Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel α-conotoxin (α-TxIA) in the venom of Conus textile. α-TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.

Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog.cyclic peptide | permeability | N-methylation P eptides are potentially valuable compounds for drug development, offering many advantages over other molecular classes (1-4). Specifically, their ability to mimic endogenous bioactive molecules allows them to bind potently and selectively to "difficult" drug targets, including protein-protein interactions that are too challenging for small-molecule therapeutics. However, the widespread use of peptides in the clinic has been slow in coming, in large part because of their generally low stability in vivo, high clearance, and poor oral bioavailability.The low oral bioavailability of peptides is attributed to a disparity between their physicochemical properties and those traditionally expected for "drug-likeness" (5, 6), leading to a perception that peptides are good drug leads but poor drugs. However, this perception is being challenged by a growing number of peptides that seem to be stable (7) and well absorbed within the gastrointestinal tract (8) and examples in which cyclic peptides have shown orally delivered bioactivity in animal disease models, including inflammatory pain (9) and neuropathic pain (10), prompting us to devise new rules for predicting pharmacokinetic properties of this compound class. Arguably the most famous example of a peptide with poor drug-likeness but reasonable oral bioavailability is cyclosporin A, widely used as the immunosuppressant drug cyclosporine (11). Two structural features of cyclosporin A in particular are thought to contribute to its oral bioavailability, namely its macrocyclic architecture and backbone N-methylation.Cyclization imparts increased rigidity to a parent peptide, which not only improves its stability against proteolytic degradation but also directs it into specific conformations that m...

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-

Disulfide-rich cyclic peptides have exciting potential as leads or frameworks in drug discovery; however, their use is faced with some synthetic challenges, mainly associated with construction of the circular backbone and formation of the correct disulfides. Here we describe a simple and efficient Fmoc solid-phase peptide synthesis (SPPS)-based method for synthesizing disulfide-rich cyclic peptides. This approach involves SPPS on 2-chlorotrityl resin, cyclization of the partially protected peptide in solution, cleavage of the side-chain protecting groups, and oxidization of cysteines to yield the desired product. We illustrate this method with the synthesis of peptides from three different classes of cyclic cystine knot motif-containing cyclotides: Möbius (M), trypsin inhibitor (T), and bracelet (B). We show that the method is broadly applicable to peptide engineering, illustrated by the synthesis of two mutants and three grafted analogues of kalata B1. The method reduces the use of highly caustic and toxic reagents and is better suited for high-throughput synthesis than previously reported methods for producing disulfide-rich cyclic peptides, thus offering great potential to facilitate pharmaceutical optimization of these scaffolds.

The -conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The -conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The ␤ and ␣ 2 ␦-1 auxiliary subunits influence the expression and characteristics of the ␣ 1B subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the -conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the ␤3 subunit had little influence on the on-and off-rates of -conotoxins, coexpression of ␣ 2 ␦ with ␣ 1B significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The The N-type (Ca v 2.2) voltage-gated calcium channels play an important role in the control of neurotransmitter release from nerve terminals (1, 2) and are important drug targets for the treatment of pain (3, 4) and ischemic brain injury (5). Native N-type Ca 2ϩ channels are hetero-oligomers that comprise a pore-forming ␣ 1 subunit (␣ 1B ) and at least two auxiliary subunits, ␤ and ␣ 2 ␦, which modulate the ␣ 1 subunit function (6, 7). Two splice variants of the ␣ 1B subunit have been identified that occur predominantly in the central (␣ 1B-d ) and peripheral (␣ 1B-b ) nervous systems (8, 9). Additional splice variants that lack large parts of the domain II-III linker region, including the synaptic protein interaction site, have also been isolated from human brain cDNA libraries (10). Multiple isoforms of the ␤ and ␣ 2 ␦ subunits also exist that can interact with the ␣ 1 subunit to produce N-type Ca 2ϩ channels with different gating properties, allowing the fine tuning of synaptic transmissions (11).A distinguishing feature of N-type channels is their high sensitivity to block by -conotoxins, which are relatively small (ϳ25 residue) polypeptides isolated from the venom of the marine snail of the genus Conus (12, 13). -Conotoxins have been used as research tools to help define the distribution and physiological roles of specific N-type calcium channels (14 -17) and have potential therapeutic value as intrathecal treatments for pain. MVIIA from Conus magus is being tested in clinical trials as a treatment for neuropathic pain, but dose-limiting side effects are a concern (18 -20). -Conotoxin CVID from Conus catus (21), which appears to have a wider therapeutic window (22), is also in clinical trials (23).The modulatory effects of auxiliary subunits on the biophysical properties of Ca 2ϩ channel ␣ 1 subunits have been well characterized. When expressed alone, ␣ 1 subunits produce functional channels with kinetic properties that differ substantially from those of the native channel (24, 25). The most commonly observed effect of the ␤ and ␣ 2 ␦ subunits when they are co-expressed with the ␣ 1 subunit in a heterologous expression system such as Xenopus oocytes or HE...