Cholinesterase Inhibitory Arisugacins LâQ from a Penicillium sp

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Cholinesterase Inhibitory Arisugacins L−Q from a Penicillium sp. Isolate Obtained through a Citizen Science Initiative and Their Activities in a Phenotype-Based Zebrafish Assay Wentao Dai,†,⊥ Imelda T. Sandoval,§,⊥ Shengxin Cai,† Kaylee A. Smith,† Richard Glenn C. Delacruz,§ Kevin A. Boyd,§ Jessica J. Mills,§ David A. Jones,*,§ and Robert H. Cichewicz*,†

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†

Natural Products Discovery Group, Institute for Natural Products Applications and Research Technologies, Department of Chemistry & Biochemistry, Stephenson Life Science Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States § Functional and Chemical Genomics Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104, United States S Supporting Information *

ABSTRACT: Phenotype-based screening of a fungal extract library yielded an active sample from a Penicillium sp. isolate that impaired zebrafish motility. Bioassay-guided purification led to the identification of 14 meroterpenoids including six new metabolites, arisugacins L−Q (4, 5, 8, and 12−14), seven known arisugacins (1−3, 6, 7, 9, and 10), and one known terreulactone (11). Their structures were determined using a combination of NMR and HRESIMS data, evidence secured from theoretical and experimental ECD spectra, and the modified Mosher’s method. The purified compounds were tested in zebrafish embryos, as well as in vitro for cholinesterase inhibition activities. Compound 12 produced defects in myotome structure (metameric muscle, which is critical for locomotion) in vivo and showed the most potent and selective acetylcholinesterase inhibitory activity with an IC50 of 191 nM in vitro. The phenotype assay was also used to reveal bioactivities for several previously reported arisugacins, which had failed to show activity in prior cell-based and in vitro testing. This study demonstrates that utilization of the zebrafish phenotype assay is an effective approach for the identification of bioactive extracts, is compatible with the bioassay-guided compound purification strategies, and offers a valuable tool for probing complex natural product sources to detect bioactive small molecules with potential therapeutic or other commercial applications.

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pathologies associated with several disease including Alzheimer’s disease,2,3 Parkinson’s disease,4 myasthenia gravis,5 and glaucoma.6 In addition to the treatment of human diseases, AChE inhibitors have found widespread uses as insecticides (e.g., aldicarb, carbofuran), fungicides (e.g., butylate, pebulate), and herbicides (e.g., ferbam, mancozeb).1 Many types of plantderived natural products have been identified as AChE inhibitors with additional reports emerging from fungi and marine source suggesting that nature is a valuable resource to

he cholinesterases are an important class of enzymes that catalyze the hydrolysis of ester bond linkages in cholinecontaining molecules. This group of enzymes is further subdivided into two major families, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), whose names reflect the enhanced rates at which these enzymes hydrolyze acetylcholine (ACh) and butyrylcholine, respectively.1 Biomedically, acetylcholinesterases play several important roles because of their abilities to rapidly hydrolyze ACh (a key neurotransmitter) and thus terminate nerve signal transmission. Consequently, the therapeutic inhibition of AChE using drugs such as donepezil, rivastigmine, and galantamine is an effective strategy for symptomatic relief from clinical © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 19, 2019

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DOI: 10.1021/acs.jnatprod.9b00563 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Zebrafish phenotype screen revealed bioactive fungal samples. (A) Distribution of assay outcomes from screening of fungal extracts. (B) Examples of phenotypes observed include growth arrest (1−3), edema (black arrow), abnormal head (green arrow) and tail (blue arrow) development, and undulated notochord (red arrow).

mine for AChE-inhibitor scaffolds.7−13 Among the handful of reported fungus-derived natural products that disrupt AChE activity, the territrems, arisugacins, and terreulactones stand out as a structurally distinct group of meroterpenoids that exhibit potent and selective AChE inhibition. To date, seven territrems, 14−17 12 arisugacins, 18−22 and six terrulactones16,23,24 have been reported, with the most potent inhibitors exhibiting IC50 values extending into the low nanomolar range.18,25 Zebrafish have been used as a model organism for phenotype-driven drug discovery since 2000.26,27 As one of the few groups to investigate chemical libraries in an unbiased phenotype screen, we have shown from previous work that we can utilize zebrafish, in juxtaposition with zebrafish genetics, to rapidly progress toward an understanding of a molecule’s putative mechanism of action.28 Zebrafish have several desirable characteristics that make them ideal as chemical testing platforms including high fecundity, small size (amenable to testing in microtiter-plate-based formats), and transparent embryos that rapidly develop ex utero. This latter point is important because it allows for the visualization and scoring of diverse phenotypes, which can provide critical insights into the mechanistic effects of many chemicals. Compared to simpler, cell-based assays, these complex phenotypic cues can help with the early stage prioritization of hits and provide an additional tool for filtering through

subtle, but meaningful differences in how molecules behave in intact organisms. Notably, zebrafish share a high degree of genetic and physiological similarity with humans; approximately 70% of human genes have an orthologue in zebrafish, enabling drug screening in zebrafish to have direct therapeutic relevance to many molecular pathways involved in a host of human diseases.27,29−35 Given the immense number of mechanistically uncharacterized bioactive compounds that are produced by fungi, a collaborative effort was launched to assess a pilot-scale set of fungal natural product extracts as a resource for integrating into a zebrafish-focused bioactive compound discovery program. In the course of this study, an extract prepared from a Penicillium sp. isolate was found to induce a paralysis phenotype in zebrafish embryos, indicative of central nervous system (CNS) modulators,36 and it was prioritized for further investigation. Bioassay-guided investigation led to the identification of 14 meroterpenoids, including six new natural products. The purification, structure determination, and biological characterization of the metabolites, including results from the zebrafish-based phenotype screen, are described in this report.

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RESULTS AND DISCUSSION Studies were performed using a circumscribed set of 384 fungal extracts prepared from fungi secured from the Great Lakes B



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Figure 2. Zebrafish phenotype screen revealed fungal extracts that impaired motility. Morphological phenotypes exhibited by a crude extract (CrEx) and representative subfractionated samples (SubFx) that impaired motility comparable to phenotype resulting from treatment with the known AChE inhibitor AZM. DMSO served as a negative control. Traces summarizing movement of zebrafish embryos (black dots) in response to external stimulus (probe is represented by a blue line) are shown in the circles associated with each picture.

Chart 1

on is often associated with perturbation of CNS functions.36 One of the bioactive extracts that induced paralysis (derived from a Penicillium sp. isolate) was chosen for further analysis since its effects were rather striking, resulting in limited movement of the zebrafish embryos when stimulated externally with a probe. This phenotype was similar to results we had observed when zebrafish embryos were exposed to azinphosmethyl (AZM), a known AChE inhibitor (Figure 2, Supplementary Figure 1). For chemical purification studies, the Penicillium sp. isolate was grown on Cheerios breakfast cereal, and the resulting mycelial mass was extracted with EtOAc, affording a crude extract that was subjected to bioassay-guided fractionation. This resulted in the purification of metabolites 1−3, which were identified as arisugacin D (1), arisugacin J (2), and arisugacin I (3) based on comparisons of their 1H NMR spectra, optical rotation values, and LC-ESIMS data to authentic samples and published data.20,21 Compound 1 had

(Michigan, USA) and soil samples from Oklahoma (USA) that were obtained through the Citizen Science Soil Collection Program operated out of the University of Oklahoma.37 Whereas a majority of the extracts had no effect on normal development, 46 samples (12%) resulted in a wide range of viable phenotypes that included growth arrest, head and tail defects, edema, and behavioral abnormalities (Figure 1). In addition, 45 extracts (12%) were lethal to the embryos (Figure 1). To examine this further, a dose curve analysis from 10 to 0.1 μg/mL was performed using 34 of the samples that caused death, which yielded 16 extracts that caused nonlethal developmental abnormalities at lower concentrations (Supplementary Table 2). This demonstrated that embryonic lethality as an assay outcome should not be dismissed, as it can lead to viable phenotypic aberrations upon optimization of experimental conditions such as dosing and time of treatment. Fungal extracts that affected motility were prioritized because of the phenotype’s clinical relevance; this phenomenC



Article

Table 1. 1H NMR Data for New Compounds 4, 5, 8, and 12−14 δH (ppm), mult (J in Hz) no. 1 2 4 5 8 12

4a,d

5b,d

5c,e

8c,d

12c,d

13c,d

14a,d

7.81 d (8.9) 7.03 d (8.9) 7.03 d (8.9) 7.81 d (8.9) 6.42 s 2.71 d (17.9)

7.54 d (8.9) 6.89 d (8.9) 6.89 d (8.9) 7.54 d (8.9) 5.95 s 2.57 d (17.1)

7.79, d (8.5) 7.00, d (8.5) 7.00, d (8.5) 7.79, d (8.5) 6.70, s 2.16, d (16.7)

7.80 d (8.9) 7.04 d (8.9) 7.04 d (8.9) 7.80 d (8.9) 6.72 s 2.65 d (18.1)

7.81 d (9.0) 7.04 d (9.0) 7.04 d (9.0) 7.81 d (9.0) 6.70 s 2.72 d (17.2)

7.73 d (8.7) 7.00 d (8.7) 7.00 d (8.7) 7.73 d (8.7) 6.67 s 3.17 d (7.4)

3.14 d (17.9)

2.71 d (17.1)

2.58, d (16.7)

2.95 d (18.1)

3.39 d (17.2)

7.82 d (9.0) 7.04 d (9.0) 7.04 d (9.0) 7.82 d (9.0) 6.73 s 2.31 dd (13.0, 16.8) 2.67 dd (4.5, 16.8) 1.78 dd (4.5, 13.0) 1.73 m

13 15

16

1.61 ddd (3.3, 5.6 11.5) 2.38 ddd (4.1, 11.5, 15.9) 1.73 ddd (3.3, 5.6, 12.3) 1.94 ddd (4.1, 12.3 15.9)

1.74 m

1.57, m

1.63 m

2.51 ddd (4.8, 10.6, 15.4) 1.85 m

2.25, ddd (2.8, 11.5, 15.0) 1.61, m

2.23 ddd (4.9, 13.4, 15.9) 1.63 m

1.87, ddd (3.1, 12.7, 15.0)

1.96 ddd (3.5, 12.1, 15.9)

1.60 ddd (3.4, 6.2, 12.7) 2.22 ddd (3.5, 12.7, 16.2) 1.74 ddd (3.5, 6.2, 13.4) 1.92 ddd (3.4, 13.4, 16.2)

17 19

4.91 brd (11.4)

20

2.05 m 2.16 ddd (2.8, 11.4, 14.4) 3.66 brdt (2.5)

21 23 24 25 26 27 OH13 OH17 OH19 OH21

1.04 s 1.10 s 1.44 s 1.19 s 3.86 s 6.72 brs

1.44 m 2.39 ddd (3.9, 11.0, 15.5) 1.70 m 1.91 m

1.11, m 2.11, m

4.74 dd (7.2, 7.3)

1.58, m

2.54 dd (7.3, 15.2) 2.74 dd (7.2, 15.2)

4.09 dd (5.2, 11.8)

3.70, dd (2.1, 8.9)

0.98 1.11 1.41 1.18 3.86

s s s s s

6.81 brs

5.74 brs

3.63 brsf

ndg

5.44 brsf

nd

2.65 dd (4.3, 16.1) 2.79 dd (9.6, 16.1)

2.11 m 1.62 m

1.10 s 0.96 s 1.38 s 1.10 s 3.81 s 6.63 brsf

0.96 s 1.11 s 1.37 s 1.24 s 3.82 s 6.44 brs

6.06, brsf

6.46 brsf

6.73 brs

2.09 m

1.74 m 1.86 dd (2.3, 12.5) 7.33 d (10.2)

5.11 dd (6.1, 7.7) 1.94 dd (6.3, 9.4)

5.81 d (10.2)

2.04 m

3.94 dd (4.3, 9.6)

0.88, s 0.81, s 1.34, s 1.04, s 3.78, s 6.65, brsf

5.32 dd (6.4, 7.4) 1.99 dd (5.5, 8.7)

1.09 1.02 1.30 1.10 3.81

s s s s s

5.06 dd (7.7, 7.1) 1.61 s 1.55 s 1.78 s 1.57 s 3.85 s

5.05, brsf 4.16, brsf

5.10, brs

a Observed in acetone-d6. bObserved in CDCl3. cObserved in DMSO-d6. dRecorded at 500 MHz. eRecorded at 600 MHz. fAssignments based on ROESY. gnd: not detected.

deshielded (δC 65.4), suggesting that it was attached to a hydroxy group. Analysis of the HMBC and COSY data provided substantiation for the structure of the remaining portions of the molecule, confirming that 4 was a new tetrahydroxy arisugacin (Figure 3). Turning to address the configuration of the stereogenic carbon atoms in 4, 1H−1H ROESY correlation data played a key role in establishing the metabolite’s relative configuration (Figure 4). Two distinct sets of ROESY correlations were observed arising from the hydrogens occupying opposite faces of the molecule’s dodecahydro-1H-benzo[f ]chromene core. Specifically, ROESY correlations among H3-26 ↔ H3-24, H325, and H-12 (δH 2.71); H3-25 ↔ H-16 (δH 1.94); and H3-24 ↔ H-21 provided evidence that those atoms were located on one face of the molecule. A second set of ROESY correlations among H2-12 (δH 3.14) ↔ H-19 and OH-13, as well as between H3-23 ↔ OH-17, H-21, and H-16 (δH 1.73), indicated those atoms occupied the opposite face of the

been reported as a selective AChE inhibitor with an IC50 value of 3.5 μM.20 This initial observation, combined with the impaired motility phenotype from our zebrafish screen, inspired us to further pursue the structure identification of other low-abundance arisugacin analogues contained within the bioactive extract because some members of this class of compounds are known to exhibit potent and selective AChE inhibitory activities.38 Switching to a focus on the LC-ESIMS evidence, we pursued the identification of several structurally related metabolites from the Penicillium sp. extract. Arisugacin L (4) was obtained as a white, amorphous powder, and its molecular formula was established as C27H34O8 based on HRESIMS data, which corresponded to 11 units of unsaturation. An inspection of the 1H and 13C NMR data for 4 (Tables 1 and 2) revealed that this compound shared many structural features with known metabolite 2.21 Considering that 4 contained one additional oxygen atom, further inspection of the data revealed that C-19 in 4 was more D


Journal of Natural Products Table 2.

13

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C NMR Data for New Compounds 4, 5, 8, and 12−14 (100 MHz, δ ppm)a δC, mult

no.

4b

5c

8d

12d

13d

14b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

127.6 CH 115.1 CH 162.4 C 115.1 CH 127.6 CH 125.4 C 158.2 C 97.4 CH 163.4 C 99.6 C 164.4 C 29.9 CH2 77.9 C 82.4 C 29.9 CH2 26.5 CH2 84.7 C 49.1 C 65.4 CH 37.2 CH2 78.9 CH 42.2 C 23.8 CH3 24.7 CH3 24.8 CH3 17.4 CH3 55.8 CH3

127.2 CH 114.2 CH 161.8 C 114.2 CH 127.2 CH 123.6 C 158.7 C 96.6 CH 164.1 C 96.7 C 165.6 C 25.7 CH2 77.6 C 82.2 C 29.5 CH2 26.0 CH2 80.1 C 43.6 C 26.0 CH2 27.3 CH2 73.7 CH 44.5 C 22.8 CH3 18.7 CH3 24.6 CH3 21.0 CH3 55.6 CH3

126.8 CH 114.5 CH 161.1 C 114.5 CH 126.8 CH 123.6 C 157.0 C 96.6 CH 162.4 C 97.4 C 163.4 C 27.4 CH2 76.3 C 81.4 C 28.5 CH2 25.3 CH2 80.7 C 47.9 C 67.1 CH 44.7 CH2 212.8 C 52.8 C 23.3 CH3 21.7 CH3 23.4 CH3 16.0, CH3 55.4, CH3

126.8 CH 114.5 CH 161.0 C 114.5 CH 126.8 CH 123.6 C 157.0 C 96.6 CH 162.4 C 97.3 C 163.3 C 26.7 CH2 75.6 C 80.8 C 28.4 CH2 25.4 CH2 81.5 C 57.1 C 209.6 C 45.8 CH2 72.4 CH 42.9 C 20.7 CH3 25.5 CH3 23.5 CH3 18.6 CH3 55.4 CH3

127.0 CH 114.5 CH 161.2 C 114.5 CH 127.0 CH 123.4 C 157.6 C 96.5 CH 163.1 C 97.8 C 162.9 C 16.8 CH2 45.8 CH 80.5 C 39.2 CH2 19.3 CH2 52.1 CH 39.0 C 157.5 CH 125.3 CH 203.5 C 44.0 C 27.3 CH3 21.4 CH3 20.7 CH3 18.1 CH3 55.5 CH3

127.8 CH 115.3 CH 162.4 C 115.3 CH 127.8 CH 125.7 C 158.4 C 98.4 CH 167.5 C 102.9 C 165.5 C 23.3 CH2 123.4 CH 135.4 C 40.7 CH2 27.4 CH2 125.2 CH 135.6 C 40.6 CH2 27.6 CH2 125.4 CH 131.7 C 26.0 CH3 17.9 CH3 16.6 CH3 16.3 CH3 56.0 CH3

a Multiplicities were assigned based on 1H, 13C, and HSQC NMR (13C 100/1H 500 MHz) experiments. bObserved in acetone-d6. cObserved in CDCl3. dObserved in DMSO-d6.

Figure 3. 1H−1H COSY (blue bold lines) and key HMBC (red arrows) correlations for compounds 4, 5, 8, and 12−14.

membered ring systems.20,21,25 Using a modified Mosher esterification strategy, C-21 was determined to have an R configuration (Figure 5); thus, the absolute configuration of 4 was assigned as 13S,14R,17R,18S,19R,21R. Arisugacin M (5) was obtained as a white, amorphous powder. The molecular formula was assigned as C27H34O7 based on its HRESIMS data indicating 11 units of unsaturation. An analysis of the 13C NMR and HSQC data

molecule. This left one hydrogen atom, H-21, whose relative orientation remained ambiguous; however, an examination of its splitting pattern (appeared as a broad triplet) and coupling constant (J = 2.5 Hz) indicated this hydrogen occupied an equatorial position. Taking these data into consideration, the relative configuration of 4 was determined to be 13S*,14R*,17R*,18S*,19R*,21R*, which is similar to several known arisugacin analogues containing three trans-fused sixE



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Figure 4. 3D molecular models of compounds 4, 5, 8, and 12−14 showing key 1H−1H ROESY correlations (blue dashed arrows).

Figure 5. ΔδS−R values obtained in pyridine-d5 for the MTPA esters of compounds 4, 5, and 12.

of C-21 was determined to be S by the modified Mosher’s method (Figure 5); therefore, the overall absolute configuration of 5 was assigned as 13S, 14R, 17R, 18S, and 21S. Arisugacin N (8) was obtained as a white, amorphous powder. The molecular formula was established to be C27H32O8, on the basis of the HRESIMS data. The 1H and 13 C NMR data for 8 showed substantial similarities to 4 except for a new carbonyl signal (δC 212.8) representing a ketone that appeared in place of a hydroxymethine (C-21 in 4). Analysis of the 1H−1H COSY and HMBC correlation data provided confirmation that, other than the noted change to C-21, the atom-bond arrangements in 8 were the same as those observed for 4 (Figure 3). Next, 1H−1H ROESY data [H3-26 ↔ H3-24 and H3-25; H3-24 ↔ H-20 (δH 2.74); H3-25 ↔ H-12 (δH 2.65); H-19 ↔ H-12 (δH 2.95), OH-13, OH-17, and H3-23; as well as H3-23 ↔ H-20 (δH 2.54)] were used to confirm a

revealed that the hydroxy group observed on C-19 in 4 was missing in 5 and, instead, a new methylene signal was present (δC 26.2). This conclusion was supported by an analysis of the HMBC and COSY data, which also confirmed the proposed bond-line structure of 5 (Figure 3). 1 H−1H ROESY correlation data were instrumental in solving the relative configuration of 5 (Figure 4). Two sets of correlations emerged based on which face of the dodecahydro-1H-benzo[f ]chromene scaffold the hydrogen atoms occupied. The first series of observed correlations included H3-26 ↔ H3-25, H3-24, and H-19 (δH 1.11), as well as between H3-25 ↔ H-16 (δH 1.87). On the opposite face of 5, ROESY correlations were observed between H-19 (δH 2.11) ↔ OH-13, H-21, and OH-17, as well as H3-23 ↔ H-16 (δH 1.61). These data pointed toward a 13S*,14R*,17R*,18S*,21S* relative configuration. The absolute configuration F



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13S*,14R*,17R*,18S*,19R* relative configuration for 8. All attempts to create a C-19 Mosher ester derivative failed, while later efforts to collect electronic circular dichroism (ECD) data for 8 after assay data collection was complete revealed that our remaining stock of the metabolite had degraded. However, in view of the consistent configurations observed for the C-13, C14, C-17, and C-18 stereogenic centers in this metabolite series (1−7), as well as the similar optical rotation values observed for these metabolites,20,21 we propose that the absolute configuration for 8 is likely 13S,14R,17R,18S,19R. Arisugacin O (12) was obtained as a white, amorphous powder. HRESIMS provided molecular ions indicative of the molecular formula C27H32O8. This molecular formula was the same as that obtained for 8, and comparisons of their 1H and 13 C NMR data (Tables 1 and 2) indicated that the structures of 8 and 12 were very similar. A comparative analysis of the HMBC data for 8 and 12 revealed that the C-19 hydroxymethine in 8 was oxidized to a ketone in 12 [H3-26 (δH 1.24) → C-19 (δC 209.6)], while the C-21 ketone in 8 now appeared in a reduced state as a hydroxymethine in 12 [H3-23 (δH 0.96) → C-21 (δC 72.4)] (Figure 3). The relative configuration of 12 was determined to be 13S*,14R*,17R*,18S*,21R* based on an analysis of 1H−1H ROESY correlation data (Figure 3). Specifically, ROESY correlations were detected among protons H3-26 ↔ H3-25, H3-24, and H-21; H3-25 ↔ H-15 (δH 1.60) and H-12 (δH 2.72); H3-24 ↔ H-21; H-12 (δH 3.39) ↔ OH-13; and H-15 (δH 2.22) ↔ OH-17. The absolute configuration of C-21 was determined to be R by the modified Mosher’s method (Figure 5), enabling us to assign the absolute configuration of 12 as 13S, 14R, 17R, 18S, 21R. Arisugacin P (13) was obtained as a white, amorphous powder, and its molecular formula was determined to be C27H30O5 by analysis of the HRESIMS data, which meant the molecule contained 13 units of unsaturation. The HSQC data revealed that in comparison to compounds 8 and 12 metabolite 13 possessed two new deshielded methine carbon atoms (δC 157.5 CH and 125.3 CH), which, based on 13C NMR (Table 2) and HMBC (Figure 3) data, were determined to be part of an α,β-unsaturated carbonyl system associated with ketone C-21. Further investigation of the HMBC data revealed that 13 was structurally similar to the previously described meroterpenoid terreulactone B,25 which had been isolated from an Aspergillus terreus. The major difference between these metabolites was that both the C-13 and C-17 hydroxy groups reported in terreulactone B were missing and, instead, metabolite 13 featured two new upfield spins (δC 45.8 and 52.1) (Table 2). The relative configuration of 13 was determined to be 13S*,14R*,17R*,18S* based on 1H−1H ROESY correlation data (Figure 4). Specifically, correlations among protons H3-26 ↔ H-16 (δH 1.62), H3-24, and H3-25; H3-25 ↔ H-12 (δH 2.31); H3-23 ↔ H-17 and H-16 (δH 1.74); and H-12 (δH 2.67) ↔ H-13 were instrumental in facilitating the assignment of the molecule’s configuration. To evaluate the absolute configuration of 13, the experimental and calculated ECD spectra of 13 were compared (Figure 6). The calculated ECD spectrum displayed Cotton effects that were similar to the experimentally derived ECD spectrum, supporting a 13S,14R,17R,18S absolute configuration for compound 13. Arisugacin Q (14) was obtained as a white, amorphous powder. HRESIMS data supported a molecular formula of C27H34O4, requiring 11 units of unsaturation. The 1H, 13C, and HSQC NMR experiments (Tables 1 and 2) provided data for a

Figure 6. Calculated and experimental ECD data for compound 13.

molecule that largely mirrored the structure of compound 3,21 which was the first arisugacin reported to possess a largely uncyclized sesquiterpenoid moiety. Notable differences in 14 compared to 3 included the presence of carbon spin resonances attributable to an additional olefinic bond (δC 125.4, 131.7) in 14, which coincided with the loss of two oxygen atoms. The 1H−1H COSY correlation data revealed couplings between the H2-20 (δH 2.04) and H-21 (δH 5.06), while HMBC correlations from H-20 → C-22, H3-23 → C-21, and H3-24 → C-21 were used to secure the structure assignment for the modified portion of this compound. Further investigation using the 2D NMR data (Figure 3) helped establish the complete structure of 14 (Figure 3). The geometry of the two double bonds (Δ13 and Δ17) was determined as E,E on the basis of ROESY correlations among H2-12 ↔ H3-26, H-13 ↔ H2-15, H2-16 ↔ H3-25, and H-17 ↔ H2-19 (Figure 4). In addition to the new compounds, several additional metabolites were detected and subsequently purified including the known compounds epi-arisugacin E (6),22 arisugacin F (7), 20 arisugacin C (9), 20 arisugacin G (10), 20 and terreulactone C (11).25 The structures of these compounds were ascertained based on comparisons of 1H and 13C NMR and HRESIMS data with previously reported data. Following the purification of these compounds, all metabolites were evaluated in zebrafish embryos. New compounds 5 and 12−14 induced paralysis, with compound 12 being the most active at a test concentration of 1 μM (Table 3). Additionally, embryos exposed to compound 12 exhibited defects in myotome structure, as shown by in situ hybridization for xirp2a, closely phenocopying embryos treated with the known AChE inhibitors azinphos-methyl and physostigmine (Figure 7A and B).1,39 This is consistent with previous reports of AChE inhibition leading to severe abnormalities in muscle development.36 Treatment with the known BuChE inhibitor tetraisopropyl-pyrophosphoramide (iso-OMPA), did not result in compromised motility or aberrant myotome development (Figure 7A and B).40 Using an in vitro assay to investigate whether the new arisugacins directly inhibit AChE, we found that compounds 5 and 12 showed the strongest inhibition, with IC50 values of 3.9 and 0.19 μM, respectively (Table 3 and Figure 7C). Compounds 4 and 8 did not impair motility in zebrafish embryos; however, these compounds did inhibit in vitro AChE activity, which suggested that certain pharmacological parameters were not met to cause bioactivity in vivo (Table 3). The known compounds 1, 3, 9, and 10 produced a paralysis phenotype in zebrafish, with compounds 3 and 10 causing myotome structural abnormalities, as well as demonstrated abilities to inhibit AChE in vitro (Figure 8, Table 3). Neither G



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Table 3. Bioactivity of Compounds compound

zebrafish assay, active concentrationa (μM)

iso-OMPAc AZMd physostigmined 1 2 3 4 5 6 7 8 9 10 11 12 13 14

− 1 100 10 − 10 − 50 − − − 5 5 − 1 5 50

AChE inhibitionb at 50 μM (%) BuChE inhibitionb at 50 μM (%) 1.3 12.2 99.8 60.1 83.4 34.1 29.2 89.4 75.4 3.0 83.6 95.1 26.9 96.6 98.0 40.3 4.3

15.1 3.3 93.2 4.7 0 4.7 0 4.7 7.0 4.7 0 32.6 11.6 9.3 0 0 0

AChE inhibition IC50 (μM) >100 nt 0.0078 nt nt >100 >100 3.9 nt nt 4.6 1.4 >100 nt 0.191 66 >100

a

Active concentration refers to the lowest compound concentration that induced paralysis in zebrafish embryos treated at 7 h postfertilization (hpf) and evaluated for phenotype at 72 hpf. A dash (−) signifies no bioactivity in vivo at 100 μM or lower. bInhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was determined at sample concentration of 50 μM. cBuChE inhibitor. dAChE inhibitor. nt, not tested. Iso-OMPA and AZM stand for tetraisopropylpyrophosphramide and azinphos-methyl, respectively.

Figure 8. Bioactivity of known arisugacin compounds with no previously reported AChE bioactivity. (A) Phenotypes of 72 h postfertilization (hpf) embryos that exhibited impaired motility following early compound treatment. (B) In situ hybridization for xirp2a in 72 hpf embryos. Figures in boxes are enlargements of the trunks revealing abnormalities in myotome (black arrow) structure. (C) IC50 values of arisugacins C, G, and I were obtained in an in vitro AChE inhibition assay.

Figure 7. New arisugacin compounds show acetylcholinesterase inhibitory activity. (A) Phenotypes of 72 h postfertilization (hpf) embryos that exhibited impaired motility following early compound treatment. (B) In situ hybridization for myotome marker xirp2a (xin actin binding repeat containing 2a) in 72 hpf embryos. Figures in boxes are enlargements of the trunks revealing abnormalities in myotome (black arrow) structure. (C) IC50 values of the new arisugacin analogues were obtained in an in vitro AChE inhibition assay.

compound had been previously shown to be biologically active, but they were reported to possess weak AChE inhibitory properties in prior in vitro assays.20,21 Compound 11, which H



Article

110 Å, 250 × 10 mm, 4 mL/min). All solvents were of ACS grade or better. Fungal Isolate and Fermentation. The fungal isolate was obtained from a soil sample submitted by a citizen scientist from Lawton, Oklahoma, to the University of Oklahoma Citizen Science Soil Collection Program (sample number 10263). The isolate was identified as a probable Penicillium sp. (most closely associated to Penicillium oxalicum) based on BLAST analysis of its ribosomal internal transcribed spacer (ITS) sequence data (GenBank accession number MK722362) to data contained in the NCBI database. Fermentation was performed by inoculating fungal mycelia and spores on a solid-state medium composed of Cheerios breakfast cereal supplemented with a 0.3% sucrose solution. The fungus was grown for 4 weeks at room temperature (25 °C). Extraction and Isolation. The fungal biomass was extracted with EtOAc (×3), and the organic-soluble material was concentrated under vacuum to generate the crude extract (130 g). The extract was separated into five fractions (Fr.1−Fr.5) by silica gel vacuum liquid chromatography [eluted with hexane−CH2Cl2 (1:1), CH2Cl2, CH2Cl2−MeOH (10:1), CH2Cl2−MeOH (1:1), and MeOH]. Fr.3 (eluted with 10:1 CH2Cl2−MeOH) was subjected to HP20ss vacuum liquid chromatography (eluted with 30%, 50%, 70%, 90%, 100% MeOH in H2O) to yield five subfractions. The fourth subfraction recapitulated the paralysis phenotype in the zebrafish assay, and it was further fractionated by semipreparative HPLC (eluted with 40:60 ACN−H2O with 0.1% formic acid in H2O), yielding compounds 1 (1.9 mg), 2 (3.7 mg), 3 (1.5 mg) 4 (4.5 mg), 5 (7.9 mg), and 6 (2.6 mg). Subfraction 5 (100% MeOH) was also bioactive, and it was further fractionated by semipreparative HPLC (70:30 ACN−H2O with 0.1% HCOOH) to afford compounds 7 (2.2 mg), 8 (3.7 mg), 9 (7.9 mg), 10 (10.1 mg), 11 (4.0 mg), 12 (2.2 mg), 13 (1.1 mg), and 14 (2.1 mg). Arisugacin M (4): amorphous, white powder; [α]20D +69.3 (c 0.225, MeOH); 1H and 13C NMR, see Table 1 and Table 2; HRESIMS [M + H]+ m/z 487.2341, [M + Na]+ m/z 509.2155 (calcd for C27H35O8, 487.2326, C27H34O8Na, 509.2146). Arisugacin N (5): amorphous, white powder; [α]20D +64.8 (c 0.395, MeOH); 1H and 13C NMR, see Table 1 and Table 2; HRESIMS [M + H]+ m/z 471.2393, [M + Na]+ m/z 493.2210 (calcd for C27H35O7, 471.2377 C27H34O7Na, 493.2197). Arisugacin O (8): amorphous, white powder; [α]20D +64.9 (c 0.185, MeOH); 1H and 13C NMR, see Table 1 and Table 2; HRESIMS [M + H]+ m/z 485.2165, [M + Na]+ m/z 507.1982 (calcd for C27H33O8, 485.2170, C27H32O8Na, 507.1989). Arisugacin L (12): amorphous, white powder; [α]20D +4.5 (c 0.11, MeOH); 1H and 13C NMR, see Table 1 and Table 2; HRESIMS [M + H]+ m/z 485.2164, [M + Na]+ m/z 507.1989 (calcd for C27H33O8, 485.2170, C27H32O8Na, 507.1989). Arisugacin P (13): amorphous, white powder; [α]20D +100 (c 0.05, MeOH); 1H and 13C NMR, see Table 1 and Table 2; HRESIMS [M + H]+ m/z 435.2181, [M + Na]+ m/z 457.1993 (calcd for C27H31O5, 435.2166, C27H30O5Na, 457.2064). Arisugacin Q (14): amorphous, white powder; 1H and 13C NMR, see Table 1 and Table 2; HRESIMS [M + H]+ m/z 423.2517, [M + Na]+ m/z 445.2341 (calcd for C27H35O4, 423.2530, C27H34O4Na, 445.2349). Preparation of MTPA Esters. Compounds 4, 5, and 12 (0.2 mg each) were dissolved separately in pyridine (500 μL) and transferred into NMR tubes. Aliquots (2 μL) of (S)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) were added to the samples, and the reaction mixtures monitored every 2 h by 1H NMR. (S)-MTPA esters were prepared in a similar way using (R)-α-methoxy-α-trifluoromethylphenylacetic acid. (R)-MTPA Ester of 4: 1H NMR (500 MHz, pyridine-d5) δ 5.49 (H21), 5.39 (H-19), 4.08 (H-12b), 3.37 (H-12a), 2.88 (H-15b), 1.87 (H-15a), 2.55 (H-20b), 2.55 (H-20a), 2.01 (H-16b), 1.86 (H-16a), 1.54 (H-25), 1.46 (H-26), 1.28 (H-23), 1.19 (H-24); ESIMS m/z 703.35 [M + H]+. (S)-MTPA ester of 4: 1H NMR (500 MHz, pyridine-d5) δ 5.53 (H21), 5.45 (H-19), 4.11 (H-12b), 3.38 (H-12a), 2.85 (H-15b), 1.84

has been previously reported to be a potent and selective AChE inhibitor, did not induce phenotypic changes in the zebrafish (Table 3).25 All compounds showed minimal inhibition of BuChE; however, only compound 12 stood out as highly selective toward AChE (Table 3). There were several compounds that had consistent bioactivity in both the phenotype-based zebrafish screen and in vitro AChE inhibition assay (Table 3). However, there were samples that showed opposing trends such as compound 11, which did not affect motility in zebrafish, while physostigmine induced a phenotype only at 100 μM. Both compounds inhibited AChE in vitro at low nanomolar concentrations, which indicates that within this class of molecules additional structural features are important for modulating in vivo efficacy. In contrast, AZM caused paralysis in zebrafish embryos at 1 μM, but showed weak inhibition of AChE in vitro. This is consistent with the mechanism of toxicity of AZM; its conversion to the oxon form by cytochrome P450 has been found to be responsible for its AChE inhibitory activity, which is not readily apparent in an in vitro assay.41 Furthermore, we have observed bioactivity associated with compounds 3 and 10 in developing zebrafish embryos, whereas both metabolites were weakly active in the in vitro assays. These examples illustrate the unique advantage of using a whole organism-based phenotype screen, as it not only provides information about the biological activities of small molecules in vivo but also affords insights about their pharmacokinetic and pharmacodynamic characteristics as well. This knowledge is critical in determining and prioritizing lead compounds and in facilitating drug discovery efforts in general. Our findings demonstrate the utility of the zebrafish assay as a tool for rapidly deconvoluting complex natural product mixtures into active components and classifying their mechanisms of action. A total of six new and eight known meroterpenoids were purified from a Penicillium sp. isolate. Four of the new compounds (5, 12−14) induced paralysis in zebrafish, with compound 12 demonstrating potent and selective AChE inhibitory activity under both in vivo and in vitro conditions. AChE inhibitors have been adopted for clinical use to treat several clinical conditions including bladder distention, glaucoma, vascular dementia, and symptoms of Alzheimer’s disease (AD).1,42 Currently, a majority of the drugs approved for AD treatment are AChE inhibitors, but the need for better therapeutics to address cognitive impairment remains.43 Our discovery of new arisugacin compounds, some with potent and specific AChE inhibitory activity, could provide leads for the development of better clinical AChE inhibitors.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Autopol III. NMR data were obtained on Varian VNMR spectrometers (500 and 600 MHz for 1H and 2D NMR, 100 MHz for 13C NMR). The LC-ESIMS analyses were performed on a Shimadzu UFLC system with a quadrupole mass spectrometer using a Phenomenex Kinetex C18 column (3.0 mm × 75 mm, 2.6 μm) and ACN−H2O (0.1% HCOOH) gradient solvent system. High-resolution mass data were collected on an Agilent 6538 QTOF mass spectrometer. Analytical and semipreparative HPLC were performed on a Waters 2998 photodiode array detector; the columns used were Phenomenex Gemini 5 μm C18 columns (110 Å, 250 × 4.6 mm, 1 mL/min; 110 Å, 250 × 10 mm, 4 mL/min) and Kinetex 5 μm biphenyl columns (110 Å, 250 × 4.6 mm, 1 mL/min; I



Article

(H-15a), 2.69 (H-20b), 2.58 (H-20a), 1.99 (H-16b), 1.84 (H-16a), 1.54 (H-25), 1.47 (H-26), 1.16 (H-23), 1.10 (H-24); ESIMS m/z 703.35 [M + H]+. (R)-MTPA ester of 5: 1H NMR (500 MHz, pyridine-d5) δ 6.03 (H21), 3.09 (H-12b), 2.92 (H-12a), 2.83 (H-15b), 1.96 (H-15a), 2.75 (H-19b), 1.42 (H-19a), 2.18 (H-20b), 2.08 (H-20a), 1.90 (H-16b), 1.86 (H-16a), 1.50 (H-25), 1.23 (H-23), 1.21 (H-26), 1.11 (H-24). (S)-MTPA ester of 5: 1H NMR (500 MHz, pyridine-d5) δ 6.02 (H21), 3.07 (H-12b), 2.90 (H-12a), 2.84 (H-15b), 1.97 (H-15a), 2.74 (H-19b), 1.39 (H-19a), 2.14 (H-20b), 1.86 (H-20a), 1.90 (H-16b), 1.86 (H-16a), 1.50 (H-25), 1.33 (H-23), 1.19 (H-26), 1.14 (H-24). (R)-MTPA ester of 12: 1H NMR (500 MHz, pyridine-d5) δ 5.96 (H-21), 4.35 (H-12b), 3.24 (H-12a), 3.48 (H-20b), 3.07 (H-20a), 2.81 (H-15b), 1.87 (H-15a), 1.96 (H-16b), 1.94 (H-16a), 1.50 (H25), 1.45 (H-26), 1.34 (H-23), 1.31 (H-24). (S)-MTPA ester of 12: 1H NMR (500 MHz, pyridine-d5) δ 5.96 (H-21), 4.35 (H-12b), 3.24 (H-12a), 3.59 (H-20b), 3.13 (H-20a), 2.80 (H-15b), 1.85 (H-15a), 1.94 (H-16b), 1.92 (H-16a), 1.49 (H25), 1.45 (H-26), 1.24 (H-23), 1.19 (H-24). ECD Calculation for Compound 13. Conformational analyses were performed using Spartan’10.44 Geometry, frequency, and ECD calculations were carried out at the DFT level with Gaussian 09.45 SpecDis 1.60 was used to average single ECD spectra after Boltzmann statistical weighting.46 Zebrafish Maintenance. Wild-type (WT) AB/TU Danio rerio (zebrafish) were maintained as previously described.47 Fertilized embryos were collected following natural spawning in 1× E3 medium (286 mg/L NaCl, 13 mg/L KCl, 48 mg/L CaCl2·2H2O, 40 mg/L MgSO4, 0.01% methylene blue) and allowed to develop at 28.5 °C. All zebrafish protocols were approved by the OMRF IACUC (protocol #17-03) on February 22, 2017. Zebrafish Phenotype Screen. Fungal extracts were tested based on our previously reported zebrafish phenotype screen.28 Extracts and fractions were prepared in DMSO as 2 mg/mL stock solutions. Pure fungal compounds were prepared as 10 mM stock solutions in DMSO. Azinphos-methyl, tetraisopropyl pyrophosphoramide, and physostigmine were prepared as 100 mM stock solutions in DMSO. Further dilutions were carried out in DMSO as required. Chemicals and control compounds were obtained from the following: DMSO and AZM from Sigma-Aldrich (St. Louis, MO, USA); iso-OMPA and physostigmine from Santa Cruz Biotechnology (Dallas, TX, USA). In Situ Hybridization. In situ hybridizations were performed as previously described using digoxigenin-labeled riboprobes for xirp2a (xin actin binding repeat containing 2a).48 Embryos were cleared in a 2:1 benzyl benzoate−benzyl alcohol solution and documented using an Olympus SZX12/DP71 imaging system (Olympus Corporation, Japan). An RNA reference sequence deposited in ZFIN (zfin.org) was used in the design of the riboprobe. Acetylcholinesterase and Butyrylcholinesterase Inhibition Assays. AChE inhibition was determined using QuantiChrom acetylcholinesterase inhibitor screening kit following the manufacturer’s protocol (BioAssay Systems-Hayward, CA, USA). Briefly, test compounds were incubated with AChE from Electrophorus electricus (Sigma-Aldrich) for 15 min. Absorbance was measured at T = 0 min and T = 15 min after the addition of assay buffer containing AChE substrate and 5,5′-dithiobis(2-nitrobenzoic acid). The same kit and protocol were used to measure butyrylcholinesterase inhibition by substituting butyrylcholinesterase from equine serum and Sbutyrylthiocholine iodide as the substrate. Both reagents were purchased from Sigma-Aldrich. Raw absorbance readings were corrected against the “no enzyme” control and normalized relative to the “no inhibitor” control. Data were plotted and analyzed using Graph Pad Prism v7.02.

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NMR (1H, 13C, 1H−1H COSY, HSQC, HMBC, and ROESY), HRESIMS data of 4, 5, 8, 12−14, information about the fungal isolate and fungal extract, zebrafish phenotype screening results (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: 405-271-4527. *E-mail: [email protected]. Tel: 405-325-6969. ORCID

Robert H. Cichewicz: 0000-0003-0744-4117 Author Contributions ⊥

W.D. and I.T.S. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The LC-ESIMS instrument used for this project was provided in part by a Challenge Grant from the Office of the Vice President for Research, University of Oklahoma, Norman Campus, and an award through the Shimadzu Equipment Grant Program (R.H.C.). We are grateful for the contribution of the soil sample from citizen scientist T. Hambrick from which the Penicillium sp. isolate was obtained. This work was supported by the National Institutes of Health under R01GM107490 (R.H.C.) and R01CA116468 (D.A.J.), Samuel Waxman Cancer Research Foundation (D.A.J.), Oklahoma Center for Adult Stem Cell Research (D.A.J.), and Oklahoma Medical Research Foundation (D.A.J.).

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REFERENCES

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