marine drugs Review Synthetic Approaches to Zetekitoxin AB, a Potent Voltage-Gated Sodium Channel Inhibitor Kanna Adachi, Hayate Ishizuka, Minami Odagi * and Kazuo Nagasawa * Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology (TUAT), 2-24-16, Naka-cho, Koganei city, Tokyo 184-8588, Japan; [email protected] (K.A.); [email protected] (H.I.) * Correspondence: [email protected](M.O.); [email protected] (K.N.) Received: 7 December 2019; Accepted: 24 December 2019; Published: 26 December 2019 Abstract: Voltage-gated sodium channels (NaVs) are membrane proteins that are involved in the generation and propagation of action potentials in neurons. Recently, the structure of a complex made of a tetrodotoxin-sensitive (TTX-s) NaV subtype with saxitoxin (STX), a shellfish toxin, was determined. STX potently inhibits TTX-s NaV, and is used as a biological tool to investigate the function of NaVs. More than 50 analogs of STX have been isolated from nature. Among them, zetekitoxin AB (ZTX) has a distinctive chemical structure, and is the most potent inhibitor of NaVs, including tetrodotoxin-resistant (TTX-r) NaV. Despite intensive synthetic studies, total synthesis of ZTX has not yet been achieved. Here, we review recent efforts directed toward the total synthesis of ZTX, including syntheses of 11-saxitoxinethanoic acid (SEA), which is considered a useful synthetic model for ZTX, since it contains a key carbon–carbon bond at the C11 position. Keywords: saxitoxin; zetekitoxin AB; voltage-gated sodium channel; guanidine alkaloid 1. Introduction 1.1. Voltage-Gated Sodium Channel Isoforms Voltage-gated sodium channels (NaVs) are membrane proteins involved in neuronal excitation and transmission [1]. Ten subtypes, NaV1.1–1.9 and NaVX, have been identified based on sequence determination (Table1)[ 2]. These subtypes can be grouped into two types depending upon their sensitivity to the pufferfish toxin, tetrodotoxin (TTX) [3–7]: tetrodotoxin-sensitive NaVs (TTX-s NaVs 1.1–1.4, 1.6, and 1.7) are significantly inhibited by TTX, while tetrodotoxin-resistant NaVs (TTX-r NaVs 1.5, 1.8, 1.9) are not [8–23]. Subtype-selective modulators of NaVs are required for studies to establish the biological functions of these subtypes. Some of the subtypes are also considered to be potential drug targets; for example, NaV1.7 and 1.8 are potential targets for pain treatment [24–29]. Therefore, there is great interest in the development of drugs targeting specific subtypes [30,31]. Mar. Drugs 2020, 18, 24; doi:10.3390/md18010024 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, 24 2 of 19 Mar. Drugs 2020, 18, x FOR PEER REVIEW 2 of 19 Table 1. Isoforms of a voltage-gated sodium channel (NaV) and their classifications. NaV1.9 PNS, DRG Pain sensation 59,600 TTX:Na tetrodotoxin;V Isoform CNS: central Primary nervous Locations system; PNS: peripheral Related nervous Diseases system; TTXDRG: IC dorsal50 (nM) root ganglion;TTX-sensitive SMCs: smooth muscle cells. NaV1.1 CNS, PNS, heart Epilepsy 5.9 Na 1.2 CNS Epilepsy 7.8 1.2. Saxitoxin AsV A NaV Modulator NaV1.3 Embryonic CNS, injured DRG Nerve injury 2.0 SaxitoxinNaV 1.4(STX, 1) is a guanidine Skeletal alkaloid muscle with potent and Myotonia specific inhibitory activity 4.5 towards NaVs (FigureNa 1)V1.6 [32,33]. It has CNS, long PNS, been SMCs, known DRG as a shellfish CNS toxin. disorders In 1937, Sommer and 3.8 co-workers Na 1.7 PNS, DRG Pain sensation 5.5 found that toxin-freeV bivalves, including the dinoflagellate Gonyaulax catenella, became poisoned in TTX-resistant seawater, and they revealed that the real producer of STX (1) is algae [34,35]. Then, STX (1) was first NaV1.5 Heart, embryonic CNS Cardiac arrhythmias 1970 isolated fromNa VAlaska1.8 butter clams PNS,by Schantz’s DRG group in 1957 Pain [36,37]. sensation Rapport’s group 1330 subsequently isolated theNa sameV1.9 toxin from the same PNS, DRGshellfish, and named Pain it sensationsaxitoxin [38]. Structural 59,600 elucidation was TTX:troublesome. tetrodotoxin; Initially, CNS: central tri- nervous or tetracyclic system; PNS: structures peripheral nervous were system;proposed DRG: based dorsal rootupon ganglion; the molecular SMCs: formulasmooth and muscle the presence cells. of two guanidines as functional groups. Finally, the structure of STX (1) was independently determined by the two groups by means of X-ray analysis in 1975 [39,40]. STX (1) 1.2.consists Saxitoxin of ten As carbons, A NaV Modulator seven nitrogens, and four oxygens, and all the carbons except for C11 are connectedSaxitoxin with (STX, heteroatoms.1) is a guanidine STX (1) contains alkaloid five- with and potent six-membered and specific cyclic inhibitory guanidines, activity which towards have NadifferentVs (Figure pKa1 )[values32,33 of]. It8.7 has and long 12.4, been respectively; known as athe shellfish five-membered toxin. In 1937,one is Sommer less basic, and presumably co-workers founddue to thatits less toxin-free planar structure bivalves, [41]. including the dinoflagellate Gonyaulax catenella, became poisoned in seawater,STX ( and1) binds they revealedto the pore-forming that the real producerregion of ofthe STX alpha-loop (1) is algae of [34Na,35V ].and Then, blocks STX the (1) influx was first of isolatedsodium cation from Alaska in a similar butter manner clams by to Schantz’stetrodotoxin group (TTX) in 1957 [42,43]. [36, 37Recently,]. Rapport’s Yan’s group group subsequently determined isolatedthe X-ray the structure same toxin of the from complexes the same of shellfish,STX (1) with and Na namedVPas itderived saxitoxin from [38 American]. Structural cockroach elucidation and washuman-derived troublesome. Na Initially,V1.7 by using tri- or cryoEM tetracyclic (Figure structures 2) [44,45]. were They proposed found that based the uponcarbamoyl the molecular group at formulaC13 in STX and (1 the) interacts presence with of twoGly1407 guanidines and Thr1409 as functional in domain groups. III, the Finally, two guanidines the structure interact of STX with (1) wasGlu364 independently in domain I and determined Glu930 in by domain the two II, groups and the by geminal means diol of X-ray interacts analysis with in Asp1701 1975 [39 in,40 domain]. STX (IV.1) consistsInterestingly, of ten residues carbons, 1409 seven and nitrogens, 1410, located and fourin the oxygens, P2 loop andof domain all the III carbons in NaV except1.7, were for mutated C11 are connectedto Thr and withIle from heteroatoms. Met and Asp, STX respectively, (1) contains five- which and may six-membered explain why cyclic STX ( guanidines,1) has a weaker which affinity have diforff erentNaV1.7 pKa compared values of with 8.7 andother 12.4, subtypes respectively; (Met1409 the and five-membered Asp1410 are one conserved is less basic, in the presumably other subtypes due toof itsTTX-s) less planar[46,47]. structure [41]. Figure 1. Structure of saxitoxin (STX, 1). Figure 1. Structure of saxitoxin (STX, 1). STX (1) binds to the pore-forming region of the alpha-loop of NaV and blocks the influx of sodium cation in a similar manner to tetrodotoxin (TTX) [42,43]. Recently, Yan’s group determined the X-ray structure of the complexes of STX (1) with NaVPas derived from American cockroach and human-derived NaV1.7 by using cryoEM (Figure2)[ 44,45]. They found that the carbamoyl group at C13 in STX (1) interacts with Gly1407 and Thr1409 in domain III, the two guanidines interact with Glu364 in domain I and Glu930 in domain II, and the geminal diol interacts with Asp1701 in domain IV. Interestingly, residues 1409 and 1410, located in the P2 loop of domain III in NaV1.7, were mutated to Thr and Ile from Met and Asp, respectively, which may explain why STX (1) has a weaker affinity for NaV1.7 compared with other subtypes (Met1409 and Asp1410 are conserved in the other subtypes of TTX-s) [46,47]. Mar. Drugs 2020, 18, x FOR PEER REVIEW 3 of 19 Mar. Drugs 2020, 18, 24x FOR PEER REVIEW 3 of 19 Figure 2. (A) Top view of the structure of the STX–NaV1.7 complex. (B) Specific interactions in the STX–NaV1.7 complex [45]. 1.3. Natural Analogs of Saxitoxin, Including Zetekitoxin AB (ZTX) To date, more than 50 kinds of natural analogs of saxitoxin have been reported, of which most are modified at N1 (R1), C11 (i.e., R2 and R3), or C13 (R4) in the common structure shown in Figure 3A [48]. For example, neosaxitoxin (neoSTX, 2) is hydroxylated at N1, decarbamoylsaxitoxin (dcSTX, 3) has a hydroxyl group at C13, and gonyautoxins I–III (GTX I–III, 5–7, respectively) have a sulfate ester at C11; all of these analogs show similar NaV-inhibitory activity to STX (1). Among the STX (1) derivatives, zetekitoxin AB (ZTX, 8) has an unusual structure [49]. ZTX (8) Figure 2. (A) Top view of the structure of the STX–NaV1.7 complex. (B) Specific interactions in the was isolatedFigure 2. from (A) Top skin view of the of thePanamanian structure of dart-poison the STX–Na frogV1.7 complex.Atelopus (zetekiB) Specific in 1969 interactions by Mosher in theand co- STX–Na 1.7 complex [45]. V V workersSTX–Na [50,51].V1.7 complexIt has extremely [45]. potent Na -inhibitory activity (more than 600-fold greater than that 1.3.of STX Natural (1)), Analogswith IC50 of values Saxitoxin, of 6.1 Including pM, 65 Zetekitoxin pM, and AB280 (ZTX)pM for NaV1.2, NaV1.4, and TTX-r subtype 1.3.NaV Natural1.5, respectively Analogs of [49]. Saxitoxin, Thus, there Including is great Zetekitoxin interest ABin the (ZTX) mode of action of ZTX (8), but studies are hamperedTo date, by more the thanfact 50that kinds Atelopus of natural zeteki analogs is designated of saxitoxin as an have endang been reported,ered species.