DOI:10.1002/cbic.201800754 Minireviews Natural Voltage-Gated Sodium Channel Ligands: Biosynthesis and Biology April L. Lukowski[c] and Alison R. H. Narayan*[a, b, c] Natural product biosynthetic pathways are composed of en- rotoxin receptor sites on VGSCs associated with three different zymes that use powerful chemistry to assemble complex mole- classes of molecules:guanidinium toxins,alkaloid toxins, and cules. Small molecule neurotoxinsare examples of natural ladder polyethers. Each of these types of toxins have unique products with intricate scaffolds which often have high affini- structuralfeatures which are assembled by biosynthetic en- ties for their biological targets. The focus of this Minireview is zymes and the extentofinformation known aboutthese en- small molecule neurotoxinstargeting voltage-gated sodium zymes varies amongeach class. The biosynthetic enzymes in- channels (VGSCs)and the state of knowledge on their associat- volved in the formation of these toxins have the potentialto ed biosynthetic pathways. There are three small molecule neu- become useful tools in the efficient synthesis of VGSC probes. 1. Introduction Natural neurotoxinsare the productsofNature’s most experi- for sodium ions. The passage of sodium ions into the excitable enced chemists and pharmacologists. These structurally com- cell is permissible when the pore is in its “open” state andnot plex small molecules have been isolated from bacteria, plank- permitted when the channel is in the “closed” conformational ton, amphibians, plants, and even birds and serve as incredibly state. Abnormalregulation of VGSCs by mutations or ligands potent weapons for self-defense and prey capture.Due to the can result in extended depolarization by excessive flow of necessity of rapidlydisabling potentialpredators and subduing sodium ions into the cell or loss of current.[2] Such phenotypes prey,naturalneurotoxins have evolvedtotarget membrane- can have significant physiological consequences. bound ion channels.[1] One such biological target is the focus Small-molecule neurotoxins are known to interactwith of this Minireview,voltage-gated sodium channels (VGSCs), VGSCs at three distinctlocations (Figure1).[5] Receptor site I which are located in neurons and distributedthroughout car- on the flexible loops connecting segments5and 6ofeach diac, skeletal, and muscle tissues. domain is targeted by the guanidinium toxins:tetrodotoxin VGSCs are transmembrane protein complexes which facili- (1), chiriquitoxin(2), saxitoxin (3), gonyautoxins(e.g.,gonyau- tate cellular communication and regulate membrane potentials toxin 3, 4), and zetekitoxin AB (5). These molecules have the in response to sodium ions.[2] Fundamental structural studies effect of blockingthe pore and preventing ion conductance, on bacterial VGSCs[3] and recent cryo-EMstudies on insect and resultinginnumbness, tingling,and paralysis.[6] Alkaloidtoxins human VGSCs have revealed the architectureofthe protein.[4] such as aconitine (6), batrachotoxin (7), grayanotoxin (8), and VGSCs are composed of alarge a-subunit containing the ion veratridine (9)are known to interactwith receptor site II, locat- conducting pore and smaller regulatory b-subunits. The a-sub- ed at the top of segment 6ofdomains Iand IV.[7] Molecules unit is comprised of four homologous domains with each that interact with receptorsite II bind to VGSCswhile in the domain consistingofsix transmembrane segments (Figure 1). open state, resulting in prolongedactivation and cell depolari- These four domains are connected by flexible loops and to- zation.[8] These alkaloid toxins have several modes of disrupt- gether assemble the ion conducting channel, which is selective ing channel function, including reducing ion specificity and al- tering the kinetics of activation andinactivation,leadingto [a] Prof. A. R. H. Narayan overstimulationofthe central nervoussystem.[9] Ladder poly- Life Sciences Institute, University of Michigan ether compounds, including brevetoxin (10)and ciguatoxin 210 Washtenaw Ave.,Ann Arbor,MI48109 (USA) E-mail:[email protected] (11), bind at receptor site V, which bridges domain Isegment 6 [b] Prof. A. R. H. Narayan and domain IV segment 5(Figure 1, shown in blue). These mol- Department of Chemistry,University of Michigan ecules primarily alter activation and inactivationkinetics of the 930 NUniversityAve.,Ann Arbor,MI48109(USA) channel.[9] [c] A. L. Lukowski, Prof. A. R. H. Narayan The highly potent nature of neurotoxins for VGSC targets PrograminChemical Biology, UniversityofMichigan has inspired numerous drug design campaigns to treat central 210 Washtenaw Ave.,Ann Arbor,MI48109 (USA) nervous system disorders, pain, and muscle spasms.[10] Small The ORCID identification numbers for the authors of this article can be found under https://doi.org/10.1002/cbic.201800754. molecule neurotoxins have the benefitoftarget specificity ob- This article is part of the young researchers’ issue ChemBioTalents. To view served with protein and peptideneurotoxins coupled with the the complete issue, visit http://chembiochem.org/chembiotalents added advantage of an array of drug formulation options that ChemBioChem 2019, 20,1231 –1241 1231 2019 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Minireviews are unsuitable for larger,more unstablemolecules.[11] However, in rat brain extracts.[12a,13] Sincethe elucidation of its structure constructing and diversifying the intricate scaffolds of natural in 1975,[14] saxitoxin (3)has become afundamentaltool for the small molecule neurotoxinsremains asignificant challenge and study of VGSCswhere it is commonly used in atritiated form has hindered full exploration of thesemolecules’ therapeutic to assay the relative affinity of compounds to bind at receptor potential. Understanding the biosynthetic pathways employed site I.[15] by organisms to assemble these structurally complex mole- PSTswere first identified in the Alaskan butter clam, Saxido- cules can inform and inspire synthetic and biocatalytic ap- mus giganteus,which bioaccumulates these toxins produced proaches towardthese small molecule neurotoxins and related by cyanobacteria and dinoflagellates.[16] PST biosynthesis could compounds. Here, we presentthe known elements and pro- present afascinating evolutionary convergence across phyla posals for the biosynthetic pathway that give rise to each which is not completely understood.[17] Studies investigating small molecule VGSC ligand. the biosyntheticpathway toward PSTshave been conducted in both cyanobacteriaand dinoflagellates.[18] The biosynthetic pathway has been studied in significantly more detail in cyano- bacteria than the dinoflagellate pathway,although there is evi- 2. Receptor Site I dence that some biosynthetic steps may be conserved. Specifi- 2.1. Saxitoxins and gonyautoxins cally in the timing of sulfation events that convert saxitoxin into gonyautoxins[19] and the formation of the first proposed Saxitoxins and their sulfate-containing derivatives, gonyautox- biosynthetic intermediate, 12.[20] ins, belong to alarger class of molecules collectively referred Several hypotheses on the biosynthetic route towardPSTs to as paralytic shellfish toxins (PSTs). Over 50 natural products have been reported.[21] Gene clusters encoding enzymes in- have been isolated, and these molecules sharea5-5-6 tricyclic volved in saxitoxin biosynthesis were first discovered by Neilan bisguanidinium ion-containing core and vary in oxygenation and co-workersinCylindrospermopsis raciborskii.[22] Since the pattern and incorporation of sulfate, carbamate, and acetate initial discovery,clusters have also been identified in Microseira [12] groups (Figure 2). Asubset of these compounds have been [23] [24] [25] wollei, Aphanizomenon sp. NH-5, Aphanizomenon gracile, evaluated for VGSC affinity.These studies have shown that the Dolichospermum circinale,[24] Raphidiopsis brookii[26] and Syctone- affinity is dependent on the decoration of the scaffold, where [27] ma crispum. These gene clusters are relatively large, ranging saxitoxin (3)isoften used as abenchmark with a Kd of 4.3 nm in size from 25.9 to 54 kb, and consist of genes encoding the following core enzymes present in all clusters: sxtA, sxtB, sxtD, Alison Narayan earned her Ph.D. at the sxtE, sxtG, sxtH, sxtI, sxtM, sxtP, sxtQ, sxtR, sxtS, sxtT,and sxtU University of California, Berkeley (USA), (Table 1). The complete biosynthetic pathway has not been where she developed novel methods elucidated;however,invitro characterization studies and inter- for the synthesis of nitrogen heterocy- mediate analysishave begun to define crucial steps.[27,28] cles and the total synthesis of complex Identificationofputative biosynthetic intermediates present natural products in the group of Prof. in dinoflagellateand cyanobacterial culture extracts has also Richmond Sarpong. As aLife Sciences contributed to hypotheses on the pathway that delivers PSTs. Research Foundation Postdoctoral Using mass spectrometry analysisincombination with synthet- Fellow,Alison studied natural product ically generatedstandards, Yotsu-Yamashita and co-workers biosynthesis and biocatalysis with Prof. have identified several new potential biosynthetic intermedi- David Sherman. In 2015, Alison ates, including 12 (Figure 3), and the identification of 16 in di- became an assistant professor in the noflagellate extracts (Figure 3B,inset).[20,29] Characterization of Department of Chemistry and the Life Sciences Institute at the Uni- SxtA supports 12 as abiosynthetic intermediate.[28a] However, versity