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.