The naturally-occurring cyclic cystine-knot microprotein trypsin inhibitors MCoTI-I and MCoTI-II have been synthesised using both thia-zip native chemical ligation and a biomimetic strategy featuring chemoenzymatic cyclisation by an immobilised protease. Engineered analogues have been produced containing a range of substitutions at the P 1 position that redirect specificity 10 towards alternative protease targets whilst retaining excellent to moderate affinity. Furthermore, we report an MCoTI analogue that is a selective low-µM inhibitor of foot-and-mouth-disease virus (FMDV) 3C protease, the first reported inhibitor of this important viral enzyme.

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNa V 1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNa V 1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNa V 1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNa V 1.7 channel inhibitor.Voltage-gated ion channels (VGICs) 4 are transmembrane proteins responsible for voltage-dependent movement of ions across cell membranes. They are involved in a wide range of physiological processes, including action potential generation in excitable cells, muscle and nerve relaxation, regulation of blood pressure, and sensory transduction. Many disorders are associated with VGIC abnormalities, and hence VGICs are actively pursued as drug targets for the treatment of a range of neuromuscular, neurological, or inflammatory disorders (1-3). For instance, the human voltage-gated sodium channel subtype 1.7 (hNa V 1.7) is involved in pain sensation, and molecules that selectively inhibit this channel might therefore be useful as leads for the development of novel analgesics (4). However, high sequence identity and structural homology exist between the nine subtypes of voltage-gated sodium channels, and given the critical role of Na V channels in the normal electrical activity of neurons, skeletal muscles, and cardiomyocytes, subtype selectivity is crucial but often difficult to achieve with small molecule inhibitors (2).Disulfide-rich peptide toxins isolated from animal venoms (e.g. spiders, snakes, and cone snails) have attracted much attention as potential analgesics because of their well defined three-dimensional structure and ability to inhibit voltage-gated sodium (Na V ), potassium (K V ), and calcium (Ca V ) ion channels with high potency and selectivity (5-9). Because of their stability, selectivity, and potency, these disulfide-rich peptides have been extensively characterized and are vigorously being pursued as drug leads as well as pharmacological tools to characterize VGICs (5).In general, peptide toxins that inhibit VGICs can be divided into two main groups based on their inhibitory strategy as fol-

MCoTI-II is a member of a class of microproteins known as cyclotides that possess a macrolactam-cystine knot scaffold imparting exceptional physiological stability and structural rigidity. Modification of residues in the active loop and engineered truncations have resulted in MCoTI-II analogues that possess potent activity against two therapeutically significant serine proteases: beta-tryptase and human leukocyte elastase. These results suggest that MCoTI-II is a versatile scaffold for the development of protease inhibitors against targets in inflammatory disease.

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The first total synthesis of MCoTI-II, a cysteine knot microprotein and potent trypsin inhibitor, is described; a synthetic strategy has been developed that combines efficient backbone construction via optimised solid phase peptide synthesis with one-pot 'thia-zip' native chemical ligation and refolding to yield the natural product.