The isolation and pharmacological characterisation of novel insect-selective and MIT-like peptides from mygalomorph spider venoms

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NO FULL TEXT AVAILABLE. Access is restricted indefinitely. ----- The overriding aim of the work described in this thesis was to isolate and pharmacologically characterise novel bioactive peptides from mygalomorph spider venom, particularly those with insect-selective toxicity or actions on gastrointestinal motility. In the 21st century, humans are faced with two important challenges associated with arthropod control: decreased food production due to the increase in phytophagous pests, and disease vector control. The aim of this project was to explore the potential application of spider venom toxins in the development of novel insecticides for the control of insecticide-resistant pests. Additionally, the project also aimed to explore possible pharmacological effects of spider venom peptides on gastrointestinal motility, due to recent leads on the discovery of polypeptides that identified from the skin secretions of amphibian and snake venom exhibiting prokinetic actions. We designed a protocol to screen and characterise insect-selective toxins from the venom of the Australian funnel-web spider Atrax robustus. This protocol involved chromatographic fractionation of the venom followed by screening of individual components on chick biventer cervicis neuromuscular preparation and an acute toxicity assay in house crickets (Acheta domestica). Lead compounds with selective insecticidal neurotoxicity then underwent peptide sequencing and their mode of action directly examined on insect neurons or cells expressing ion channels, using the patch-clamp technique. Using this approach, we discovered two insect-selective neurotoxins: ⍵-ACTX-Ar1a and η-ACTX-Ar1a. ⍵-ACTX-Ar1a is an insect-selective voltage-gated Ca²⁺ channel blocker. This 37- residue neurotoxin shows high homology to several previously characterized members of the ⍵-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis, leading to death in acute toxicity tests in house crickets. Electrophysiological experiments showed that ⍵-ACTX-Ar1 a and its homolog ⍵-ACTX-Hv1 a from Hadronyche versuta, reversibly block both mid-low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Caᵥ) currents. This block occurred in the absence of alterations in the voltage-dependence of Caᵥ channel activation, and was voltage-independent, suggesting that ⍵-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 μM ⍵-ACTX-Ar1a failed to significantly affect global Kᵥ channel currents. However, 1 μM ⍵-ACTX-Ar1a caused a modest 18% block of insect Naᵥ channel currents, similar to the minor block of Naᵥ channels reported for other insect Caᵥ channel blockers such as ⍵-agatoxin IVA. η-ACTX-Ar1a (hybrid-ACTX-Ar1a) is a 39-residue peptide toxin showing homology to known members of both ⍵-ACTX-1 and ҝ-ACTX-1 families. It is the first atracotoxin known to target two channels, namely insect Cav and Kea channels. Crickets injected with η-ACTX-Ar1a exhibited biphasic excitatory and inhibitory phenotypes. Electrophysiological experiments revealed that η-ACTX-Ar1a blocked both M-LVA and HVA insect calcium channels with a higher potency when compared to ⍵-ACTX-1 family toxins. It also blocked heterologously expressed the a-subunit of the large-conductance Kea (BKca) channel of the cockroach, encoded by the slowpoke gene (pSlo) with a decreased potency when compared to ҝ-ACTX-Hv1 c. Nevertheless, the dual-target synergistic property reveals a novel self-synergising action. In addition, it displayed a pore blocking mode of action on these two channels. Mutagenesis studies of the close homolog η-ACTX-Hv1 a revealed that this toxin's insectophores define a significant overlap with the insectophores of ⍵-ACTX-1 and ҝ-ACTX-1 family toxins. NMR studies revealed the topological restriction of the insectophore which should increase the probability of successfully designing small-molecule mimetics to increase oral or topical absorption. This project also attempted to identify the target of atracotoxin-Hvf17 (ACTX-Hvf17), a 68- residue non-toxic peptide previously isolated from the venom of the Australian funnel-web spider Hadronyche versuta. This non-toxic peptide shows sequence homology to a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These peptides stimulate gastrointestinal smooth muscle contractility via interaction with prokineticin receptors and thus ACTX-Hvf17 may stimulate gastrointestinal smooth muscles. Using isolated rat stomach fundus and guinea-pig ileum organ bath preparations, we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 μM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The polypeptide also lacked activity on other isolated smooth muscle preparations, including rat aorta. Furthermore, a FLIPR Ca²⁺ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed β-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family polypeptides, variations in the amino-acid sequence, particularly the N-terminus and surface charge, result in a loss of activity on prokineticin receptors.
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