Glutamate (Metabotropic) Group III Receptors

In the FP assay, 12 outcompeted 9 from -Cbtx similarly to 1 1, despite slightly reduced affinity compared to 1 (12= 6, 0

In the FP assay, 12 outcompeted 9 from -Cbtx similarly to 1 1, despite slightly reduced affinity compared to 1 (12= 6, 0.003, Figure ?Figure22E). livestock is also emerging as a substantial problem.3 Treatment requires rapid transport to a medical facility and intravenous administration of antivenom, a mix of immunoglobulin G antibodies isolated from, e.g., horse or sheep immunized with snake venom.1 As the only effective treatment, antivenoms save numerous lives but have the disadvantages of time-consuming and expensive production, poor access for those in need, and alarming numbers of adverse reactions.4?6 There is therefore an urgent need for novel treatments and new strategies, reflected in growing attention from the World Health Organization.7,8 In the case of elapid snakes, such as cobras and kraits, three-finger -neurotoxins such as -cobratoxin (-Cbtx) and -bungarotoxin (-Bgtx) are the most prominent and lethal venom components.9?11 -Cbtx and -Bgtx potently inhibit postsynaptic nicotinic acetylcholine receptors (nAChRs), which depresses neuromuscular signaling, causing paralysis K145 hydrochloride and loss of respiration.12 The effectiveness of conventional antivenoms is limited by immunogenicity, abundance of -neurotoxin antibodies in the immunized animal at the time of production,13 and which type of snake inflicts the bite. Newer antibody-based strategies have emerged, with a focus on smaller, less immunogenic, and venom-specific antivenoms. Smaller camelid antibody fragments raised against cobra venom were shown to neutralize the effects of -Cbtx in mice and may allow for greater tissue penetration than conventional antivenoms.14 Phage display of a library of human-derived antibody single-chain variable fragments against -Bgtx from krait venom led to one fragment that neutralized toxin injected into mice.15 In the future, combinations of such lead entities could enable development of safe and effective treatments, as reflected in the recent application of cocktails of human monoclonal antibodies targeting mamba toxins.16 These showed promising neutralization of dendrotoxins in mice, although some improvement is needed in neutralizing -neurotoxins. As an alternative to antibody-based strategies, we sought to discover short peptide-based neutralizers of -Cbtx by screening phage display peptide libraries for -Cbtx binders. Hits were then tested for neutralization of -Cbtx inhibition of nicotinic nAChRs using electrophysiology. This led Rabbit Polyclonal to ARMCX2 to the identification of an effective 8-mer peptide, for which we solved the peptide:-Cbtx X-ray cocrystal structure, revealing the detailed binding mode. The identification and characterization K145 hydrochloride of short peptide neutralizers of -Cbtx could lead to the development of targeted, fully synthetic, next-generation antivenoms for snakebite envenoming. Results High-Throughput Identification of Peptide Binders of -Cbtx Seeking novel binders of -Cbtx, we screened na?ve phage display libraries containing 1 109 disulfide-constrained 7-mers and linear 7-mers, 12-mers, 16-mers, or 20-mers against immobilized -Cbtx from (monocled cobra). Isolation and sequencing of the most prevalent phages after five rounds of biopanning identified two 16-mers, peptides 1 and 2 (Table 1). Additionally, deep sequencing of phages from all rounds identified six additional prevalent peptides, 3C8, that were also considered further (Table 1; see Experimental Section). Table 1 Phage Display Hits, Derivatives Thereof, and Their Prevention of -Cbtx Inhibition of nAChRs = 2). = 6), only measured for two lead peptides. cAfter five rounds of selection by biopanning. dFrom deep sequencing of all phages. ePEG, polyethylene glycol. fNal, 3-(2-naphthyl)-l-alanine. Characterization of Peptides with YM Motifs That Modulate -Cbtx Inhibition of Nicotinic Acetylcholine Receptors We next tested these binders for functional modulation of -Cbtx activity in electrophysiological assays. We heterologously expressed nicotinic 7-homomeric nAChRs in oocytes and measured acetylcholine-gated currents in response to acetylcholine alone, in the presence of -Cbtx, or in the presence of -Cbtx K145 hydrochloride preincubated with the identified peptides. As expected, -Cbtx alone (40.