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. 2025 Oct 22;88(11):2635–2644. doi: 10.1021/acs.jnatprod.5c00978

Discovery of Nav1.7 Inhibitors through the Screening of Marine Natural Product Extracts

Adetola H Adewole †, Bhuwan Khatri Chhetri ‡, Ghada M Abdelwahab †, Riya Bhanushali †, Anne Marie Sweeney-Jones ‡, Madison Greene †, Jaehoon Shim §, Carter K Asef ‡, Patric Vaelli ∥, Lee Barrett §, Facundo M Fernández ‡,#, Cassandra L Quave ⊥, Julia Kubanek †,‡,#,*
PMCID: PMC12670511  PMID: 41124213

Abstract

Automated high-throughput screening of a prefractionated extract library of marine macroorganisms identified 239 hits (hit rate 2.5%), including a marine algal extract that blocked the Nav1.7 channel in a fluorescent-based flux assay. Bioactivity-guided chemical investigation led to the isolation of two glycoglycerolipids (1 and 2). In preliminary screening, 1 showed stronger Nav1.7 inhibition, while secondary screening using patch-clamp electrophysiology, which measures ion movement across membranes, revealed 2 as more potent. This study identified some ion channel modulators from diverse taxonomic origins, including red algae, sponges, and corals, many of which are underexplored and represent promising leads for future drug discovery.

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Voltage-gated ion channels are transmembrane proteins that play a key role in generating and propagating electrical signals. Their activity is controlled by changes in membrane potentials, with channel opening allowing the movement of ions. − Voltage-gated sodium channels (Nav), a class of transmembrane ion channels, mostly found in the peripheral nervous system, are promising targets for analgesic activity. Their exploitation presents a potential alternative to opioids, which act via interaction with G-protein coupled receptors and are subject to addiction and overdose risks.

Natural products have played a pivotal role in drug discovery, and previous studies have screened natural product extracts for small molecules targeting voltage-gated ion channels. Nav1.7 and Nav1.8 are voltage-gated sodium channel isoforms with distinct contributions to pain signaling. Nav1.7 acts as a threshold channel that initiates action potential, whereas Nav1.8 mediates repetitive firing and thereby sustains their propagation. − Cai et al. reported that two clerodane diterpenoids, (−)-hardwickiic acid and hautriwaic acid, isolated from the aerial parts of Salvia wagneriana, a flowery shrub, and Croton setigerus, an ornamental plant, respectively, blocked sodium channels without affecting calcium or potassium channels. Naringenin, a flavonoid abundant in citrus fruits, selectively inhibited Nav1.8, and Khanna and colleagues proposed its potential use in pain management. Two meroterpenoids, acetoxydehydroaustin A and austin, isolated from the fungus Verticillium albo-atrum, activated sodium currents in the central neurons of the cotton bollworm Helicoverpa armigera. The acetylated derivative of an ent-kaurane diterpenoid fungal metabolite, 1-O-acetylgeopyxin A, was also found to inhibit Nav1.7.

Beyond sodium channel activity, conotoxins, a group of disulfide-rich, neuropeptides isolated from venomous snails of the genus Conus, have been shown to selectively block the voltage-gated calcium channel Cav2.2, making them attractive leads in drug development. A synthetic derivative of ω-conotoxin, Ziconotide, was approved in 2004 by the FDA as a nonopioid analgesic. Leconotide, another ω-conotoxin, exhibited potency and high selectivity but failed to advance beyond Clinical Phase IIa trials due to its low safety profile at higher doses. , Physalin F, a steroidal derivative from Physalis acutifolia, a flowering plant in the Solanaceae family native to the Southwestern United States, exhibited antinociceptive activity in neuropathic pain models by selectively blocking voltage-gated calcium channels Cav2.2 and Cav2.3, without affecting sodium or potassium channels. Capnellene, a tricyclic sesquiterpene isolated from the soft coral Capnella imbricata, was found to induce pain-relieving effects in neuropathic mice models. In that work, Wen and co-workers attributed the analgesic effect to the substantial inhibition of COX-2 protein expression, a protein that can excite the nerves that transmit pain signals. In their review, Khanna and colleagues compiled approximately 40 plants and fungi-derived natural products that modulate voltage-gated sodium and calcium channels. They highlighted the structural diversity of these compounds, which include alkaloids, terpenes/terpenoids, phenolics, and flavonoids (including their glycosides). The authors noted that while certain natural products act selectively on sodium channels and others on calcium channels, a subset exhibits activity on both channels.

Natural products modulate ion channels through diverse mechanisms. Conotoxins from cone snail venoms, saxitoxins from shellfish, and tetrodotoxin from pufferfish act as antagonists by blocking voltage-gated channels. − In contrast, batrachotoxins from poison dart frogs and the diterpenoid alkaloid aconitine function as agonists by keeping sodium channels in an open state. − Furthermore, veratridine, a naturally occurring neurotoxin, was proposed to act as an allosteric modulator, altering channel activity without binding to the primary active site.

The marine environment’s rich biodiversity represents an untapped resource for novel chemical scaffolds, with ongoing studies aimed at discovering natural products with potent pharmacological properties. While previous investigations into sodium channel modulators have examined both terrestrial and marine sources, our study focused on marine extracts with the aim of identifying novel sodium channel inhibitors, thereby expanding the chemical space.

In our pursuit of biologically active secondary metabolites, selected natural product mixtures from our International Cooperative Biodiversity Group (ICBG) library of over 9000 unique marine algal and invertebrate extract fractions collected from Fiji and the Solomon Islands were screened against a voltage-gated sodium channel assay as a potential pain target. This broad screening approach was employed to explore their potential activity as sodium channel inhibitors. Our goal was to identify hits that could establish how prevalent sodium channel modulators are among marine natural products, serving as a starting point for further investigation. The tropical red alga Halymenia sp., an understudied species in the Halymeniaceae family, was prioritized among hits for bioactivity-guided fractionation to discover Nav1.7 blockers, leading to the isolation of two bioactive glycoglycerolipids. This selection was further motivated by our field observations, which revealed relatively intact tissues despite the genus being recognized as a food source, suggesting the presence of deterrent secondary metabolites, as well as by the limited knowledge of its chemistry.

Results and Discussion

As part of our ongoing efforts to identify and isolate biologically active and/or novel natural products, 9360 midpolar prefractionated extracts from 3238 marine organisms in the Georgia Tech ICBG library were prioritized based on taxonomic diversity and subjected to high-throughput screening focused on ion channel modulation. In the preliminary screen, extract fractions were evaluated using a fluorescence-based thallium (Tl+) flux assay, targeting the sodium channel Nav1.7, expressed in a stable cell line. Extract fractions exhibiting significant Nav1.7 blocking activity and a z-score of −2 or lower were classified as hits (Figure ). Using these criteria, a hit rate of 2.5% was observed.

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Preliminary Nav1.7 screening of 9360 marine extract fractions, expressed as z-scores, which quantify the deviation of each test sample’s fluorescence from the mean fluorescence of the control in the thallium flux assay. A cutoff of z ≤ −2 was applied to designate hits, shown as red dots, while turquoise dots represent fractions with negligible or no activity. The distribution guided the prioritization of promising leads for further investigation.

Some of the most active hits were recorded from midpolar fractions obtained from organisms in the phylum Cnidaria, particularly those belonging to the genera Sinularia, Sarcophyton, and Palythoa (Figure S1, Table S1). Cnidarian venoms, which are often toxic to humans, serve ecological roles in predation and offense and exhibit cytotoxic, hemolytic, and other biological activities with potential therapeutic applications. − Palythoa spp., zoanthid corals, produce palytoxin, a highly potent natural toxin. , Although human cases are rare, palytoxin poisoning can occur following the consumption of contaminated seafood. Other exposure routes include direct contact with the coral or inhalation of dust, vapor, or aerosols in aquarium settings or marine environments. , Lacano-Pérez et al. reported that the venom from Palythoa caribaeorum inhibits voltage-gated sodium (Nav1.7) and calcium (Cav2.2) channels, suggesting the venom’s potential as a modulator of these ion channels. Beyond the genus Palythoa, other cnidarians including Sinularia spp. and Sarcophyton spp. are known to be a rich source of secondary metabolites, particularly bioactive terpenoids. , More than 200 novel compounds have been isolated from various Sinularia species, with cembrane terpenoids being among the most commonly reported structural classes. − The broad pharmacological potential of Sinularia extracts and isolated compounds is well documented in reviews. ,,

Fractions belonging to Cribrochalina sp. and some other sponges (phylum Porifera) were also active in our preliminary screen (Figure S1). In a previous study, 23 linear and branched monoacetylene lipids were isolated from Caribbean Cribrochalina vasculum, some of which were selectively cytotoxic. Two acetylenic alcohols isolated from Cribrochalina vasculum inhibited the phosphorylation of IGF-1Rβ and reduced its target signaling molecules IRS-1 and PDK1, indicating their potential in antitumor treatment. , Other secondary metabolites reported from Cribrochalina spp. include alkaloids, isoquinolines, phosphorylated sterol sulfates, cyclic and linear peptides, and additional acetylenic alcohols. − Gibsmithia hawaiiensis, a gelatinous red alga, was also among the most active hits. The chemistry and biological activity of this species and its genus remain unexplored, with no prior published reports.

Among the hits in the current study, Halymenia sp., an understudied red alga, was selected for further investigation. Over 150 species of the Halymenia genus have been described with wide distribution across the Indian and Pacific Oceans. Previous reports have shown that Halymenia sp. releases allelopathic compounds that prevent biofilm formation and interfere with bacterial quorum sensing. , Although a report considered Halymenia dilatata as a food source for certain herbivorous marine organisms including sea urchins and some fish species, our field observations revealed relatively intact tissues with little sign of fouling or scarring by herbivores, suggesting it could be chemically defended. In previous studies, the hexane-soluble fraction of Halymenia durvillei exhibited moderate cytotoxicity against MDA-MB-231 breast cancer cells., while crude methanol extract and fractions of Halymenia palmata demonstrated moderate to weak mosquito larvicidal activity. Although the active constituents were not identified in either study, GC-MS profiling tentatively identified long-chain fatty acids, their esters, and other hydrocarbons in both H. durvillei and H. palmata. Sulfated polysaccharides from Halymenia floresii inhibited herpes simplex virus type 1 in vitro, with an EC50 of 3.3 μg/mL. In addition, sulfated galactans isolated from Halymenia dilatata showed antibacterial activity against pathogens infecting tilapia.

Following its identification as an active hit in the thallium flux assay (Figure A), the dichloromethane-soluble fraction of Halymenia sp. was prioritized for chromatographic separation to isolate its bioactive constituents. The extract fraction was observed to be active against Nav1.7 and could be a source of potential analgesic leads.

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(A) Dose–response curve for two replicates of the dichloromethane-soluble fraction derived from extracts of Halymenia sp., tested for inhibition of the Nav1.7 channel in the thallium flux assay. Fluorescence was measured to evaluate the electrical conductivity of Na+ ions across the channel, with a decrease in normalized sodium flux indicating greater inhibition. The two replicates originated from the same algal collection but were extracted and fractionated separately, highlighting the consistency of bioactivity within the extract, with an average IC50 value of 31.7 μg/mL. (B) Primary screening results of the dichloromethane-soluble subfractions of Halymenia sp., presented as a heatmap. Darker colors indicate greater inhibition of Nav1.7 channel. F2.2.1 to 10 means that combined fractions 1 through 10 were screened for biological activity. Fractions 2.2 and 2.7.14 were measured in μg/mL, whereas others were reported as μg/mL equivalents.

Bean and co-workers reported that inhibition of Nav1.7 channel leads to a decrease in the electrical excitability of primary sensory neurons thereby producing analgesia. Our study employed normalized fluorescence to evaluate the electrical conductivity of sodium channels, with a decrease in fluorescence signifying enhanced inhibition. The assay results revealed a concentration-dependent inhibition of the Nav1.7 channel by selected marine extract fractions (Figure ). The tarantula venom peptide, ProTx-II, known for selectively inhibiting Nav1.7 was used in a 125I-ProTx-II binding assay previously described by Schmalhofer et al., as a validation technique (data not shown).

The dichloromethane-soluble fraction of Halymenia sp. was subjected to preparative thin-layer chromatography (prep TLC), yielding subfractions F2.1 to F2.8. F2.2 was further separated by reversed-phase high-performance liquid chromatography (HPLC) to generate F2.2.1 to F2.2.60, while HPLC fractionation of F2.7 produced subfractions F2.7.1 to F2.7.40. The subfractions were combined based on chromatographic profiles and screened in the thallium flux assay (Figure B). Among these, subfractions F2.7.14 and F2.2.25 to F2.2.30 exhibited the highest bioactivity, resulting in the isolation of two bioactive glycoglycerolipids, 1-palmitoyl-2-oleoyl-3-(β-D-galactosyl)-glycerol (1) and sulfoquinovosyldiacylglycerin (2).

Compound 1 was described in a previous study by our group as a glycosylated glycerolipid, with molecular formula C43H80O10 (m/z 774.6086 [M+NH4]+). Most aspects of its molecular structure were elucidated by NMR spectroscopy, mass spectrometry (MS), and microcrystal electron diffraction (MicroED). At that time, the position of the isolated olefin within the oleoyl chain could not be determined, as three-bond heteronuclear multiple bond correlation (HMBC) signals were only observed for adjacent carbons, with no signals detected for the methyl at C-18’’ or the carbonyl at C-1’’, ruling out its position within three bond lengths of the terminal carbons. Although crystals suitable for MicroED analysis were obtained, disorder within the crystal lattice, likely due to the flexibility and conformation of the lipid chain, prevented precise identification of the positioning of the double bond in that portion of the molecule. In the current study, we turned to a recent innovation in MS to pinpoint the location of the 1,2-disubstituted olefin. Triboelectric nanogenerators (TENG), which convert mechanical energy to electric current, have been used as an effective power supply (high voltage and low current) for nanoelectrospray ionization (nanoESI). TENG-driven nanoESI ion sources produce pulsed corona discharges that trigger gas phase reactions which aid in structure elucidation. , With the aid of TENG-MS, the olefinic site was unambiguously localized in the negative ionization mode using the method described by Fernández and co-workers. TENG-MS yielded diagnostic fragments at m/z 155.1076 and 171.1024, corresponding to the ethylene and aldehyde fragments from C-9’’/C-10’’ bond cleavage, affording identification of the site of unsaturation on the MS3 spectrum (Figures A and S2). An oxidized species of the C18:1 fatty acid fragment was observed at m/z 297.2434 in the MS2 fragment ion spectrum (Figure S3). Adduct ions corresponding to [M+Ac–H]− and [MO+Ac–H]− were observed in the MS1 spectrum (Figure S4). Compound 1 was thus established as 1-palmitoyl-2-oleoyl-3-(β-D-galactosyl)-glycerol. Its first isolation was from the roots of Arisaema amurense Maxim., Araceae and later from other vascular plants, including Lycium barbarum L., Solanceae and Aralia elata (Miq.) Seem., Araliaceae, as well as from the brown alga Sargassum horneri (Turner) C. Agardh, Sargassaceae. − Even though glycoglycerolipids are associated with biological activities such as antitumor, , antiviral , and anti-inflammatory, , Ma et al. reported that 1 failed to inhibit triglyceride accumulation in 3T3-L1 adipocytes while Jung et al. observed a weak cytotoxicity against murine leukemia (P388) and human colon adenocarcinoma (DLD-1) cell lines.

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(A) Proposed fragmentation pattern for 1-palmitoyl-2-oleoyl-3-(β-D-galactosyl)-glycerol (1) via TENG-MSn, revealing the position of the fatty acid olefin at C-9. An epoxide ring is formed during TENG ionization, enabling subsequent fragmentation at the double bond position. (B) Structure of sulfoquinovosyldiacylglycerin (2).

The molecular formula of 2 was determined to be C41H78O12S by accurate mass electrospray ionization MS (m/z 812.5558 [M+NH4]+). The loss of a palmitoyl group resulted in an MS fragment ion being observed at m/z 539.2889. A mass fragment ion depicting the aglycone moiety (m/z 551.5038) was observed and a further loss yielded a fragment ion at m/z 313.2736, thereby confirming the presence of the second saturated C-16 acyl chain.

For 2, a large 1H NMR signal at δH 1.23 ppm was indicative of overlapping saturated methylene groups, suggesting the presence of long-chain fatty acyl chains. The chemical shifts and coupling constants observed for the saccharide were consistent with α-d-glucose. The sulfonate position was assigned based on the observation of the 1H NMR signal for C-6’’’ (δC 54.7 ppm) further upfield. HMBC correlations confirmed the position of attachments of both fatty acid ester chains and sugar to the glycerol. Compound 2 was established as sulfoquinovosyldiacylglycerin and the observed spectroscopic data was consistent with literature (Figure B). Glycoglycerolipid 2 has been previously isolated in its sulfonic acid form and sodium salt from brown and green algae, Dictyochloris fragrans and Caulerpa racemosa, , respectively, and from bacteria in the Rhizobiaceae family, lichen, and vascular plants. ,,, It selectively inhibited P-selectin’s adhesion to its ligand with an IC50 of 5 μM, induced apoptosis in lymphoblastic leukemia MOLT-4 cell lines in a dose-dependent manner, showed anthelmintic activity against Raillietina sp. and moderate antiviral activity against HSV-1 and HSV-2 clinical strains. ,,, Prior to the current study, secondary metabolites isolated from red algae of the Halymenia genus have been limited to a few sterols, N-acylsphingosines, and sulfated polysaccharides. ,,, This study is the first to report the isolation of glycoglycerolipids from Halymenia species.

Pure 1 and 2 were subjected to the thallium flux assay, revealing 1, with an IC50 value of 6.9 μM, as a moderate Nav1.7 inhibitor (Figure A). Our primary screening measured membrane potential-dependent fluorescence changes caused by ion flux, whereas patch-clamp electrophysiology, considered the gold standard for assessing ion channel activity, directly measures ion currents. Pure 1 and 2 were thus further evaluated using automated patch-clamp electrophysiology on human Nav1.7 (hNav1.7) channel expressed in HEK cells as a secondary screening. Inhibition was assessed at four concentrations by measuring Na+ currents before and after compound application, compared with tetrodotoxin (TTX) as positive control. State-dependent block was evaluated using Qube patch-clamp electrophysiology by measuring inhibition at membrane potentials of −100 mV (tonic block) and −70 mV (inactivated state block). Surprisingly, given its poor activity in the thallium flux assay, 2 demonstrated greater modulation of membrane potentials than 1, as indicated by a reduction in Na+ current across tested concentrations in both states (Figures B, S11–S14). The dissociation constants (Kd), which measure the affinity of a drug for its target receptor, were 49.9 μM for 1 and 25.2 μM for 2 in the inactivated state. Although 2 exhibited a slightly lower Kd, both compounds demonstrated state-dependent inhibition. Notably, their potency increased against the inactivated state of Nav1.7, showing a 5-fold increase compared to the resting state. Additionally, 2 displayed clear dose-dependent activity. This binding behavior aligns with the mechanism observed in local anesthetics such as lidocaine. ,

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(A) Dose–response curves of 1 and 2 against Nav1.7 channel using the thallium flux assay. The y-axis represents relative fluorescence as a measure of ion flux. In this assay, 1 exhibited greater potency compared to 2. (B) Dose–response curves of 1 and 2 against human Nav1.7 (hNav1.7) channels using the Qube automated patch-clamp assay, with greater inhibition indicated by a decrease in current (I). I max is the control current in 0.1% DMSO before compound addition. The dissociation constant (Kd) was determined for each compound in the tonic block (−100 mV) and inactivated (−70 mV) states using a modified Hill’s equation.

Some functional groups of 1 and 2 have been previously found pharmacologically relevant in studies related to pain management. For instance, transient receptor potential vanilloid 1 (TPRV1), a nonselective cation channel associated with pain, itch, and inflammation, was inhibited by oleic acid, a naturally occurring omega-9 unsaturated fatty acid, resulting in reduced pain and itch in mice. Polysorbate 80, a nonionic emulsifier derived from oleic acid and polyethoxylated sorbitan, was also shown by Kim and Choi in their patch-clamp study to inhibit action potential generation in neurons and block Nav1.7 in a concentration-dependent manner. Notably, 1 contains an oleoyl chain, which may contribute to the activity observed in the preliminary screening.

When screening a library of known synthetic selective Nav1.7 inhibitors, aryl sulfonamides, McKerrall et al. reported that a one-unit increase in lipophilicity led to a 75-fold increase in Nav1.7 inhibition. Similarly, some lipid-soluble neurotoxins, such as brevetoxins, ciguatoxins, veratridine, and aconitine, exert their primary pharmacological effect by targeting voltage-gated sodium channels through agonistic, partial agonist, and allosteric mechanisms. , Although glycoglycerolipids are structurally distinct from these modulators, their amphipathic nature may contribute to voltage-gated sodium channel modulation.

In conclusion, our screening of a large marine extract library led to the identification of extract fractions from diverse taxa, including red algae, cnidarians, and sponges, that inhibit the voltage-gated sodium channel Nav1.7. This highlights the broad phylogenetic range of marine organisms as valuable sources of novel sodium channel modulators. Isolated natural products from one of the active hits showed preliminary pharmacological activity that presents an opportunity for further exploration. These findings underscore the value of marine biodiversity in the search for novel therapeutics and support continued exploration of marine-derived compounds in drug discovery.

Experimental Section

General Experimental Procedures

Optical rotatory dispersion measurements were performed on a Jasco DIP-360 digital polarimeter using methanol as the solvent. NMR spectra were acquired in DMSO-d 6 on an 18.8 T Bruker Avance IIIHD spectrometer with a 3 mm triple resonance broadband cryoprobe. Chemical shifts were reported in parts per million relative to the solvent residual peaks δH 2.50 and δC 39.52. NMR spectroscopic data were processed and analyzed using MestReNova 11.0.4. High-resolution mass spectrometry (MS) and TENG-MS were performed using a ThermoFisher Orbitrap ID-X instrument. Preparative thin layer chromatography (TLC) separations were performed using Silicycle (20 × 20 cm, 200 μm thickness) TLC plates. High-performance liquid chromatography (HPLC) separations were conducted on a Waters 1525 binary pump equipped with a Waters 2996 photodiode array detector and an Altech 800 evaporative light scattering detector.

Specimen Collection and Identification

Marine organisms were collected as part of an NIH ICBG program in Fiji and the Solomon Islands over a 12-year period (2006–2018). Each specimen was extracted three times using methanol, followed by vacuum liquid chromatography (VLC) fractionation using HP20SS Diaion resin at a dry extract-to-resin ratio of 1:20. The crude extract was adsorbed onto the resin, washed with water to remove salts, and sequentially eluted using 50% aqueous methanol (Fraction A), 80% aqueous methanol (Fraction B), methanol (Fraction C), and acetone (Fraction D), yielding four extract fractions. The midpolar fractions (B and C) were selected to form the extract library for further studies. Each extract fraction was prepared at volumes of 100, 300, and 600 nL, plated into a 384-well plate, and screened using the thallium flux assay.

Halymenia sp. (sample G-0815) was collected from Mango Bay locale, Vitu Levu Island, Fiji Islands (S 18°14.184’, E 177°46.86′) in April 2010 at depths ranging from 5 to 40 m. It had a soft texture with mucus and was pink-red in color. The collection was identified as an uncertain species of Halymenia based on the morphological features and comparison to the previous description. Voucher specimen and morphological samples were preserved in formaldehyde and ethanol, respectively, at the Georgia Institute of Technology and at the University of the South Pacific (USP) in Suva, Fiji. The bulk sample was stored in a −80 °C freezer.

On the day of collection, in the field, 20 g wet weight of Halymenia sp. was extracted with methanol, followed by fractionation using Diaion HP-20SS as stationary phase once extracts were returned to the lab at USP. Elution was carried out sequentially using 50% aqueous methanol, 80% aqueous methanol, methanol and acetone, resulting in four extract fractions. These fractions were screened against a human embryonic kidney (HEK) cell line expressing Nav1.7 to identify bioactive fractions.

Separately and at a later date, additional material from the same collection of Halymenia sp. (428.2 g wet weight) was extracted exhaustively using MeOH, MeOH/CH2Cl2 (1:1) and CH2Cl2, resulting in 17.4 g of combined extract after drying in vacuo. The crude extract was suspended in 10% aqueous methanol and partitioned with hexanes to afford the hexanes-soluble fraction (F1). The aqueous portion was adjusted to 40% aqueous methanol and partitioned with CH2Cl2, resulting in the CH2Cl2-soluble fraction (F2). MeOH was evaporated from the aqueous phase and the residual aqueous portion was partitioned with ethyl acetate (EtOAc), yielding the EtOAc-soluble fraction (F3). The remaining H2O-soluble fraction was designated as F4.

The CH2Cl2-soluble fraction (0.7 g) was subjected to silica gel Prep TLC in multiple batches and eluted with MeOH/EtOAc (1:4), affording 8 fractions (F2.1 – F2.8). Fraction F2.7 (15.4 mg) was subjected to HPLC separation on a normal phase silica column (5 μm, 4.6 × 250 mm) using ethyl acetate to MeOH/EtOAc (3:2) over 20 min (solvent flow rate 1 mL/min). This resulted in 40 fractions (F2.7.1 – F2.7.40) that were collected at 30-s intervals. F2.7.14 contained pure 1 (1.5 mg) which was quantified with quantitative NMR spectroscopy using a capillary filled with benzene-d 6 as an internal standard and calibrated against caffeine. It also afforded crystals suitable for MicroED analysis, a portion of which was used for bioassay. An adjacent HPLC fraction, F2.7.15, also consisted of pure 1 that was used for NMR, HRMS, and TENG-MS analysis. The partial structural elucidation of 1, including its NMR and HRMS data, was documented in our previous study.

Fraction F2.2 was separated on the HPLC using a reversed phase C18 silica column (5 μm, 4.6 × 250 mm) using a solvent gradient of 1:1 MeOH/H2O to 1:9 CH3CN/IPA over 15 min, held for 7 min at 1:9 CH3CN/IPA, followed with 1:9 CH3CN/IPA to 1:1 MeOH/H2O over 3 min and isocratic 1:1 MeOH/H2O for 5 min (solvent flow rate 0.9 mL/min). 60 fractions (F2.2.1 – F2.2.60) were generated with F2.2.25 – F2.2.30 containing pure 2 (1.7 mg).

Sulfoquinovosyldiacylglycerin (2)

Yellow solid; [α]25 D + 23 (c 0.14, MeOH); 1H and 13C data, Table S2; HRMS m/z [M+NH4]+ calcd for C41H78O12S 812.5557, found 812.5558.

TENG-MS Analysis

1-palmitoyl-2-oleoyl-3-(β-D-galactosyl)-glycerol (1) (250 μM) was solubilized in DMSO and diluted into 6:1:1 acetone/H20/MeOH with 200 mM ammonium acetate, a solution which enhances oxidation during TENG ionization. The diluted sample was introduced into the TENG ion source through nanoemitter glass tips pulled in-house using borosilicate thin-wall glass capillaries. Additional details can be found elsewhere.

Thallium Flux Assay

A day before screening, each HEK cell line (in duplicate) is plated in a 384-well plate (Greiner 781090) at a density of 20,000 cells/well and grown for ∼24 h in 37 °C incubator. We used DMEM+10%FBS+P/S with selective antibiotics for subtype specific Nav expressing cell lines. On the day of screening, one compound plate (Greiner 784201) was filled with 30 μL of Live Cell Imaging Solution containing 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 20 mM HEPES pH 7.4 (A59688DJ, Thermo Fisher). Next, 300 nL of small molecules were pin-transferred to each compound plate. Cell plates were taken out of the incubator and media was removed and washed with 20 μL of Buffer twice using Bravo liquid handler (Agilent), and 20 μL of FluxORII Green dye (F20017, Thermo Fisher Scientific) was added to each well. Cell plates were incubated at RT for 30 min in the dark. After incubation, 10 μL of compounds from the compound plate was added to the HEK cells for 15 min at RT in the dark (one compound plate per two cell plates).

Stimulus buffer composed of KHCO3, KCl, CaSO4, MgSO4, Glucose, HEPES, TiSO4 was added to the assay plates and read using the FDSS7000EX (Hamamatsu Photonics) where 10 μL of Stimulus buffer is added to each well at the appropriated depth and speed of 15s/μL, making a final volume of 40 μL. Continuous readings (Ex480:Em540) were taken at .23s intervals in the wells. Raw data is normalized by background signal of each well. The initial slopes were calculated within 10 s time zone 3 s after the addition of stimulus. Z-scores were then calculated based on plate average of experimental wells. A compound is considered a hit if the z-score is lower than −2. Both replicates must meet criteria to be considered a hit.

Human Nav1.7 Cell Line

A stable line of HEK293 cells expressing human Nav1.7 voltage-gated sodium channel alpha subunit (1) were maintained in EMEM media (Corning) containing 10% FBS, 1% penicillin/streptomycin (Sigma), and 700 μg/mL Geneticin (Sigma) under 5% CO2 in a 37 °C incubator.

Cell Preparation for Electrophysiology

Cells were cultured to approximately 70% confluency to maximize cell viability and channel expression. On the day of recording, cells were washed twice with divalent-free phosphate-buffered saline (Corning) and incubated in Detachin enzyme solution (Genlantis) for 3 min at 37 °C. The cell enzyme mixture was diluted 1:10 in serum-free EX-CELL media (Sigma) and centrifuged at 65 G for 2 min. The cell pellet was resuspended in serum-free EX-Cell media (Sigma), and cell density and viability were measured using a Countless 3 cell counter (Invitrogen). Volume was adjusted to a cell density was 1.5 – 2 × 106 cells/mL with cell viability ≥ 98%.

Automated Patch-Clamp Electrophysiology

Voltage clamp recordings of Nav1.7 were measured using a Qube 384 automated patch-clamp system (Sophion Biosciences). All recordings were performed using multihole recording chips that permit simultaneous recording of up to ten cells in the same well. The currents from all cells within a well were summed to form the macroscopic Na+ current, and the leak generated from patch holes without cells was measured using 10 mV hyperpolarizing pulses and subtracted accordingly. The extracellular recording solution contained in mM: 145 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 using NaOH and osmolarity between 300 and 305 mOsm/L. The intracellular solution contained in mM: 140 CsF, 10 NaCl, 1 EGTA, 10 HEPES, and 10 glucose with pH adjusted to 7.2 with CsOH and osmolarity of 320 mOsm/L. All compounds were prepared in the same extracellular solution with the addition of 0.1% Pluronic-F68 (Sigma), a surfactant poloxamer that enhances solubility of lipophilic compounds. All recordings were performed with temperature control at 25 °C.

Data acquisition and operation of the Qube was controlled with Sophion Viewpoint software (v9.0.42). Whole-cell recordings were obtained using the “Sophion Standard” protocol, in which cells were captured on each patch hole with a negative suction pressure of −80 mbar. Once the patch hole resistance increased, indicating presence of a cell, the pressure was switched to −10 mbar for holding and seal formation. After gigaseal formation of ≥ 800 MΩ, whole-cell configuration was achieved by applying two negative pressure pulses from −10 mbar to −250 mbar for two seconds, then to −350 mbar for another two seconds. All cells were held at a membrane potential of −100 mV, and sodium currents were evoked using protocols designed to evaluate state-dependent interactions between the channel and test compounds. Currents were digitized and filtered at 50 kHz with an eighth-order Bessel filter and a corner frequency of 16.66 kHz. After data acquisition, the current and voltage command waveforms were exported from Sophion Analyzer software (v9.0.42) and imported into Igor Pro (version 6.7, WaveMetrics) for analysis.

Data Analysis

Raw current traces were corrected for linear capacitative and leak currents, which were determined using 10 mV hyperpolarizing steps applied from resting potential of −100 mV and both scaled and subtracted accordingly. Maximum inward Na+ currents were measured using 10 ms test pulses to +10 mV. To measure tonic block of resting state channels, cells were held at −100 mV membrane potential prior to the test pulse. Inactivated state-dependent block was evaluated in two ways: first, cells were held at −70 mV for 2.5 s before test pulse; second, cells were held at −60 mV for 2.5 s, then hyperpolarized to −100 mV for 40 ms before the test pulse. The former approach allows measurement of channel block at the approximate half maximal voltage (V 1/2) for Nav1.7, while the latter permits drug exposure to a greater fraction of inactivated channels but requires a hyperpolarization period before the test pulse to allow some recovery from fast inactivation. Channel block was determined by calculating the ratio of inward Na+ current after drug exposure to the current at the end of the control period. N values for each group include wells with currents ≥ 3 nA, and error bars represent standard deviation (SD). Dose–response analysis was used to estimate the dissociation constant (Kd) for each compound using the fit to a Hill-Langmuir equation equation modifed to account for a nonzero baseline, I= IMin+(IMax-IMin)/(1+[Drug]/Kd), where I is the Na+ current following 7 min of drug exposure at a concentration [Drug], IMax is the control current in 0.1% DMSO before drug addition, and IMin is a baseline to account for a small remaining current in saturating drug concentrations. All data analyses were completed in Igor Pro (v6.7).

Supplementary Material

np5c00978_si_001.pdf (5MB, pdf)

Acknowledgments

This study was supported by the US National Institutes of Health, National Center for Complementary and Integrative Health 1R01AT011990 to C.L.Q. and J.K. The previous support of the US National Institutes of Health, International Cooperative Biodiversity Groups U19-TW007401 to J.K. for the generation of the ICBG library is also appreciated. F.M.F. acknowledges support from National Institutes of Health grant 1R01CA218664. The authors thank Clifford J. Woolf and Bruce P. Bean (Harvard Medical School) for the valuable discussions during this study and NIH grant 5R35NS105076 to C. J. Woolf, which supported the screening program. We appreciate the Georgia Tech NMR Center and Systems Mass Spectrometry Centers for providing access to instrumentation and technical expertise. We thank the governments of Fiji and Solomon Islands for their permission to collect samples in their territorial waters. The authors acknowledge biorender.com for the use of an icon in the graphical abstract, created under a license held by the Georgia Institute of Technology.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c00978.

  • Photographs of selected marine organisms with biologically active extracts and their collection sites, NMR and HRMS spectra of isolated compounds, voltage protocol, and primary data from the automated patch-clamp electrophysiology study (PDF)

∇.

A.H.A and B.K.C contributed equally.

The authors declare no competing financial interest.

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