Structural Analysis of Saxitoxin and Neosaxitoxin Toxins with Potential Therapeutic Targets ^†

Abstract

Historically, saxitoxin (STX) and its analogs, like neosaxitoxin (NeoSTX), have been restricted to scientific research due to their potent toxicity, despite their potential as therapeutic agents. This study aimed to evaluate the pharmacological versatility of STX and NeoSTX through molecular docking. Using the SwissTargetPrediction server, 18 potential human therapeutic targets were identified, with the 5-HT6 receptor being the primary target. Molecular docking assays were performed with AutoDock Vina version 1.1.2, and the lowest-energy pose for NeoSTX (−5.7 kcal/mol) suggested a stronger binding affinity compared to STX (−5.5 kcal/mol). The analysis of the binding mode revealed key interactions, including hydrogen bonds with Thr199 and hydrophobic contacts with Phe285. These findings provide robust hypotheses regarding the therapeutic potential of NeoSTX as a 5-HT6 receptor antagonist, supporting the development of novel bioactive agents derived from these marine toxins.

1. Introduction

Historically, several toxins have been explored as potential bioterrorism agents [1]. These include marine toxins produced by dinoflagellates and cyanobacteria, which are known for their ecological impact and association with outbreaks of paralytic shellfish poisoning (PSP), present in fresh water from Southern America [2]. Due to their potent toxicity and difficulty in detection, some of these molecules, including saxitoxin (STX), have been listed in the annexes of the Chemical Weapons Convention (CWC)—an international treaty implemented by the United Nations (UN)—prohibiting their use for warfare purposes and restricting their application exclusively to scientific research [3].

Nevertheless, STX and some of its analogs, such as neosaxitoxin (NeoSTX), possess significant chemical diversity and biological functionality, making them promising reservoirs for the discovery of new therapeutic agents [4] (Figure 1). STX is recognized as one of the most potent blockers of neuronal voltage-dependent sodium channels (NaVs), causing rapid muscle paralysis. Structurally, it is a hydrophilic toxin formed by a tricyclic guanidine structure, a feature that confers high polarity and affinity for NaVs [5].

Conversely, NeoSTX differs from STX by the substitution of a hydroxyl group with a carbamoyl group in the tricyclic ring of the molecule, slightly altering its pharmacokinetic profile. Consequently, NeoSTX is being investigated as an analgesic, anti-inflammatory, and even anesthetic agent [6,7]. In this regard, molecular docking becomes an essential tool for understanding the therapeutic potential of these substances. This in silico approach allows the prediction of ligand–receptor binding modes, the identification of intermolecular interactions, and the estimation of binding affinity [8]. This capability provides robust hypotheses, even before conducting in vitro or in vivo assays [8].

Therefore, this study aims to evaluate, using docking assays, the binding profile of STX and NeoSTX to various therapeutic targets and to discuss the potential in vitro consequences of these interactions. Furthermore, we intend to provide a strategy for exploring the pharmacological versatility of these marine toxins, thereby proposing new approaches for the development of bioactive agents derived from these structures.

2. Methods

The structures of Saxitoxin (STX) and Neosaxitoxin (NeoSTX) were used as ligands, retrieved from the Protein Data Bank (PDB) database [9]. To identify novel human therapeutic targets, we utilized the SwissTargetPrediction server (http://www.swisstargetprediction.ch/ (accessed on 19 June 2024)) [10,11,12], which employs the principle of ligand-based similarity searching. The 3D structure of 5-HT6 was selected from PDB ID 7XTB [13] at 3.30 Å resolution as a receptor. Although the structure exhibited a relatively high resolution for a membrane protein, its validation metrics were satisfactory, including acceptable side chain and Ramachandran outliers. The 3D structure of STX (used as a ligand) was obtained from PDB ID 600F [14], and the 3D structure of NeoSTX (used as a ligand) was obtained from PubChem ID 21117946 [15]. Subsequently, molecular docking simulations were prepared using the AutoDock Vina 1.1.2 program [16,17]. The receptor and ligand were prepared using AutoDock Tools version 1.5.6. Polar hydrogens and partial charges were added using the Gasteiger method.

The docking grid box was constructed using AutoGrid version 4 integrated into AutoDock Tools with a spacing of 0.25 Å and was centered on the structure’s co-crystallized ligand (SRO). A docking simulation was performed in AutoDock Vina, setting the exhaustiveness parameters to 150 and an energy range of 5. The analysis of the protein–ligand interaction profiler was performed from the Protein-Ligand Interaction Profiler (PLIP) server online (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index/ (accessed on 9 July 2024)) [18].

3. Results

The potent paralytic toxins, STX and NeoSTX, were used as ligands. The 2D structures of the toxins were uploaded to the SwissTargetPrediction server, which identified 18 potential therapeutic targets in humans, belonging to different classes (

Table 1. Potential human therapeutic targets for STX and NeoSTX identified by the SwissTargetPrediction server analysis.

Targets	UNIPROT ID
Serotonin 6 (5-HT6) receptor	P50406
Carbonic anhydrase II	P00918
Carbonic anhydrase I	P00915
Carbonic anhydrase IX	Q16790
Carbonic anhydrase VII	P43166
Carbonic anhydrase XII	O43570
Carbonic anhydrase XIV	Q9ULX7
Steryl-sulfatase	P08842
C-X-C chemokine receptor type 3	P49682
Carbonic anhydrase III	P07451
Carbonic anhydrase VI	P23280
Carbonic anhydrase IV	P22748
Carbonic anhydrase VB	Q9Y2D0
Carbonic anhydrase VA	P35218
Carbonic anhydrase XIII (by homology)	Q8N1Q1
Tyrosinase	P14679
Nuclear receptor subfamily 4 group A member 1	P22736
Protein tyrosine kinase 2 beta	Q14289

). Considering these identified targets, the PDB was subsequently searched for structures featuring STX and targets reported by SwissTargetPrediction server online. According to Table 1, the primary potential therapeutic target identified was the 5-HT6 receptor. The 5-HT6 receptor is one of several subtypes of serotonin (5-hydroxytryptamine, or 5-HT) receptors, and a crucial biogenic neurotransmitter in the Central Nervous System (CNS). It stands out for its function and location, making it an important target for the development of new drugs [19].

The redocking reported estimated free-energy values of the 10 poses from −6.3 kcal/mol to −4.3 kcal/mol (

Table 2. Docking scores from AutoDock Vina for the top 10 poses of the native ligand, SRO, and STX and NeoSTX ligands on the 5-HT6 receptor.

Poses	Score (kcal/mol)
	SRO—Native Ligand	STX	NeoSTX
1	−6.3	−5.5	−5.7
2	−6.1	−5.2	−5.4
3	−6.0	−5.0	−5.1
4	−5.9	−4.9	−4.8
5	−5.9	−4.7	−4.6
6	−5.7	−4.6	−4.5
7	−5.7	−4.6	−4.4
8	−4.3	−4.5	−4.2
9	−5.4	−4.2	−4.0
10	−4.3	−4.1	−3.7

). The lowest-energy solution pose was −6.3 kcal/mol. This pose showed three specific hydrogen bonds between the indole group of SRO and Thr196’s side chain; between the amine group’s nitrogen atom on SRO and the side chain of Asp106; and the oxygen atom on SRO and Asn288. A hydrophobic interaction was identified between the purine ring of SRO and the side chain of Ala192. And finally, a π-stacking interaction between the indole group of SRO and Phe285 was identified (Figure 2).

The estimated free-energy values of the 10 poses reported by AutoDock Vina ranged from −5.5 kcal/mol to −4.1 kcal/mol (Table 2). The lowest-energy solution was calculated to be −5.5 kcal/mol (Figure 2). This pose exhibited a specific hydrogen bond between the amide group’s nitrogen atom on STX and the side chain of Leu183.

Additionally, hydrogen bonds were identified between the guanidine group of STX and Thr196’s side chain and between the amide group’s oxygen atom on STX and Asn288’s side chain. Furthermore, a hydrophobic interaction was identified between the purine ring of STX and the side chain of Phe285.

For the NeoSTX ligand, docking on the same target resulted in estimated free-energy values for the 10 poses ranging from −5.7 kcal/mol to −3.7 kcal/mol (Table 2). The lowest-energy pose exhibited a binding affinity of −5.7 kcal/mol. This pose showed a specific hydrogen bond between the purine ring of NeoSTX and the side chain of Leu183. Two additional hydrogen bonds were formed between the guanidine group of NeoSTX and the side chain of Thr199, and between the amide group’s oxygen atom and Asn288. A fourth hydrogen bond was observed between the amide group’s nitrogen atom on NeoSTX and Val189’s side chain. Furthermore, residues Ser130, Ala192, and Thr199 established specific hydrogen bonds with the oxygen atoms attached to the pyrrolidine ring. Finally, a hydrophobic interaction between the purine ring of NeoSTX and the side chain of Phe285 was identified.

4. Conclusions

As a conclusion, the toxins exhibited affinity for different potential receptor targets, which were identified by in silico target prediction. Molecular docking showed that STX and NeoSTX interact with Leu183, Asn288, and Phe285 residues; although NeoSTX interacted with more residues than the HTR6 receptor, maybe its structure allows a large number of interactions. However, the interactions were similar between SRO, STX, and NeoSTX with Asn288 and Phe285 residues.

We show that the wealth of existing complexes and of molecular docking results provides deep insights into the design principles for drug targeting.

These findings provide robust hypotheses about the therapeutic potential of STX and NeoSTX as 5-HT6 receptor antagonists, supporting the development of new bioactive agents derived from these marine toxins. Molecular dynamics simulations will be performed for the most promising complexes, and atomic force microscopy tests will be performed to identify the interaction between STX and neosaxitoxin with the targets.