intensity was quantified and normalized to a DMSO control. Average and standard error of YopE protein band quantification are shown. The results are averages of at least two independent experiments (Table S3). For some points, the error bars are shorter than the height of the symbol and therefore not visible. Red lines indicate 50% secretion with respect to DMSO, below which the secretion inhibition is greater than 50%. *YopE secretion for 6 at a log concentration of 1.2 μM was not determined due to insufficient material.
INP0010 (21) was shown to be active against multiple molecular targets at a single concentration of 40 μM.23 Additionally, although no values were reported for clioxanide (19), it was shown to substantially inhibit YopE secretion at concentrations of 2−20 μM.21 The effects of 1−7 on the secretion of Pseudomonas aeruginosa T3SS effector proteins were also examined. P. aeruginosa was grown, similar to Y. pseudotuberculosis, in lowcalcium media to induce type III secretion, and then compounds 1−7 (60 μM) were added. The secreted proteins were collected and run on SDS-PAGE gels. No compounds inhibited the P. aeruginosa T3SS compared to the DMSO control (Figure S48). This different outcome, in comparison to that reported above, suggests that the T3SS inhibitory activity of 1−7 may be either specific to Y. pseudotuberculosis or simply not effective against P. aeruginosa. Indeed, the Pseudomonas cell envelope is notoriously impervious to small molecules.24 Alternatively, other T3SS inhibitors, such as piericidin A1, have been shown to specifically inhibit certain T3SS classes.25 Other T3SS-utilizing pathogens will need to be screened in order to determine if the activity of 1−7 is selective against Y. pseudotuberculosis. As an important next step, experiments were conducted to confirm that the decrease in YopE secretion was not the result of the enisorines (1−5) and (+)-1-O-methylhemibastadinols (6
similarities are greatly overshadowed by other different structural features. First, the “A”-ring substituents are quite heterogeneous among 1−5, vs 6 and 7, vs 19−21. Second, the acyclic linkers connecting the “A”- to “B”-rings in each of these three preceding sets are quite distinct. Third, the “B”-ring substituents at C-16 for 1−5 are not replicated by the presence of similar acyclic moieties in 6 and 7 or 19−21. Finally, the juxtaposition of N-containing moieties at C-13 to the ring “B” of 6 and 7 is vastly different vs those present in 1−5 or 19−21. Based on these key differences it is clear that the enisorines (1− 5) and (+)-1-O-methylhemibastadinols (6 and 7) do not belong to any previously reported class of T3SS inhibitors and therefore cannot add to the SAR information on any known classes of inhibitors. Therefore, we suggest that compounds 1− 7, possessing a 2-(4-oxyphenyl)ethan-1-amine core, constitute their own class of T3SS inhibitors. A direct comparison of the T3SS inhibitory activity of compounds 1−7 to that of other reported T3SS inhibitors (i.e., 19−21) is somewhat difficult. There are no standard measurements for this inhibitory activity, and a variety of proteins are often employed to assess potency. Nonetheless, we can conclude that the potency of 3, 5, and 6 appears better or comparable to a few other reported T3SS inhibitors. For example, in the same assay piericidin A1 inhibited YopE secretion by >50% at 71 μM,6 and salicylidene acylhydrazide 3262
DOI: 10.1021/acs.jnatprod.7b00694 J. Nat. Prod. 2017, 80, 3255−3266
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Figure 6. Effects of enisorines A−E (1−5) and (+)-1-O-methylhemibastadinols (6 and 7) on Yersinia pseudotuberculosis growth and metabolic activity. Y. pseudotuberculosis (pYV−) was grown in low-calcium media at 37 °C in the presence of compounds 1−7 (60 μM), DMSO, or a kanamycin (50 μg/mL) control. (A) Bacterial growth was measured by absorbance at 600 nm over 8 h, or (B) MTT was added to the pYV− culture after 8 h of incubation and the absorbance at 570 nm was used as a measurement of bacterial metabolic activity. (C) Wild-type Y. pseudotuberculosis (WT) was grown at room temperature in 2×YT in the presence of compounds 1−7 (60 μM), DMSO, or a kanamycin (50 μg/mL) control. Bacterial growth was measured by absorbance at 600 nm over 15 h, or (D) MTT was added to the WT cultures after 15 h of incubation and the absorbance at 570 nm was used as a measurement of bacterial metabolic activity.
and 7) affecting Y. pseudotuberculosis growth or metabolic activity (Figure 6). WT Y. pseudotuberculosis was incubated with compounds 1−7 (60 μM), and absorbance at 600 nm was measured every hour to determine if growth was affected (Figure 6C). Additionally, bacterial metabolism was evaluated by adding yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl2)-2,5-diphenyltetrazolium bromide) to the culture after 8 h of incubation with compounds 1−7, and formazan abundance was measured at 570 nm. In both experiments compounds 1−7 did not inhibit growth or metabolic activity relative to the DMSO control (Figure 6D). In addition, we tested the effects of compounds under active secretion conditions. As active type III secretion in WT Y. pseudotuberculosis causes growth arrest, we used a Y. pseudotuberculosis strain lacking the T3SS-encoding virulence plasmid, pYV (pYV−) (Figure 6A and B). Similar to what was observed in the WT strain, there was no measurable decrease in growth or metabolic activity compared to the DMSO control. In all experiments kanamycin was used as a negative control and was shown to inhibit growth and metabolic activity in Y. pseudotuberculosis. The overall outcomes of this study demonstrate the value of accessing repositories rich in Indonesian sponges as a source of bioactive compounds. Our success in prioritizing samples for further study was enhanced by employing data obtained from two orthogonal screens. The structure elucidation challenges presented in this project were effectively dealt with by merging insights from contemporary 2D NMR data with conclusions gained from accurate MS2 analysis. Additionally, important value was added by the identification of seven new bromo- and
iodo-containing sponge-derived compounds (1−7) possessing a 2-(4-oxyphenyl)ethan-1-amine core. All of these compounds exhibited some degree of T3SS inhibition in Y. pseudotuberculosis without affecting the growth or metabolic activity of the pathogen. At this juncture three compounds, enisorines C (3) and E (5) and (+)-1-O-methylhemibastadinol 2 (6), can be considered as leads for further study as inhibitors of the Y. pseudotuberculosis T3SS, as they were shown to inhibit YopE secretion by >50% at 30 μM. This degree of potency is comparable to other know T3SS inhibitors such as clioxanide (19) and INP0010 (21), a salicylidene acylhydrazide. With the rise in new antibiotic-resistant bacteria there is a need for new classes of antibiotics. Targeting virulence factors such as the T3SS should be further explored as a powerful tool for the discovery of new small-molecule antimicrobials.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation was determined on a JASCO P-2000 polarimeter. UV spectra were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Experimental ECD spectra were recorded on a JASCO J-1500 spectrometer in CH3OH using a 1 mm cell length, a 4 nm bandwidth, and a scanning speed of 50 nm/min. IR spectra were measured on a Spectrum One FT-IR spectrophotometer (PerkinElmer). All NMR experiments were run on a Varian UNITY INOVA spectrometer (600 MHz for 1H, 150 MHz for 13C) (Agilent Technologies) outfitted with a 5 mm triple-resonance (HCN) cold probe. Chemical shifts (δ) were referenced to C6D6 residual peaks at δH 7.16 and δC 128.06.26 High-accuracy mass spectrometry measurements were obtained using a Thermo Velos Pro electrospray ionization hybrid ion trap-orbitrap MS. All HPLC was done in 3263
DOI: 10.1021/acs.jnatprod.7b00694 J. Nat. Prod. 2017, 80, 3255−3266
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Table 1; HAESIMS m/z 491.1542 [M + H] + (calcd for C24H32BrN2O4, 491.1540); MS2 data, Figure S8. Enisorine D (4): white solid; UV (CH3OH) λmax (log ε) 230 (3.65), 280 (3.12) nm; IR (film) νmax 3339, 2944, 2833, 2520, 2237, 2045, 1607, 1494, 1452, 1431, 1111, 1032, 876, 824, 688 cm−1; NMR data, Table 1; HAESIMS m/z 555.0502 [M + H] + (calcd for C23H29Br2N2O4, 555.0489); MS2 data, Figure S9. Enisorine E (5): white solid; UV (CH3OH) λmax (log ε) 211 (3.02), 230 (3.74), 279 (3.16) nm; IR (film) νmax 3339, 2945, 2833, 2521, 2233, 2045, 1607, 1493, 1451, 1113, 1033, 876, 824, 688 cm−1; NMR data, Table 1; HAESIMS m/z 603.0360 [M + H]+ (calcd for C23H29BrIO4N2, 603.0350); MS2 data, Figure S10. (+)-1-O-Methylhemibastadinol 2 (6): white solid; [α]23D +40 (c 1.25, CH3OH); UV (CH3OH) λmax (log ε) 210 (3.89), 222 (3.88), 280 (3.18) nm; ECD (1.8 mM, CH3OH) λmax (Δε) 213 (+16.4), 285 (+2.3) nm; IR (film) νmax 3401, 2944, 2833, 2521, 2230, 2045, 1646, 1608, 1451, 1115, 1033, 876, 824, 688 cm−1; NMR data, Table 2; HAESIMS m/z 549.8875 [M + H]+ (calcd for C18H19Br3NO4, 549.8859). (+)-1-O-Methylhemibastadinol 4 (7): white solid; [α]23D +29 (c 0.85, CH3OH); UV (CH3OH) λmax (log ε) 209 (3.52), 224 (3.41), 282 (2.69) nm; ECD (1.6 mM, CH3OH) λmax (Δε) 202 (+13.5), 282 (+0.9) nm; IR (film) νmax 3368, 2927, 2833, 2521, 2234, 2045, 1607, 1493, 1451, 1404, 1111, 1048, 1033, 876, 825, 699 cm−1; NMR data, Table 2; HAESIMS m/z 597.8732 [M + H]+ (calcd for C18H19Br2IN O4, 597.8720). Computational Details. Optimal geometry and ECD calculations were performed using Turbomole v7.1 and the TD-DFT-B3LYP/ TZVPP level of theory (Figures S51 and S52).29 The spectra were simulated by overlapping Gaussian functions centered at the wavelengths of the respective electronic transitions and corresponding rotatory strengths transformed into Δε values. Cytotoxicity Assay. Cytotoxic soft agar disk diffusion assays were performed as previously described.12,30 Briefly, 300 μg of the W and F fractions and 120 μg of the F1−F6 fractions were loaded onto filter disks and plated onto a matrix containing one of the following human cell lines: H-116 = colon adenocarcinoma; H-125 = lung adenocarcinoma; MCF-7 = breast adenocarcinoma; LNCaP = prostate adenocarcinoma; OVCAR-5 = ovarian carcinoma; U251N = glioma cells; PANC-1 = prostrate adenocarcinoma; MDA = breast adenocarcinoma; CEM = leukemia cells. The plates were incubated for 7 to 10 days, and the zone of inhibition around the disk was measured. Each fraction was assayed against a minimum of six different cell lines and given a hit priority score from 3 to 1 based on their observed cytotoxicity. Any extract fraction that exhibited a zone of inhibition of ≤3 mm (low cytotoxicity) in all cell lines it was screened against was given a high hit priority score of 3. Any fraction that exhibited a zone of inhibition of >3 mm but
