A novel heterodimeric three-finger neurotoxin, irditoxin, was isolated from venom of the brown treesnake Boiga irregularis (Colubridae). Irditoxin subunit amino acid sequences were determined by Edman degradation and cDNA sequencing. The crystal structure revealed two subunits with a three-finger protein fold, typical for "nonconventional" toxins such as denmotoxin, bucandin, and candoxin. This is the first colubrid three-finger toxin dimer, covalently connected via an interchain disulfide bond. Irditoxin showed taxon-specific lethality toward birds and lizards and was nontoxic toward mice. It produced a potent neuromuscular blockade at the avian neuromuscular junction (IC(50)=10 nM), comparable to alpha-bungarotoxin, but was three orders of magnitude less effective at the mammalian neuromuscular junction. Covalently linked heterodimeric three-finger toxins found in colubrid venoms constitute a new class of venom peptides, which may be a useful source of new neurobiology probes and therapeutic leads.

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle ␣ 1 ␤␥␦-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 Å by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet. Three-finger toxins (3FTXs)3 form one of the most abundant, well recognized families of snake venom proteins. They share a similar structure and are characterized by three ␤-stranded finger-like loops, emerging from a globular core and stabilized by four conserved disulfide bridges. An additional disulfide linkage may sometimes be present in the first (non-conventional toxins) or second (long-chain ␣-neurotoxins and -toxins) loop (1-5). All 3FTXs are monomers except for -toxins, which are noncovalent homodimers isolated from Bungarus venoms. Minor structural differences in the three-finger fold, viz. the number of ␤-strands, overall morphology of the loops, and differential lengths of turns or C-terminal tails (6), lead to the recognition of varied targets and modulate the toxicity and specificity (7). Hence, 3FTXs affect a broad range of molecular targets, including ␣ 1 -nicotinic acetylcholine receptors (nAChRs; short-and longchain ␣-neurotoxins), ␣ 7 -nAChRs (long-chain ␣-neurotoxins), and ␣ 3 -and ␣ 4 -nAChRs (-toxins) (4, 5); muscarinic acetylcholine receptors (muscarinic toxins) (8); L-type calcium channels (calciseptine and FS2 toxin) (9, 10); integrin ␣ IIb ␤ 3 (dendroaspin) (11, 12); integrin ␣ v ␤ 3 (cardiotoxin A5) (13); acetylcholinesterase (fasciculins) (14); phospholipids and glycosphingolipids (cardiotoxins) (15); and blood coagulation protein factor VIIa (16). As the interaction with such a broad spectrum of target proteins results in a variety of pharmacological effects, the understanding...

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means is often required to determine the biologically relevant target(s) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3 or Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is dramatically elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.

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