Toxineering: New Tool for Harnessing the Power of Nature’s Poisons
Although spiders, snakes, and other poisonous predators get a bad rap, the diverse and highly specific peptides found in their venoms have proven quite useful for uncovering the function of ion channels and developing drugs that target them. In a paper published online February 11 in Current Biology, a team of researchers led by Michael Nitabach, Yale Medical School, New Haven, US, describes a new approach to identifying novel peptide toxins, a method that could bolster the design of new drugs targeting ion channels.
Using this approach, termed “toxineering,” the researchers identified a tarantula spider peptide, protoxin-I, as the first high-affinity peptide antagonist of the transient receptor potential ankyrin 1 (TRPA1) channel. Nitabach’s team also used toxineering to create a novel protoxin-I mutant with improved specificity for TRPA1, underscoring the value of the approach for pharmaceutical discovery.
“This is the beginning of an exciting strategy to sort through the offerings of nature and then work to improve those leads,” said Steve Goldstein of Brandeis University, Waltham, Massachusetts, US, who was not involved in the study.
Predator-derived peptide toxins have long been used to elucidate the structure and function of ion channels, including in pain pathways (see PRF related news story and PRF news story). The cone snail toxin-derived N-type calcium channel blocker ziconotide is FDA approved for the treatment of intractable pain.
Among pain-transducing channels, TRPA1 plays a special role in chronic inflammatory pain, itch, and peripheral neuropathies, making it a promising potential therapeutic target (for a recent review, see Brederson et al., 2013).
Taking aim at TRPA1
In the current paper, Nitabach and colleagues capitalized on a 2004 discovery that naturally occurring soluble toxin peptides can be fused to membrane-bound proteins without a loss of function (Ibañez-Tallon et al., 2004). Once expressed in cells, these chimeric “tethered-toxins” (t-toxins) remain at the cell surface instead of diffusing away, resulting in a very high local concentration of toxins.
To identify toxin peptides that act at TRPA1, co-first authors Junhong Gui and Boyi Liu screened over 100 peptide t-toxins derived from spider venoms using a recombinant cDNA library created from both published and unpublished sequences. After co-expressing TRPA1 and individual membrane-tethered toxins in Xenopus oocytes, the researchers found one tarantula venom peptide—protoxin-I—that inhibited TRPA1-mediated inward currents induced by the agonist mustard oil.
To ensure that the observed inhibition was not an artifact of the membrane tethering, Gui, Liu, and colleagues confirmed that the pharmacological activity of membrane-bound protoxin-I is similar to that of the native peptide. Consistent with previous findings using the native peptide, the protoxin-I fusion protein also acted as a voltage-gated sodium channel Nav1.2 antagonist.
The researchers then created a t-toxin library of protoxin-I mutants in which each successive amino acid in the peptide was mutated to the chemically inert residue alanine. Co-expressing each cDNA sequence with TRPA1 and Nav1.2 “enabled us to map on the surface of the toxin what residues are actually at the binding interface between the toxin and its two targets,” said Nitabach. Screening revealed that protoxin-I antagonizes the two receptors by binding to extracellular loops of the S1-S4 gating domain.
Improving nature
The cDNA-based toxineering approach has several advantages over traditional biochemical fractionation methods, Nitabach added. Toxineering eliminates the need for venom and does not require in vitro protein folding reactions to ensure synthetic toxin bioactivity. In addition, although still in the early stages, the approach could in theory be significantly scaled up. If so, toxineering would provide a functional screening alternative to the high-throughput, entirely binding-based phage display approach previously developed by Goldstein and colleagues (Takacs et al., 2009).
One of the most important benefits of the new approach is that the high local concentration of toxin created by tethering permits the detection of even low-affinity interactions with the receptor, said Goldstein. The effect is a broadening of the pool of potential drug targets to include those that would otherwise be missed.
However, toxineering’s biggest advantage lies in its ability to generate novel peptides with improved specificity. Although most of the protoxin-I mutants made by the researchers acted non-specifically at both TRPA1 and Nav1.2 channels, one was able to block TRPA1 alone, while another selectively antagonized Nav1.2.
With the binding surface data in hand, similar mutagenesis and screening of tethered protoxin-I libraries could be used to yield variants with even higher affinity and specificity for TRPA1 that may be of therapeutic value, write the authors.
The purpose of natural toxins is to stun and immobilize prey, so they are therefore non-specific by design—a problem for drug discovery. “To take those toxins and turn them into medications, what we need to do is identify a toxin that binds and then optimize it so that it is specific for only the receptor of interest. Toxineering does both,” said Goldstein.
Allison Curley, PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, Pennsylvania, US.
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