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Herbicidins from Streptomyces sp. CB01388 Showing Anti-. Cryptosporidium Activity. Jian-Jun Chen,. †,#. Mostafa E. Rateb,. †,#. Melissa S. Love,. ...
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Herbicidins from Streptomyces sp. CB01388 Showing AntiCryptosporidium Activity Jian-Jun Chen,†,# Mostafa E. Rateb,†,# Melissa S. Love,‡ Zhengren Xu,† Dong Yang,†,§ Xiangcheng Zhu,⊥,□ Yong Huang,⊥ Li-Xing Zhao,¶ Yi Jiang,¶ Yanwen Duan,⊥,□ Case W. McNamara,‡ and Ben Shen*,†,∥,§ †

Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33458, United States California Institute for Biomedical Research, La Jolla, California 92037, United States § Natural Products Library Initiative at The Scripps Research Institute, The Scripps Research Institute, Jupiter, Florida 33458, United States ⊥ Xiangya International Academy of Translational Medicine, Central South University, Changsha, Hunan 410013, People’s Republic of China □ Hunan Engineering Research Center of Combinatorial Biosynthesis and Natural Product Drug Discovery, Changsha, Hunan 410013, People’s Republic of China ¶ Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, People’s Republic of China ∥ Department of Molecular Medicine, The Scripps Research Institute, Jupiter, Florida 33458, United States ‡

S Supporting Information *

ABSTRACT: A high-content imaging assay was used to screen the fraction collection of the Natural Product Library at The Scripps Research Institute for inhibitors of Cryptosporidium parvum. A chemical investigation of one strain, Streptomyces sp. CB01388, resulted in the isolation of six herbicidins (1−6), one of which is new (herbicidin L, 1). Five of the six herbicidins (1−3, 5, 6) showed moderate inhibitory activity against C. parvum, with 1 and 6 comparable to the FDA-approved drug nitazoxanide, and 2-6 showed no toxicity to the host HCT-8 cells and human HEK293T and HepG2 cells. These findings highlight the herbicidin scaffold for antiCryptosporidium drug development.

promised patients, e.g., HIV-infected people, thus highlighting the unmet medical need for these vulnerable patient populations.4 Due to the technical challenges to work with this notoriously intractable parasite, early drug discovery efforts for cryptosporidiosis treatment have been limited to a few targeted mechanisms, 5−8 e.g., fatty acyl-CoA binding protein (CpACBP1),5 calcium-dependent protein kinases (CDPKs),6 and inosine-5′-monophosphate dehydrogenase (IMPDH),7 in the parasite. The drug discovery process has been facilitated with the establishment of the whole cell phenotypic screening platforms for inhibitors of C. parvum proliferation within human intestinal epithelial HCT-8 cells,9,10 leading to the discovery of lead compounds, e.g., clofazimine 9c and KDU731,10 which showed submicromolar inhibitory activities. Most screens have been focused against libraries containing compounds with known biological effects, especially those with

Cryptosporidium species are gastrointestinal parasites belonging to the phylum Apicomplexa and are one of the most common causes of diarrhea in humans and some domestic animals.1 Infection of the parasites occurs through the oral−fecal route initiated by ingestion of water or food contaminated with environmentally resilient Cryptosporidium oocysts. Cryptosporidium parvum and Cryptosporidium hominis are two clinically relevant species that cause cryptosporidiosis by invading the epithelial cell of the intestine and triggering severe watery diarrheal symptoms. According to the World Health Organization, infectious diarrhea accounts for nearly 800 000 deaths every year worldwide, mostly among young children in developing countries and immunocompromised patients.2 A recent epidemiological study has revealed that Cryptosporidium has emerged as a leading cause of nonviral diarrhea in young children and is strongly associated with death and stunted development.3 Only one FDA-approved drug, nitazoxanide (NTZ), is currently used for the treatment of cryptosporidiosis. However, NTZ has been shown to be less efficacious in malnourished children and shows no effect in immunocom© XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 6, 2017

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antiparasite activities for drug repurposing.9a,b,10 While several known natural products, e.g., monensin and cyclosporine, have also been identified as inhibitors of Cryptosporidium proliferation within a human intestinal epithelial cell line,9c there is no report on screening a natural product library for cryptosporidiosis therapeutics. Natural products have an indisputable track record in drug discovery and remain a rich source of new drug leads and smallmolecule probes.11 Biased on natural products of Actinomycetales origin, the Natural Products Library Initiative at The Scripps Research Institute (TSRI) aims to construct a library that occupies unique chemical space and complements the small-molecule collection at TSRI.12−14 Also, natural products of bacterial origin are advantageous since the resupply of enough materials for follow-up studies will be guaranteed by large-scale fermentation once the interesting entities are identified. Three types of collections, including (i) crude extracts, (ii) medium-pressure liquid chromatography (MPLC)-generated fractions, and (iii) structurally assigned pure natural products, constitute the current natural products library (NPL) at TSRI. Typically, strains were fermented in multiple media to give the corresponding crude extracts and analyzed by HPLC for chemical profiling, the results of which were used to guide strain selection for MPLC fractionation and natural products isolation. In this context, the fraction collection of the library, which preserves most of the chemical diversity, maintains reasonable concentrations, and is compatible with most high-throughput screen platforms, is often favored for further biological assay-guided natural products discovery.

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RESULTS AND DISCUSSION By taking advantage of the established whole-cell phenotypic screen platform,9c we used the high-content imaging assay to screen 3127 fractions prepared from a collection of 159 microbial strains for active compounds that inhibit the proliferation of C. parvum within HCT-8 cells. The primary screen resulted in the identification of 98 hit fractions (from 42 strains) showing >70% inhibition, representing a hit rate of 3.1%. The number of hit fractions was further reduced to 19 (from 11 strains) by removing those with obvious cytotoxicity to the host HCT-8 cells. These 19 fractions were further subjected to dose−response testing for reconfirmation of the activity and counter-screened with HEK293T and HepG2 cell lines, for general cytotoxicity. On the basis of the selectivity toward inhibition of C. parvum, the number of active fractions from the same strain, and the chemical profiles of the active fractions upon LC-MS analysis, Streptomyces sp. CB01388 was thus chosen for further follow-up dereplication studies (Figure S1, Supporting Information). Strain CB01388 was isolated from a soil sample collected in Wuyi Mountain, Fujian Province, China. Based on the phylogenetic analysis of the concatenated partial sequences of three housekeeping genes, including 16S rRNA, rpoB, and trpB,14a strain CB01388 was classified as a Streptomyces species (Table S1 and Figure S2, Supporting Information). Streptomyces sp. CB01388 was refermented on a large scale in the same medium previously used to prepare the fractions, from which six herbicidin congeners (1−6)15 were isolated and characterized, including one new compound, herbicidin L (1) (Figure 1). The structures of the known compounds were assigned as herbicidin A (2),15k herbicidin B (3),15k 8′-epiherbicidin B (4),15k herbicidin C (5),16 and herbicidin K15j

Figure 1. High-content imaging assay using Cryptosporidium parvuminfected HCT-8 cells to identify hits inhibiting C. parvum proliferation. (A) Structures of herbicidin congeners. (B) Representative proliferation images of C. parvum from both Sterling Parasitology Laboratory (SPL) and Bunch Grass Farm (BGF) treated with DMSO or 12.5 μM 6. Two fluorescent channels are artificially colored for comparison,9c with DAPI in cyan for HCT-8 cell count (left) and FITC-conjugated Vicia villosa lectin in red for Cryptosporidium spot count (middle). The “cell area” was drawn in green around each host cell nuclei to encompass where the Cryptosporidium parasites may be located. Merged images (right) showed both the host HCT-8 cells and Cryptosporidium parasites.

(also known as 2,-O-demethylherbicidin F 15k) (6) by comparing their NMR data with those reported in the literature (Table 1 and Figure S3, Supporting Information). Compound 1 was isolated as an optically active, [α]D +58 (c 0.1, MeOH), faint yellow gum. The molecular formula of 1 was assigned as C23H29N5O11 by high-resolution electrospray B

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Table 1. NMR Spectroscopic Data (1H at 400 MHz and 13C at 100 MHz, CD3OD) for Herbicidins L (1) and A (2)a herbicidin L (1) position

δC, type

2 4 5 6 8 1′ 2′ 3′ 4′ 5′ 6′

154.0, CH 150.3, C 119.4, C 157.2, C 142.5, CH 89.1, CH 92.1, CH 74.3, CH 79.8, CH 26.4, CH2 65.5, CH

7′ 8′ 9′

93.9, C 69.8, CH 74.9, CH

10′ 11′ 1″ 2″ 3″ 4″ 5″

75.3, CH 171.2, C 167.4, C 133.3, C 144.0, CH 14.4, CH3 56.1, CH2

2′-OMe 11′-OMe

58.4, CH3 52.7, CH3

a b

δH (J in Hz) 8.21, s

8.71, s 6.17, d (1.2) 4.01, d (0.8) 4.47,b br s 4.47,b m 2.25, m 4.67, dd (7.4, 9.6) 3.84, d (3.0) 5.56, dd (1.2, 3.0) 4.52, br s

7.17, 1.94, 4.40, 4.37, 3.46, 3.76,

q d d d s s

(7.2) (7.2) (12.1) (12.1)

herbicidin A (2) δC, type 154.2, CH 150.5, C 119.5, C 157.4, C 140.9, CH 88.4, CH 91.6, CH 74.5, CH 79.1, CH 26.5, CH2 66.5, CH 93.4, C 71.9, CH 70.6, CH 78.3, CH 171.3, C 166.1, C 132.5, C 145.7, CH 15.1, CH3 56.3, CH2 58.3, CH3 52.8, CH3

δH (J in Hz) 8.22, s

7.99, s 6.06, d (1.7) 4.06, d (1.6) 4.50, d (2.1) 4.40, m 2.26, m 4.54, dd (6.1, 10.7) 5.08, d (3.2) 4.34, dd (1.2, 3.3) 4.48, br s

6.78, 1.99, 4.43, 4.39, 3.40, 3.62,

q d d d s s

(7.2) (7.2) (11.9) (11.9)

Assignments are based on COSY, HSQC, and HMBC experiments. Overlapped. Figure 2. Structural elucidation of herbicidins on the basis of 1D and 2D NMR and CD spectroscopic data analysis. (A) Key 1H−1H COSY and HMBC correlations of herbicidin L (1) and selected NOESY correlations of 1 and herbicidin A (2). (B) CD spectra of herbicidin L (1), in comparison with herbicidin A (2) and 8′-epi-herbicidin B (4).

ionization mass spectrometry. Analysis of the 1D and 2D NMR data (Table 1 and Figure S4, Supporting Information) indicated that the planar structure of 1 consists of an adenine, an undecose moiety with two methoxyl groups [δH at 3.76 (3H, s), 3.46 (3H, s) and δC at 52.7, 58.4], and a (E)-2″-hydroxymethyl2″-butenoyloxyl group [δH at 7.17 (1H, q, J = 7.2 Hz), 4.40 (1H, d, J = 12.1 Hz), 4.37 (1H, d, J = 12.1 Hz), 1.93 (3H, d, J = 7.2 Hz) and δC at 167.4, 144.0, 133.3, 56.1, 14.4].17 The presence of correlations from H-1′ to C-4/C-8 in the HMBC spectrum suggested that the adenine group and the undecose moieties were connected between C-1′ and N-9. The two methoxyl groups were determined to be connected at C-2′ and C-11′ based on the observation of the HMBC correlations from 2′-OCH3 (δH at 3.46) to C-2′ and from 11′-OCH3 (δH at 3.76) to C-11′, respectively. Considering the observed downfield shift of the H-9′ (δH at 5.56) in the 1H NMR spectrum and the HMBC correlations from H-9′ to C-1″, the (E)-2″-hydroxymethyl-2″-butenoyloxyl moiety was then placed at C-9′. The observation of an NOE correlation between H-5″ and H-4″ indicated the cis-arrangement of C-4″ and C-5″, hence supporting the assignment of the E configuration of the double bond between C-2″ and C-3″. Therefore, the planar structure of compound 1 was deduced as shown in Figure 2A. The elucidation of the 3D structure of 1 was aided by the analysis of the NOESY spectrum in comparison with that of herbicidin A (2).15k Both 1 and 2 show similar NOE correlation peaks between H-8 and H-2′/H-6′, between H-1′ and 2′-OCH 3/H-4′, and between H-3′ and 2′-OCH 3, indicating their same H-1′α, H-2′β, H-3′α, H-4′α, and H-6′β

orientations. The observation of NOE correlations between H6′ and 11′-OMe/H-8′ and between H-9′ and 11′-OMe in 1 suggests that H-8′, H-9′, and H-10′ are β-, β-, and α-oriented, respectively. Thus, 1 is an 8′-epimer at the undecose moiety of herbicidin. Only two 8′-epi-herbicidins, i.e., 8′-epi-herbicidin B (4)15k and 8′-epi-herbicidin F,15l have been characterized to date, both of which were of Streptomyces origin. In the circular dichroism (CD) spectrum (Figure 2B), 1 [257 nm (negative), 221 nm (positive), 210 nm (negative)] showed a similar Cotton effect to those of 4 [259 (negative), 228 (positive), 215 nm (negative)]15k and 8′-epi-herbicidin F [260 (negative), 223 (positive), 213 nm (negative)],15l hence supporting the similarities of their configurations. On the basis of their same biosynthetic origin and the occurrence of herbicidin congeners 2−6 with previously defined configurations, 1 was elucidated with its absolute stereochemistry as shown in Figure 1A and named herbicidin L. We next evaluated the anti-Cryptosporidium activity of the isolated herbicidins, using NTZ and floxuridine (FDU) as controls for C. parvum inhibition and puromycin as a control for host cell cytotoxicity. The high-content imaging assay allowed us to determine the inhibitory effect toward the proliferations of both C. parvum and the host HCT-8 cells (Figure 1B).9c As summarized in Table 2, 6 showed comparable activity to that of NTZ in inhibiting the proliferation of C. C

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Table 2. Anti-Cryptosporidium and Cytotoxicity Assays for Herbicidin Congeners 1−6 anti-Cryptosporidium (EC50,a μM) compound 1 2 3 4 5 6 nitazoxanide floxuridine puromycin

SPL C. parvum 0.17 16 11 >25 24 2.7 2.3 0.0050 0.60

c

± 0.04 ±2 ±1 ± ± ± ± ±

2 0.4 0.3 0.0007 0.07

HCT-8

d

0.18 ± 0.02 >25 >25 >25 >25 >25 >25 0.015 ± 0.002 6.1 ± 0.2

cytotoxicity (CC50,b μM)

BGF C. parvum 0.15 21 8.8 >25 18 2.8 2.9 0.0096 0.55

e

± 0.06 ±2 ± 1.0 ± ± ± ± ±

2 1.4 0.9 0.0009 0.07

HCT-8

f

0.14 ± 0.02 >25 >25 >25 >25 >25 >25 0.038 ± 0.004 7.0 ± 0.2

HEK293T

HepG2

>40 >40 >40 >40 >40 >40 >40 0.0084 ± 0.0009 0.42 ± 0.02

24 ± 6 >40 >40 >40 >40 >40 >40 0.0076 ± 0.0028 0.83 ± 0.06

a Half-maximal effective concentration. bHalf-maximal cytotoxic concentration. cC. parvum from Sterling Parasitology Laboratory (SPL) at the University of Arizona. dHost of SPL C. parvum. eC. parvum from Bunch Grass Farm (BGF) in Deary, Idaho. fHost of BGF C. parvum.

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parvum, while 2−5 were less potent. Furthermore, 2−6 as well as NTZ showed no cytotoxicity to the host HCT-8 cells at the highest concentration (25 μM) used in the assay. In contrast, 1, FDU, and puromycin exhibited cytotoxicity to the host cells at concentrations comparable to those required to inhibit C. parvum proliferation. It was therefore difficult to distinguish whether the anti-Cryptosporidium activities of these compounds resulted from direct inhibition of the parasites, indirect killing of the host cells or a combination of both. The general cytotoxicity of all six herbicidin congeners was further evaluated toward two human cell lines, i.e., HEK293T and HepG2. The results showed that 2−6 were not toxic to either cell line at the highest concentration (40 μM) used in the assay, while 1 was toxic to the HepG2 cells with a half-maximal cytotoxic concentration (CC50) of 24 ± 6 μM (Table 2). Herbicidins belong to a family of undecose (C11)-based adenine nucleoside antibiotics. Since the first discovery of the herbicidin skeleton from Streptomyces saganonensis in 1976, 15 additional members have been reported (Figure S3, Supporting Information), most of which varied only in the substitution patterns at C-2′, C-8′, and C-11′.15 Typically, the configurations of the undecose moiety are relatively conserved except for that at C-8′. Only two 8′-epi-herbicidin congeners, 8′-epiherbicidin B (4)15k and 8′-epi-herbicidin F,15l have been reported previously, and herbicidin L (1) represents the third member of this 8′-epi-herbicidin type. Also, 1 is the first member of herbicidin congeners with its 9′-hydroxy group being esterified, while only the 8′-hydroxy group was found to be esterified in previously discovered herbicidins. The most promising feature of the herbicidin family of natural products is their selective herbicidal activity toward dicotyledonous plants while showing no toxicity to animals. Although there are no prior reports on activities of the herbicidins with apicomplexan parasites such as Cryptosporidium, herbicides have been repurposed for the treatment of Plasmodium,18 another apicomplexan parasite that causes malaria. Similarly, antimalarial drugs have also been repurposed for use as herbicides.19 The rationale behind this repurposing practice is the evolutionary connection of apicomplexan parasites with plants, as evidenced by the presence of the apicoplast, a nonphotosynthetic plastid that is similar to the chloroplast.20 Although Cryptosporidium lacks an apicoplast, both herbicidal and anti-Cryptosporidium activities exerted by herbicidins may suggest the presence of other evolutionarily related targets.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using an AUTOPOL IV automatic polarimeter (Rudolph Research Analytical). UV spectra were recorded with a NanoDrop 2000C spectrophotometer (Thermo Scientific). IR spectra were collected with a Spectrum One FT-IR spectrometer (PerkinElmer). CD spectra were collected with a Jasco J-815 circular dichroism spectropolarimeter. NMR spectra were collected with a Bruker Avance III Ultrashield 700 and a Bruker Avance 400 MHz Ultrashield instrument. HRESIMS data were acquired on an Agilent 6230 TOF LC/MS instrument. MPLC separation was conducted on a Biotage Isolera One using a KP-C18-HS (30 g) column. HPLC was carried out on a Varian semipreparative HPLC system (Woburn, MA, USA) equipped with a Prostar 330 detector, using a GRACE Apollo C18 column (250 mm × 4.6 mm, 5 μm) for analysis and an Alltima C18 column (250 mm × 10.0 mm, 5 μm) for purification. All fermentations were carried out in New Brunswick Scientific Innova 44 incubator shakers or New Brunswick BioFlo/celliGen 115 fermentors. Diaion HP-20 resin was purchased from Sigma-Aldrich. Microbial Material Isolation and Identification. Strain Streptomyces sp. CB01388 was isolated from a soil sample collected in Wuyi Mountain, Fujian Province, China. A standard diluting plate method was applied for the purification of the strain by using agar plates with medium consisting of glycerol 10 g, asparagine 1 g, K2HPO4·H2O 1 g, MgSO4·7H2O 0.5 g, CaCO3 0.3 g, vitamin mixture (thiamine-HCl 0.5 mg, riboflavin 0.5 mg, niacin 0.5 mg, pyridoxine 0.5 mg, calcium pantothenate 0.5 mg, inositol 0.5 mg, 4-aminobenzoic acid 0.5 mg, and biotin 0.25 mg), and agar 15 g, in 1 L of H2O, pH = 7.7.21 The strain was preserved as a spore suspension (20% glycerol, v/ v) at −80 °C. After growing on ISP4 medium (Difco) at 28 °C for 7 days, the fresh spores of the strain CB01388 were harvested and cultured in tryptic soy broth (TSB, Bacto) at 28 °C for 2 days, after which the genomic DNA was isolated following standard protocols.14 Three housekeeping genes, 16S rRNA, rpoB (encoding RNA polymerase β subunit), and trpB (encoding tryptophan synthase β subunit), were amplified by PCR with One Taq Quick-Load 2× Master Mix (New England BioLabs Inc.) using primers listed in Table S1. The PCR product was recovered with QIAquick gel extraction kit (Qiagen), sequenced, and deposited into GenBank under accession numbers KT722881, KT736438, and KT793853, respectively. The sequences of the PCR products were used for BLAST on the NCBI Web site to search for the homologous gene candidates, which were then used as their representative Streptomyces spp. in the phylogenetic tree, assigning CB01388 as a Streptomyces species (Figure S2, Supporting Information).14a Fermentation and Isolation. A fresh spore solution (100 μL) of Streptomyces sp. CB01388 was inoculated into 2 L flasks containing 400 mL of TSB medium and cultured for 2 days at 28 °C. The resultant seed culture (800 mL) was then inoculated into 10 L of production medium [medium B: dextrin (40 g/L), tomato paste (7.5 g/L), NZ amine A (2.5 g/L), primary yeast (5 g/L), pH = 7],14 in a 14 D

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L fermentor, and the fermentation continued at 250 rpm at 28 °C for 7 days. Diaion HP-20 resin (500 g) was added to the resultant fermentation broth, which was stirred at room temperature overnight. The resin and the cell mass were harvested by centrifugation, washed by deionized H2O, air-dried, and extracted with methanol (1.5 L × 2). The combined methanol extracts were concentrated under reduced pressure to give 58 g of crude extract, which was suspended in 800 mL of 50% aqueous MeOH and successively extracted with hexane (700 mL × 3) and CH2Cl2 (800 mL × 3). The combined fractions were evaporated to dryness under vacuum to yield hexane (28 g), CH2Cl2 (7 g), and MeOH−H2O (23 g) fractions. The CH2Cl2 fraction (7 g) was loaded on a silica gel (150 g) chromatography column (CC) and eluted with CH2Cl2−MeOH (100:1−5:1, gradient systems) to afford six fractions (C1−C6). Fractions C5 (0.6 g) and C6 (0.8 g) were loaded on a silica gel CC and fractionated using CH2Cl2−MeOH (gradient elution with 1% to 20% MeOH) to give subfractions C5.1− C5.3 and C6.1−C6.3, respectively. Subfractions C5.3 and C5.2 were further separated using semipreparative HPLC eluted with MeCN− H2O (gradient elution in 30 min from 10% to 25% MeCN with a flow rate of 4.0 mL/min), affording compounds 2 (26 mg) and 3 (21 mg) from C5.3, and compound 6 (3 mg) from C5.2, respectively. Subfraction C6.2 was separated using semipreparative HPLC eluted with MeCN−H2O (gradient elution in 25 min from 10% to 40% MeCN with a flow rate of 4.0 mL/min) to afford compound 4 (4 mg). Subfraction C6.3 was separated using semipreparative HPLC eluted with MeCN−H2O (gradient elution in 30 min from 20% to 40% MeCN with a flow rate of 4.0 mL/min) to afford compounds 1 (1.2 mg) and 5 (0.9 mg). Herbicidin L (1): white powder; [α]27D +58 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (3.70) nm, 260 (3.40) nm; IR νmax 3323, 2948, 1693, 1423, 1199, 1107, 1052 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 552.1934 [M + H]+ (calcd [M + H]+ for C23H29N5O11 at m/z 552.1936). Herbicidin A (2): white powder; [α]27D +50 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 221 (3.65) nm, 258 (3.60) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 552.1939 [M + H]+ (calcd [M + H]+ for C23H29N5O11 at m/z 552.1936). High-Content Imaging of Cryptosporidium Proliferation within HCT-8 Cells. Two different sources of Cryptosporidium oocysts were used in this study. SPL C. parvum oocysts were purchased from the Sterling Parasitology Laboratory, University of Arizona. BGF C. parvum oocysts were purchased from Bunch Grass Farm in Deary, Idaho. The oocysts were stored at 4 °C for ≤3 months in an antibiotic solution (0.01% Tween 20 containing 100 U/mL penicillin and 100 μg/mL gentamicin). Host human ileocecal adenocarcinoma (HCT-8; ATCC CCL244) cells were maintained and seeded into 1536-well tissue-culture-treated, black-walled, clearbottomed, low-base assay plates (Greiner) at a density of 5.5 × 105 cells/mL (2750 cells/well) as previously described.9c Compound transfer was performed by an ECHO liquid handler (Labcyte) 24 h after cell seeding and prior to infection. For single-point testing in primary screening, fractions were transferred to an estimated final concentration of 1.88 μM. For dose−response testing, fractions were transferred in a 1:3 dose−response with an estimated top concentration of 6.23 μM across triplicate plates. Resupplied pure compounds for confirmation were prediluted serially 1:3 in an 11 pt dose−response and transferred with a final top concentration of 25 μM. For infection of the assay plate, 24 h after the HCT-8 cell seeding, oocysts were excysted and dispensed (3125 oocysts/well) as previously described.9c The infected cells were incubated at 37 °C for 48 h with 5% CO2 in a humidified tissue culture incubator covered with metal assay lids (The Genomics Institute of the Novartis Research Foundation) and then fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized and stained in the same way as previously described.9c Specifically, Cryptosporidium parasites were stained with 1 μg/mL fluorescein isothiocyanate (FITC)-conjugated Vicia villosa lectin (Vector Laboratories) in 1:10 diluted SuperBlock in phosphate buffered saline (PBS)-T (1× PBS with 0.1% Tween 20), supplemented with 3 μM 4′,6-diamidino-2phenyindole (DAPI) to visualize host cell nuclei for 1 h in the dark at

room temperature. The stained cells were washed with PBS-T twice and imaged with a CellInsight CX5 High Content Screening Platform (Thermo) with a 10× objective using the 384/440 nm channel for DAPI-stained nuclei and the 485/521 nm channel for FITC-lectinlabeled Cryptosporidium parasites. Images were processed by the HCS Studio Scan software, and selected object counts for HCT-8 host cells and spot counts for Cryptosporidium were analyzed in a Genedata Screener (v13.0-Standard). Both cytotoxicity against HCT-8 cells (number of nuclei relative to DMSO-treated controls) and Cryptosporidium inhibition (spot counts relative to DMSO-treated controls) were assessed based on the software-identified primary objects (HCT-8 host cells) and spots within allowed distances to the nuclei (Cryptosporidium). Mammalian Cell Cytotoxicity Assays. Compounds were prespotted into tissue-culture-treated white solid-bottomed 1536-well plates (Greiner) in a 1:3 dose−response dilution with a top concentration of 40 μM. Human embryonic kidney cells (HEK293T; ATCC CRL-3216) and human hepatocellular carcinoma cells (HepG; ATCC HB-8065) were maintained and dispensed into assay plates as previously described,9c with 375 cells/well for HEK293T cells and 700 cells/well for HepG cells, respectively. Cells were incubated at 37 °C for 72 h with 5% CO2 in a humidified tissue culture incubator covered with metal assay lids (The Genomics Institute of the Novartis Research Foundation). The cell viability assays were carried out with a CellTiter-Glo (Promega) kit according to the manufacturer’s instructions. Luminescence intensities were measured with an EnVision multilabel plate reader (PerkinElmer) and normalized to DMSO- and puromycin-treated wells. Dose−response data were fitted to a four-parameter nonlinear regression curve with Genedata to determine the CC50 of each compound.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00850. Table providing the primers used to clone the three housekeeping genes for the phylogenetic analysis; figures showing the LC-MS analysis of the hit fractions from S. sp. CB01388 in comparison with the crude extract of S. sp. CB01388, the phylogenetic analysis for assignment of strain CB01388 as a Streptomyces species, all the known herbicidin congeners, and NMR spectra of compounds 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (B. Shen): (561) 228-2456. Fax: (561) 228-2472. E-mail: [email protected]. ORCID

Jian-Jun Chen: 0000-0001-5937-5569 Dong Yang: 0000-0003-2917-0663 Yong Huang: 0000-0002-3163-1716 Ben Shen: 0000-0002-9750-5982 Author Contributions #

J. Chen and M. E. Rateb contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the Natural Products Library Initiative at The Scripps Research Institute. J.-J. C. was supported in part by the State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical E

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Engineering, Lanzhou University, and a scholarship from the Chinese Scholarship Council (201606185009). This is manuscript #29598 from the Scripps Research Institute.

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DOI: 10.1021/acs.jnatprod.7b00850 J. Nat. Prod. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jnatprod.7b00850 J. Nat. Prod. XXXX, XXX, XXX−XXX