The tri-molecular concept: exploiting the lipid membrane in the design of potent and selective inhibitors for voltage-gated sodium channels

Akello Agwa

Research output: ThesisDoctoral Thesis


Voltage-gated ion channels are physiologically important transmembrane proteins involved in several pathologies including cardiac, muscular and neuronal disorders. Efforts are underway to develop potent and selective modulators of voltage-gated ion channels for the purposes of probing the pharmacology of these channels, and for the development of safe, efficacious therapeutics.

Gating modifier toxins (GMTs) are a class of peptide toxins (30–40 amino acid residues in length) that modulate the gating mechanism of voltage-gated ion channels and are being pursued as probes and drug leads for these transmembrane proteins. GMTs can be isolated from the venom of various animals including cone snails, scorpions and spiders.

GMTs that contain a conserved inhibitor cystine knot (ICK) motif in which disulfide bridges are arranged in a Cys I–IV, Cys II–V, Cys III–VI pattern, typically display a surface profile including a hydrophobic patch and a charged ring. This amphipathic structure putatively facilitates interactions between GMTs and lipid membranes giving rise to a tri-molecular complex formed when GMTs interact with the lipid bilayer during modulation of voltage-gated ion channels.

The first aim of this thesis is to examine GMT–lipid bilayer interactions and to investigate how these interactions influence potency and selectivity for voltage-gated sodium channels. Secondly, the objective is to determine whether GMT–lipid bilayer interactions can be included in the design of potent and selective GMT modulators of NaV1.7. The third aim is to explore the pharmacological relevance of GMTs in terms of their stability, and as tools that can be used to derive novel insight about voltage-gated sodium channel pharmacology. Given that voltage-gated sodium channel pharmacology is currently a widely pursued field, bivalent GMTs, known as double-knottins, were designed and produced semi-enzymatically, in order to examine the possibility of bimodal modulation of NaV1.7.

Disulfide-rich GMTs were obtained using optimized conditions for thermodynamic and regioselective oxidative folding. Double-knottins were produced via chemoenzymatic ligation using sortase A, a bacterial enzyme. Solution state NMR was used to examine structural integrity of the peptides and previously unsolved three-dimensional NMR structures were also completed. Cell-based biological FLIPRTETRAassays were conducted to analyze the activity of GMTs in the study and these assays were complemented by electrophysiological studies conducted by collaborators. Membrane binding affinity of peptides was studied with model lipid bilayers using surface plasmon resonance and the in-depth location of tryptophan residues into model lipid bilayers was examined using fluorescence spectroscopy methodologies.

Through these studies, it was determined that not all native GMTs have affinity for lipid bilayers, but those with affinity bind preferentially to model membranes with anionic charge, primarily via electrostatic interactions. HwTx-IV, a GMT with weak affinity for lipid bilayers was re-engineered to form gHwTx-IV, an analogue with a higher electropositive potential and this peptide showed higher affinity for model membranes combined with a higher potency at NaV1.7 compared to HwTx-IV. Unconventionally, selectivity of gHwTx-IV was improved by modifications to important pharmacophore residues with the rationale that affinity for the lipid bilayer would compensate for potency whereas pharmacophore modifications would improve selectivity. [R26A]gHwTx-IV, [K27A]gHwTx-IV and [R29A]gHwTx-IV had lower affinity for the lipid bilayer compared to gHwTx-IV but higher affinity compared to HwTx-IV. The three analogues were more selective for NaV1.7 compared to off target NaVs, and the lipid bilayer had thus been included in the design of potent, selective modulators of NaV1.7. Stability of GMTs in physiologically relevant conditions was established using stability assays, and the versatility of these peptides as pharmacological tools was shown in their chemoenzymatic conversion to double-knottins that revealed cooperative modulation of NaV1.7, a feature that provides a novel bivalent approach to the modulation of NaVs for the design of therapeutics.v
Original languageEnglish
Awarding Institution
  • University of Queensland
Award date1 Feb 2019
Publication statusPublished - 2019


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