Dinoflagelates and cyanobacteria produce saxitoxin (STX), a lethal bis-guanidinium neurotoxin causing paralytic shellfish poisoning. A number of metazoans have soluble STX-binding proteins that may prevent STX intoxication. However, their STX molecular recognition mechanisms remain unknown. Here, we present structures of saxiphilin (Sxph), a bullfrog high-affinity STX-binding protein, alone and bound to STX. The structures reveal a novel high-affinity STX-binding site built from a “proto-pocket” on a transferrin scaffold that also bears thyroglobulin domain protease inhibitor repeats. Comparison of Sxph and voltage-gated sodium channel STX-binding sites reveals a convergent toxin recognition strategy comprising a largely rigid binding site where acidic side chains and a cation-π interaction engage STX. These studies reveal molecular rules for STX recognition, outline how a toxin-binding site can be built on a naïve scaffold, and open a path to developing protein sensors for environmental STX monitoring and new biologics for STX intoxication mitigation.

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme’s natural substrate, adenosine 5′-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}­methyl)­benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}­methyl)­benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.

The steroidal neurotoxin (-)-batrachotoxin functions as a potent agonist of voltage-gated sodium ion channels (Nas). Here we report concise asymmetric syntheses of the natural (-) and non-natural (+) antipodes of batrachotoxin, as well both enantiomers of a C-20 benzoate-modified derivative. Electrophysiological characterization of these molecules against Na subtypes establishes the non-natural toxin enantiomer as a reversible antagonist of channel function, markedly different in activity from (-)-batrachotoxin. Protein mutagenesis experiments implicate a shared binding side for the enantiomers in the inner pore cavity of Na These findings motivate and enable subsequent studies aimed at revealing how small molecules that target the channel inner pore modulate Na dynamics.

Improper function of voltage-gated sodium channels (Na V s), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target Na V s hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of Na V subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of Na V through a systematic structure-activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human Na V 1.7 (hNa V 1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective Na V inhibitors.sodium channel | guanidinium toxin | mutant cycle analysis M odulation of action potentials in electrically excitable cells is controlled by tight regulation of ion channel expression and distribution. Voltage-gated sodium ion channels (Na V s) constitute one such family of essential membrane proteins, encoded in 10 unique genes (Na V 1.1-Na V 1.9, Na x ) and further processed through RNA splicing, editing, and posttranslational modification. Sodium channels are comprised of a large (∼260 kDa) pore-forming α-subunit coexpressed with ancillary β-subunits. Misregulation and/or mutation of Na V s have been ascribed to a number of human diseases including neuropathic pain, epilepsy, and cardiac arrhythmias. A desire to understand the role of individual Na V subtypes in normal and aberrant signaling motivates the development of small-molecule probes for regulating the function of specific channel isoforms (1-4).Nature has provided a collection of small-molecule toxins, including (+)-saxitoxin (STX, 1) and (−)-tetrodotoxin (TTX), which bind to a subset of mammalian Na V isoforms with nanomolar affinity (5-7). Guanidinium toxins inhibit Na + influx through Na V s by occluding the outer pore above the ion selectivity filter (site 1). This proposed mechanism for toxin block follows from a large body of electrophysiological and site-directed mutagenesis studies (Fig. 1A and refs. 8-10). The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na V has been solved to date (11-16). Na V homology models, constructed based on X-ray analyses of prokaryotic Na + and K + voltage-gated channels, do not sufficiently account for experimental structure-activity relationship (SAR) data (6,(17)(18)(19)(20), and the molecular details underlying distinct differences in toxin potencies toward individual Na V subtypes remain undefined (5, 6, 21-23)....

The bis-guanidinium toxins are a collection of natural products that display nanomolar potency against select isoforms of eukaryotic voltage-gated Na + ion channels. We describe a synthetic strategy that enables access to four of these poisons, including 11-saxitoxinethanoic acid, C13acetoxy saxitoxin, decarbamoyl-saxitoxin, and saxitoxin. Highlights of this work include an unusual Mislow-Evans rearrangement and a late-stage Stille ketene acetal coupling. The IC 50 of 11-saxitoxinethanoic acid has been measured against rat Na V 1.4, and found to be 17.0 nM, similar to sulfated toxins gonyautoxin II and III.