Lycocasine A, a Lycopodium Alkaloid from Lycopodiastrum casuarinoides and Its Acid-Sensing Ion Channel 1a Inhibitory Activity

A novel Lycopodium alkaloid, lycocasine A (1), and seven known Lycopodium alkaloids (2–8), were isolated from Lycopodiastrum casuarinoides. Their structures were determined through NMR, HRESIMS, and X-ray diffraction analysis. Compound 1 features an unprecedented 5/6/6 tricyclic skeleton, highlighted by a 5-aza-tricyclic[6,3,1,02,6]dodecane motif. In bioactivity assays, compound 1 demonstrated weak inhibitory activity against acid-sensing ion channel 1a.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disorder characterized by the immune system's attack on the synovial membrane of joints, leading to inflammation and, in severe cases, permanent damage and disability [1].Epidemiological studies indicate that around 1% of the global population is afflicted with rheumatoid arthritis.The primary treatment strategy focuses on controlling synovitis, though long-term medication use does not cure the disease and often results in serious side effects [2].
Recent studies showed that acid-sensing ion channel 1a (ASIC1a) was a new therapeutic target for rheumatoid arthritis [2,3].The ASIC1a, part of the degenerin/epithelial sodium channel family, is activated under acidic conditions and plays a crucial role in managing physiological and pathological states, including inflammation, acidosis, ischemia, and hypoxia [3].Despite the existence of compounds with notable ASIC1a inhibitory activity, few are derived from plant sources [4], highlighting the significance of exploring plant-based natural products as ASIC1a inhibitors.
Our research team has been engaged in the discovery of structurally novel and biologically active Lycopodium alkaloids.Previously, we reported fourteen new Lycopodium alkaloids from L. casuarinoides and their Ca v 3.1 channel inhibitory activity [24].Further study on the chemical constituents of L. casuarinoides led to the isolation of a new Lycopodium alkaloid, lycocasine A (1) (Figure 1), and seven known Lycopodium alkaloids Our research team has been engaged in the discovery of structurally novel and biologically active Lycopodium alkaloids.Previously, we reported fourteen new Lycopodium alkaloids from L. casuarinoides and their Cav3.1 channel inhibitory activity [24].Further study on the chemical constituents of L. casuarinoides led to the isolation of a new Lycopodium alkaloid, lycocasine A (1) (Figure 1), and seven known Lycopodium alkaloids (2)(3)(4)(5)(6)(7)(8).Lycocasine A (1) exhibits a novel 5/6/6 tricyclic skeleton, characterized by a 5-aza-tricyclic[6,3,1,0 2,6 ]dodecane motif.Owing to the traditional utilization of L. casuarinoides described above, compounds 1-8 were evaluated for ASIC1a inhibitory activity and 1 displayed a weak inhibitory effect.This paper elaborates on the isolation, structural elucidation, possible biosynthetic pathway, and ASIC1a inhibitory activity of these compounds.

Molecular Docking
The interactions of lycocasine A (1) with the ASIC1a were revealed by the molecular docking.Given the lack of mammalian ASIC1a co-crystallized with potent small-molecule inhibitors, a chicken ASIC1 protein [43] (cASIC1 protein, PDB code: 6X9H, a homolog of mammalian ASIC1a protein) was selected as the receptor protein for molecular docking.In Figure 6A,B, the docking result shows one pi-alkyl interaction of OMe carbon atom with TYR341, an amino acid with which JNJ-799760 interacts in the crystal of the cASIC1/JNJ-799760 complex, as reported by Michael Maher's research team [43].Additionally, hydrogen bond interactions with ASP238, GLU239, and ASP346 and an alkyl interaction with LEU349 were also observed.Moreover, the LibDockscore (45.60) and binding energy (−35.62Kcal/mol) in the active site indicates the weak interactions between lycocasine A (1) and cASIC1 protein.These results may elucidate the weak inhibitory effect of lycocasine A (1) on ASIC1a.
various levels of lycocasine A (1) and Amiloride.(B) Dose-response of the inhibitory action of lycocasine A (1) on the peak current of ASIC1a.The data were fitted using a Hill equation and are presented as the mean ± SD (n ≥ 3).(C) Dose-response curve of the inhibitory action of Amiloride on the peak current of ASIC1a.The data were fitted using a Hill equation and are presented as the mean ± SD (n ≥ 3).

Molecular Docking
The interactions of lycocasine A (1) with the ASIC1a were revealed by the molecular docking.Given the lack of mammalian ASIC1a co-crystallized with potent small-molecule inhibitors, a chicken ASIC1 protein [43] (cASIC1 protein, PDB code: 6X9H, a homolog of mammalian ASIC1a protein) was selected as the receptor protein for molecular docking.In Figure 6A,B, the docking result shows one pi-alkyl interaction of OMe carbon atom with TYR341, an amino acid with which JNJ-799760 interacts in the crystal of the cASIC1/JNJ-799760 complex, as reported by Michael Maher's research team [43].Additionally, hydrogen bond interactions with ASP238, GLU239, and ASP346 and an alkyl interaction with LEU349 were also observed.Moreover, the LibDockscore (45.60) and binding energy (−35.62Kcal/mol) in the active site indicates the weak interactions between lycocasine A (1) and cASIC1 protein.These results may elucidate the weak inhibitory effect of lycocasine A (1) on ASIC1a.

Cell Transfection and Electrophysiological Recordings
HEK293T cells, acquired from ATCC, were cultured at 37 • C in a 5% CO 2 atmosphere using Dulbecco's Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with glucose, L-glutamine, pyruvate, 10% fetal bovine serum (FBS, VivaCell, Shanghai VivaCell Biosciences Ltd., Shanghai, China), and 1% penicillin-streptomycin (Pen-Strep, VivaCell, Shanghai VivaCell Biosciences Ltd, Shanghai, China).Cells were plated at a low density in 12-well plates 24 h prior to transfection.For transfection, Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was utilized to introduce 300 ng of ASIC1a cDNA into adherent cells, which were then analyzed for 24-48 h post-transfection.Complete-cell voltage-clamp measurements were obtained at ambient temperature (24 • C), holding the cell membrane voltage at −60 mV.The ASIC1a greatest currents were triggered by a solution pH of 6.0, keeping the holding potential at −60 mV.Borosilicate glass micropipettes, fashioned to achieve a resistance of 2-6 MΩ, were filled with an intracellular recording solution comprising 140 mM KCl, 2 mM MgCl 2 , 5 mM EGTA, 5 mM NaCl, and 10 mM HEPES (adjusted to pH 7.4 with KOH).The extracellular recording solution consisted of 145 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , and 10 mM HEPES (pH 7.4 with NaOH) or 10 mM MES (pH 6.0 with HCl).Amiloride (MedChemExpress, Monmouth Junction, NJ, USA) was used as a positive control.Current signals were amplified using a SUTTER IPA-2 amplifier, with data acquisition and analysis performed using SutterPatch 9.0 software.Data processing was carried out using GraphPad Prism version 8.0.

Molecular Docking
Molecular docking was performed using Discovery Studio 4.0 software.The crystal structure of chicken ASIC1 protein [43] (cASIC1 protein, PDB code: 6X9H, a homolog of mammalian ASIC1a protein) was obtained from the Protein Data Bank.After removing the water molecules, hydrogens and charges were added to the system.The docking site was defined based on the position of the co-crystallized inhibitor.The 3D conformations of compound 1 were optimized by Discovery Studio 4.0 and the top scoring ligand poses were saved.Docking analysis was performed by the LibDock protocol and the docking parameters were set as defaults.The docking results with the highest LibDock score were visualized and are presented in the full text [44].

Conclusions
In summary, we have identified a novel Lycopodium alkaloid, lycocasine A (1), and seven known Lycopodium alkaloids (2-8) from L. casuarinoides.Compound 1 is characterized by an unprecedented 5/6/6 tricyclic skeleton with a 5-aza-tricyclic[6,3,1,0 2,6 ]dodecane moiety.In bioactivity assays, compound 1 exhibited weak inhibition of the ASIC1a.These results not only extend the chemical diversity of Lycopodium alkaloid but also provide a solid basis for further exploration of Lycopodium alkaloids as ASIC1a inhibitors in the treatment of rheumatoid arthritis.

Lycocasine A ( 1 )
was isolated as colorless needle-shaped crystals.Its molecular formula was established as C 18 H 24 N 2 O 4 through HRESIMS m/z 333.1812 ([M + H] + ; calculated for C 18 H 25 N 2 O 4 + , 333.1809), indicating the presence of eight double-bond equivalents (DBEs).The IR spectrum displayed the presence of one OH/NH (3429 cm −1 ) The 1 H− 1 H COSY correlations identified a single spin system as follows: H 2 -10/H-11/H-12/H-7(H-6b)/H-8.The HMBC correlations from N-Me(a) to C-13 and N-Me(b) and from N-Me(b) to C-13 demonstrated their linkage via the N β atom.The HMBC cross-peaks from H-11 to C-13, from H-12 to C-13 and C-4, from H-6a to C-5 and C-4, and from H-6b to C-5, along with the 1 H− 1 H COSY correlations of H-12/H-7/H-6b, constructed the ring B. The monosubstituted ∆ 10(11) double bond was connected to ring B through C-12, as revealed by the 1 H− 1 H COSY cross-peaks of H 2 -10/H-11/H-12.The ring D was established by the HMBC cross-peaks from H 3 -16 to C-15, C-14, and C-8, and from H 2 -14 to C-13 and C-4, together with the 1 H− 1 H COSY cross-peaks of H-12/H-7/H-8.The HMBC correlations from 5-OH (δ H 5.74) to C-6, C-5, and C-4 and from the N α H proton (δ H 8.76) to C-4 and C-5, along with the chemical shift of C-5 (δ C 87.0), fixed 5-OH and N α H at C-5, which further confirmed the presence of a carbinolamine moiety.Subsequently, the HMBC correlations from the N α H proton (δ H 8.76) to C-1 (δ C 166.0), C-3, and C-4 not only indicated the presence of the amide group and ∆ 3( The 1 H− 1 H COSY correlations identified a single spin system as follows: H2-10/H-11/H-12/H-7(H-6b)/H-8.The HMBC correlations from N-Me(a) to C-13 and N-Me(b) and from N-Me(b) to C-13 demonstrated their linkage via the Nβ atom.The HMBC cross-peaks from H-11 to C-13, from H-12 to C-13 and C-4, from H-6a to C-5 and C-4, and from H-6b to C-5, along with the 1 H− 1 H COSY correlations of H-12/H-7/H-6b, constructed the ring B. The monosubstituted Δ 10(11) double bond was connected to ring B through C-12, as revealed by the 1 H− 1 H COSY cross-peaks of H2-10/H-11/H-12.The ring D was established by the HMBC cross-peaks from H3-16 to C-15, C-14, and C-8, and from H2-14 to C-13 and C-4, together with the 1 H− 1 H COSY cross-peaks of H-12/H-7/H-8.The HMBC correlations from 5-OH (δH 5.74) to C-6, C-5, and C-4 and from the NαH proton (δH 8.76) to C-4 and C-5, along with the chemical shift of C-5 (δC 87.0), fixed 5-OH and NαH at C-5, which further confirmed the presence of a carbinolamine moiety.Subsequently, the HMBC correlations from the NαH proton (δH 8.76) to C-1 (δC 166.0),C-3, and C-4 not only indicated the presence of the amide group and Δ 3(

Figure 5 .Figure 4 .
Figure 5.The effects of compound 1 and Amiloride on ASIC1a.(A) Illustrative complete-cell ASIC1a currents caused by pH 6.0 with a consistent voltage of −60 mV without (blank control) and with

Figure 5 .
Figure 5.The effects of compound 1 and Amiloride on ASIC1a.(A) Illustrative complete-cell ASIC1a currents caused by pH 6.0 with a consistent voltage of −60 mV without (blank control) and with

Figure 5 .
Figure 5.The effects of compound 1 and Amiloride on ASIC1a.(A) Illustrative complete-cell ASIC1a currents caused by pH 6.0 with a consistent voltage of −60 mV without (blank control) and with various levels of lycocasine A (1) and Amiloride.(B) Dose-response of the inhibitory action of lycocasine A (1) on the peak current of ASIC1a.The data were fitted using a Hill equation and are presented as the mean ± SD (n ≥ 3).(C) Dose-response curve of the inhibitory action of Amiloride on the peak current of ASIC1a.The data were fitted using a Hill equation and are presented as the mean ± SD (n ≥ 3).