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Abstract

Objective: The aim of this research was to isolate and identify the natural products responsible for the biological and fluorescence activity of the extract from the marine sponge Aaptos suberitoides. Methods: Fluorescence activity-guided separation was performed after validation of the inhibitory activity of the extract against human transient receptor potential ankyrin 1 (hTRPA1) and subsequent measurement of its fluorescence activity. Three previously reported marine alkaloids, aaptamine, isoaaptamine, and 9-O-demethylaaptamine, were obtained and identified based on their nuclear magnetic resonance and high-resolution mass spectrometry data. Results: Aaptamine showed emission of the fluorescent light at a wavelength of 485 nm with a quantum yield of 31%. Isoaaptamine and 9-O-demethylaaptamine showed marginal fluorescence activity but promising inhibitory activity against hTRPA1 with IC₅₀ values of 3.1 and 3.9 μM, respectively. Conclusions: This research demonstrates that natural fluorophores can be discovered in living organisms through photoactivity-guided isolation. Aaptamine, isolated as a result, provides an additional 1,6-naphthyridine fluorophore, while isoaaptamine and 9-O-demethylaaptamine suggest a chemical scaffold that could be used to study the function of ion channels.

Keywords

alkaloid aaptamine activity-guided isolation fluorescence activity transient receptor potential ankyrin 1 marine sponge

Introduction

In our search for natural products with potential as anti-pain medications, the extracts obtained from marine sponges were tested for their activity against transition receptor potential ankyrin 1 (TRPA1) as a preliminary evaluation of the extracts. TRPA1 is a nonselective cation channel involved in the transduction of stimuli at peripheral terminals of primary afferent sensory neurons and in the amplification of nociceptive transmissions at central terminals.¹ As it has been previously revealed that TRPA1 is involved in chronic pain and that TRPA1 antagonists can reduce pathophysiological pain with only minor side effects, efforts are being made to discover effective TRPA1 antagonists.^2–4 The method used to screen the extracts was the fluorometric imaging plate reader (FLIPR) assay, which measures the cytosolic calcium ion concentration detected by the fluorescent dye.⁵ Since hits may result from the photochemical properties of the extract composed of unknown constituents rather than the biological activity of those, it was necessary to validate the hit extracts for their fluorescence activity before proceeding to identify the biologically active compounds. Based on this consideration, the question arose as to whether the fluorescence activity-guided isolation of the natural product for the discovery of new fluorophores would be possible if the fluorescence activity was detected at the extract level.

Fluorescent molecules (fluorophores) are those that have a distinct response to light compared to other molecules; they absorb and emit light of a specific wavelength, and the emitted light typically has wavelengths in the range of 390 to 700 nm. The fluorophore itself can be utilized as a molecular probe if the fluorescence intensity or wavelength changes dramatically with the presence or absence of a specific biomolecule or metal. If the fluorophore is attached to a structural scaffold that can bind to the specific biomolecule, it can be used to measure the distribution of that biomolecule in cells or organisms.^6–8 There has been tremendous progress in the development and application of fluorescent probes, particularly in the fields of cell biology, drug discovery, and disease diagnosis. However, there is still a need for more diverse fluorophores with improved properties such as lower detection limits, broader applications, and the ability for reversible or real-time detection. Natural products have proven to be promising scaffolds for fluorophores; compounds such as coumarins, quinolones, and anthraquinones are representative examples.⁹

Results and Discussion

Ninety-four extracts derived from the sponges collected off the coast of Vietnam were tested in the calcium FLIPR assay (Figure 1), with 13 showing inhibition > 70% at a concentration of 10 μg/mL. Among these extracts, 5 were obtained from the sponges sharing the same morphological characteristics. One of these was selected for further investigation (Supplemental Figure S1), which was derived from the sponge specimens collected off the coast of Nha Trang Bay and identified as Aaptos suberitoides based on its morphological characteristics. This identification was later confirmed by sequencing the cytochrome c oxidase subunit 1 (CO1) mitochondrial gene (Supplemental Figure S2).

Figure 1.

Scatter plot obtained from duplicate experiments of the inhibition rate of 94 Vietnamese marine sponge extracts against TRPA1. The yellow dots are the results obtained from the cells treated with 5 µM AITC only (0% inhibition); the blue dots are those obtained from the untreated cells (100% inhibition); the pink dots represent the results obtained from the cells treated with 10 µM A-967079, then 5 µM AITC. The circles represent the results obtained from the cells treated with 10 µg/mL extract, followed by 5 µM AITC. Among them, a green circle represents the results obtained with the Aaptos suberitoides extract investigated in this research, and the red circles represent the results obtained with the extracts derived from the sponges with the same morphological characteristics as A suberitoides.

The specimens were sequentially extracted with methanol and dichloromethane, and the combined extract was partitioned between n-butanol and water to remove the inorganic salts. The n-butanol extract was the one that has been subjected to the activity screening of a panel of extracts against TRPA1 and resulted in 70% inhibition at a concentration of 10 μg/mL. The weak fluorescence emission was also detected for this extract (Figure 2). In the FLIPR assay used to measure TRPA1 inhibitory activity, the excitation and emission wavelengths were set at 485 and 525 nm, respectively, with a cut-off wavelength of 515 nm applied to prevent the excitation light from reaching the detector. Since those determined for the n-butanol extract were 367 and 482 nm, respectively, interference from the fluorescence activity of the extract, if any, was assumed to be very weak.

Figure 2.

Initial photochemical property measurement results for the extract and fractions obtained from Aaptos suberitoides. Absorbance and emission spectra at a concentration of 10 μg/mL in dimethyl sulfoxide (DMSO).

Encouraged by these results, we proceeded with further separations of this extract in the hope of identifying natural products with either a fluorescent activity or TRPA1 inhibitory activity. The n-butanol extract was further partitioned between 15% aqueous methanol and n-hexane and it was found that only the aqueous methanol fraction (F-1) exhibited fluorescence emission at a concentration of 10 μg/mL in dimethyl sulfoxide (DMSO) (Figure 2). This fraction was further separated by reverse-phase column chromatography using an octadecyl silica (ODS) resin and a gradient solvent of varying polarity (90%, 70%, 50%, and 10% aqueous methanol, then methanol and acetone). Only the fractions eluted with 50% aqueous methanol (F-2) showed fluorescence emission (Figure 2). F-1 absorbed UV in a complex manner, which is not surprising since it contains diverse metabolites. The emission was rather simple and maximal at 482 nm. The complexity of the absorption is reduced in F-2; the absorption at 262, 324, and 368 nm was clearly dominant, while the emission wavelength remained unchanged from F-1.

The photoactive fraction F-2 was separated by high-performance liquid chromatographic (HPLC) to provide 3 aaptamine derivatives—aaptamine, isoaaptamine, and 9-O-demthylaaptamine (1-3, Figure 3). The identity of the isolated derivatives was confirmed by comparison of their ¹H and ¹³C nuclear magnetic resonance (NMR) spectra with those previously reported (Supplemental Figures S3 and S4),^10,11 together with their molecular formula derived from the high-resolution mass spectrometry (HRMS) data (Supplemental Table S1). Each compound was measured for its fluorescence activity in DMSO at a concentration of 10 μM (Table 1). Aaptamine (1), isolated as the most major metabolite, showed strong fluorescent activity with a maximal absorption wavelength of 260 nm and an emission wavelength of 485 nm, suggesting that 1 is responsible for the fluorescent activity of F-2 (Figures 4 and 5). The quantum yield for 1 was 31% at a concentration of 10 μM and the Stoke's shift was 225. In contrast to aaptamine, 2 and 3 showed only marginal fluorescence activity with quantum yields too low to be measured.

Figure 3.

Structures of isolated aaptamine derivatives 1-3.

Figure 4.

Photochemical property measurement results for aaptamine (1). (A) Absorbance and (B) fluorescence spectra at a concentration of 10 μΜ in dimethyl sulfoxide (DMSO) and (C) a plot of absorbance versus concentration for the determination of the molar extinction coefficient in DMSO.

Figure 5.

Fluorescence image of isolated aaptamine derivatives (1-3) upon irradiation with UV light at 365 nm. (A) Solutions in DMSO (10 μΜ); (B) thin-layer chromatography on ODS plates eluted with 0.3% trifluoroacetic acid in 60% aqueous methanol.

Table 1.

Photochemical Properties for the Isolated Aaptamine Derivatives 1-3.^a

Compound	λ_abs (max, nm)	λ_ex (max, nm)	λ_em (max, nm)	ɛ	Ф_F (%)	Stoke's shift
1	260	366	485	730	31	225
2	255	372	510	n.d^a	n.d^b	255
3	240	477	543	n.d^a	n.d^b	303

Excitation maximum, emission maximum, and absolute quantum yield at 10 μM in dimethyl sulfoxide (DMSO). The reliability of the quantum yield measured by integrating the sphere detector was confirmed by quantum yields obtained for known fluorescent dyes [fluorescein, 92% (reported: 89%); rhodamine 6G, 92% (reported: 92%)].¹²

Not determined due to weak fluorescence emission.

The TRPA1 activity of isolated derivatives was contrasted with fluorescence activity; aaptamine (1) exhibited only marginal inhibition of TRPA1 even at a concentration of 40 μΜ, while isoaaptamine (2) and O-demethylaaptamine (3) provided IC₅₀ values of 3.1 and 3.9 μM, respectively (Table 2 and Supplemental Figure S5). Two derivatives (2, 3) with potent TRPA1 inhibitory activity inhibited the growth of HEK293 cells with IC₅₀ values of 20.5 and 21.6 μM, respectively, while aaptamine showed no growth inhibition at 40 μM (Supplemental Figure S6).

Table 2.

Inhibitory Activity Against Transient Receptor Potential Ankyrin 1 (TRPA1) and Cytotoxicity Against HEK293 Cells of the Isolated Aaptamine Derivatives (1-3).

Compound	IC₅₀ (μΜ)^a
Compound	TRPA1	HEK293
1	> 40.0	>40.0
2	3.1	20.5
3	3.9	21.6
A-967079^b	0.3	8.7
Staurosporine	n.m.^c	1.3

Values obtained from duplicate experiments.

A known TRPA1 antagonist used as a positive control.

Not measured.

It is clear that the 9-methoxy group is crucial for the fluorescent activity, while the hydroxyl group at the same position plays an important role in TRPA1 antagonistic activity. Regarding the fluorescent activity, the effect of these functional groups on the fluorescent activity can be considered to be electronic rather than steric,⁷ since all 3 aaptamine derivatives bear a rigid natphthyridine scaffold.¹³ However, it is difficult to derive a conclusive explanation from the results obtained with only 3 examples.

Aaptamine analogs are known to exhibit biological activities^14,15 including antioxidant activity,^16,17 agonistic or antagonistic activity on various proteins,^18–20 and growth inhibition of pathogenic microbes^21,22 or cancer cells.^{13,21,23–25} The most studied therapeutic area for these compounds is related to anticancer; there are reports describing their effect on the proliferation and progression of cancer cells in detail at the molecular or genetic level.^{23,24,26–29} It is noteworthy that the dramatic difference in activity between aaptamine and isoaaptamine was previously observed in the measurement of their growth inhibitory activity against cancer cells. For the first, Fedoreev et al¹¹ reported that isoaaptamine and demthylaaptamine suppressed Ehrlich's tumor cells by 95% at a concentration of 25 μg/mL, while no effect of aaptamine was observed even at a concentration of 50 μg/ mL. The same tendency was observed in the research of other groups; when compared to isoaaptamine or demethylaaptamine, aaptamine showed lower efficacy against most of the cancer cell lines tested so far, and the difference in IC₅₀ values was several orders of magnitude.^23,25,28,30 In the recent report, the cytotoxicity of aaptamine and isoaaptamine measured against 3 breast cancer cell lines showed a tendency similar to that reported here against HEK cells; the IC₅₀ values obtained for isoaaptamine ranged from 30 to 50 μM, whereas aaptamine was not active even at a concentration of 88 μM.³¹

Aaptamine and its derivative have been shown to have distinct activities on proteins located intra- or extracellularly. The first reported activity of aaptamine against a specific protein was α-adrenoreceptor antagonistic activity.³² It has been reported that aaptamine inhibits norepinephrine-induced contraction of both rabbit aorta and renal arteries at a concentration of 10 μM. In both tissues, no inhibitory effect of 9-O-demethylaaptamine or 9-O-demethyloxyaaptamine was observed even at concentrations of 30 μM. The comprehensive screening for the activity of aaptamine on various G-protein coupled receptors (GPCRs) was recently reported.¹⁹ Based on the primary screening on 168 GPCRs, aaptamine was found to be a potent antagonist of β-adrenoreceptor and dopamine receptor D4 with IC₅₀ values being 0.2 and 6.9 μM, respectively, while an agonist of chemokine receptor 7 (EC₅₀ 6.2 μM). Another study showed that aaptamine and demthylaaptamine have agonistic activity on δ and μ opioid receptors.¹⁸ The TRPA1 inhibitory activity of the aaptamine derivatives reported herein can also be added to the aforementioned activities and utilized for a deeper understanding of the functions and interactions of these proteins. In a recent report research, the fluorescent apptamine derivative was used to study its mode of action even before its photochemical properties were characterized. As 3-(phenethylamino)-demethyl-(oxy)aaptamine (PDOA) had previously been found to exhibit a potent growth inhibitory activity against Mycobacterium bovis BCG,³³ several derivatives were synthesized and one of the synthesized derivatives with the trifluoromethyl diazirine group showed inhibition comparable to PDOA.³⁴ When this compound was added to the lysate of M bovis BCG, 4 proteins of different sizes were observed to be fluorescently active, presumably due to the covalent binding of the synthesized aaptamine derivative, although these proteins could not be identified.

Conclusion

This research demonstrates that natural fluorophores can be discovered in living organisms through photoactivity-guided isolation; aaptamine (1), isolated as a result, can provide the additional 1,6-naphthyridine fluorophore. Isoaaptamine (2) and 9-O-demethylaaptamine (3), isolated together with aaptamine, exhibited potent inhibitory activity against TRPA1, providing a chemical scaffold that could be used to study the function of proteins and the dynamics of complex cellular mechanisms and signal transduction. In-depth studies should be followed for the synthesis and evaluation of diverse derivatives to establish the structure–activity relationship and to reveal the ligand–protein interactions.

Experimental

General Experimental Procedures

The NMR spectra were measured by an ASCEND 600 spectrometer (Bruker BioSpin GmbH) at 298 K, and chemical shifts are reported in δ values (ppm) from tetramethylsilane with the solvent resonances as the internal references (DMSO-d₆: δ_H 2.50/δ_C 39.5). High-resolution mass spectrometry (HR-ESIMS) data were obtained on an X500R (SCIEX, Framingham, MA). High-performance liquid column chromatography was performed on a Waters Breeze ^TM HPLC system (Waters Corporation) equipped with a 1525 binary pump and a 2998 photodiode array detector, using a YMC-Pack Pro C-18 column (YMC Co., Ltd). Thin-layer chromatography was performed using Merck RP-18 F₂₅₄ plates and spots were detected by UV irradiation. Photophysical properties were measured using a Shimadzu UV1650P (Shimadzu Corporation), a GENESIS 180 (Thermo Fisher Scientific, Inc.), and a fluorescence spectrometer Scinco FS-2 equipped with a LabSphere integrating sphere for the measurement of quantum yield. AITC (TRPA1 agonist), A-967079 (TRPA1 antagonist), staurosporine, and DMSO were purchased from Sigma-Aldrich. Organic solvents used for fractionation and HPLC were purchased from Duksan and distilled prior to use. Deutrated DMSO used in NMR spectroscopy was purchased from Cambridge Isotope Laboratories. Throughout the study, we used ultrapure water, obtained using the Mili-Q system (Millipore Corporation).

Sponge Material

The sponge specimens were collected off the shore of Nha Trang Bay, Vietnam, and kept frozen until extracted. The general morphological features are consistent with those of Aaptos suberitoides, which was confirmed later by the sequence of the cytochrome c oxidase subunit 1 mitochondrial gene (CO1). The voucher specimens (registry no. 191VN-241) are deposited at the Korea Institute of Ocean Science and Technology.

Extraction and Isolation

The freshly collected sponges were immediately frozen and stored at −25 ^◦C until use. The wet specimens were cut into pieces with sizes of ∼ 1.0 to 1.2 cm, naturally dried, and extracted with methanol and dichloromethane at room temperature. The combined extracts were partitioned between n-butanol and water, and the n-butanol fraction was partitioned again with 15% aqueous methanol and n-hexane. The 15% aqueous methanol fraction (F-1) was subjected to the reverse-phase column chromatography (YMC Gel ODS-A, 60 Å, 230 mesh) with a stepped gradient solvent system of 90%, 70%, 50%, and 10% aqueous methanol, 100% methanol, acetone, and ethyl acetate. The fraction obtained by the elution with 50% aqueous methanol (F-2) was separated by HPLC on an ODS column (YMC-Pack Pro C-18, 250 × 10 mm) using 80% aqueous acetonitrile containing 0.5% trifluoroacetic acid to afford compounds 1−3. The identity of the isolated derivatives was confirmed by comparison of their ¹H and ¹³C NMR spectra (Supplemental Figures S3 and S4) with those previously reported, together with their molecular formula derived from HRMS data (Supplemental Table S1).

Cytotoxicity Measurement

CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay (Promega) was applied for the MTS assay. After HEK-293 cells were plated in 384-well clear plates at densities of 2000 cells/well and incubated for 24 h at 37 °C in 5% (v/v) CO₂, they were treated with the compounds at a variety of concentrations (1.25, 2.5, 5.0, 10, 20, 40, and 80 μΜ) for 48 h in total. DMSO and staurosporine were used as the vehicle and positive control, respectively. The percentage of viable cells was determined from the concentration of formazan converted from tetrazolium measured by the absorbance at 490 nm using an EnVision Xcite Multilabel Reader (PerkinElmer).

TRPA1 Inhibitory Activity Measurement

The inhibitory activity of the extract, fractions, and isolated compounds against TRPA1 protein was measured by a fluorescence-based calcium immobilization assay. The HEK-293 cells overexpressing hTRPA1 were plated in a black clear-bottomed plate (1.5 × 10⁴ cells/well). Each well was loaded with FLIPR Calcium 6 assay reagent (20 μL, Molecular Devices, LLC) and incubated for 2 hours. The cells were then incubated with each of the test samples or the reference compound A-967079 for 5 minutes, and then treated with 5 μM AITC. Fluorescence measurements were performed on a Flexstation III (Molecular Devices, LLC) with excitation/emission wavelengths of 495/516 nm. The % inhibition was calculated by setting the fluorescence intensity of the cells treated with AITC alone to 0% and that of untreated cells to 100%.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231217942 - Supplemental material for Fluorescence Activity-Guided Isolation of Aaptamine Derivatives From the Marine Sponge Aaptos suberitoides and Their Inhibitory Activity Against Transient Receptor Potential Ankyrin 1

Supplemental material, sj-docx-1-npx-10.1177_1934578X231217942 for Fluorescence Activity-Guided Isolation of Aaptamine Derivatives From the Marine Sponge Aaptos suberitoides and Their Inhibitory Activity Against Transient Receptor Potential Ankyrin 1 by Suhyun Kim, Dan-Bi Sung, Jung Mi Hyun, Myung Jin Song, Kwiwan Jeong, Jong Seok Lee and Yeon-Ju Lee in Natural Product Communications

Footnotes

Acknowledgments

We would like to thank Dr Young-a Kim (Hannam University, Republic of Korea) for the morphological identification of the sponge specimens.

ORCID iDs

Yeon-Ju Lee

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the Korea Institute of Ocean Science and Technology (grant numbers PEA0121 and PEA0125).

Ethical Approval

Ethical approval is not applicable to this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

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