Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNaV1.7

The human voltage-gated sodium channel sub-type 1.7 (hNaV1.7) is emerging as an attractive target for the development of potent and sub-type selective novel analgesics with increased potency and fewer side effects than existing therapeutics. HwTx-IV, a spider derived peptide toxin, inhibits hNaV1.7 with high potency and is therefore of great interest as an analgesic lead. In the current study we examined whether engineering a HwTx-IV analogue with increased ability to bind to lipid membranes would improve its inhibitory potency at hNaV1.7. This hypothesis was explored by comparing HwTx-IV and two analogues [E1PyrE]HwTx-IV (mHwTx-IV) and [E1G,E4G,F6W,Y30W]HwTx-IV (gHwTx-IV) on their membrane-binding affinity and hNaV1.7 inhibitory potency using a range of biophysical techniques including computational analysis, NMR spectroscopy, surface plasmon resonance, and fluorescence spectroscopy. HwTx-IV and mHwTx-IV exhibited weak affinity for lipid membranes, whereas gHwTx-IV showed improved affinity for the model membranes studied. In addition, activity assays using SH-SY5Y neuroblastoma cells expressing hNaV1.7 showed that gHwTx-IV has increased activity at hNaV1.7 compared to HwTx-IV. Based on these results we hypothesize that an increase in the affinity of HwTx-IV for lipid membranes is accompanied by improved inhibitory potency at hNaV1.7 and that increasing the affinity of gating modifier toxins to lipid bilayers is a strategy that may be useful for improving their potency at hNaV1.7

A complicated complex: ion channels, voltage sensing, cell membranes and peptide inhibitors

Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Nas) have attracted particular attention as potential analgesic targets. Nas, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na1.7) to controlling electrical signalling in cardiac function (Na1.5). Understanding the structural basis of Na function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Nas by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays

Molecular Simulations of Disulfide-Rich Venom Peptides with Ion Channels and Membranes.

Disulfide-rich peptides isolated from the venom of arthropods and marine animals are a rich source of potent and selective modulators of ion channels. This makes these peptides valuable lead molecules for the development of new drugs to treat neurological disorders. Consequently, much effort goes into understanding their mechanism of action. This paper presents an overview of how molecular simulations have been used to study the interactions of disulfide-rich venom peptides with ion channels and membranes. The review is focused on the use of docking, molecular dynamics simulations, and free energy calculations to (i) predict the structure of peptide-channel complexes; (ii) calculate binding free energies including the effect of peptide modifications; and (iii) study the membrane-binding properties of disulfide-rich venom peptides. The review concludes with a summary and outlook

In silico structural evaluation of short cationic antimicrobial peptides

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Cationic peptides with antimicrobial properties are ubiquitous in nature and have been studied for many years in an attempt to design novel antibiotics. However, very few molecules are used in the clinic so far, sometimes due to their complexity but, mostly, as a consequence of the unfavorable pharmacokinetic profile associated with peptides. The aim of this work is to investigate cationic peptides in order to identify common structural features which could be useful for the design of small peptides or peptido-mimetics with improved drug-like properties and activity against Gram negative bacteria. Two sets of cationic peptides (AMPs) with known antimicrobial activity have been investigated. The first reference set comprised molecules with experimentally-known conformations available in the protein databank (PDB), and the second one was composed of short peptides active against Gram negative bacteria but with no significant structural information available. The predicted structures of the peptides from the first set were in excellent agreement with those experimentally-observed, which allowed analysis of the structural features of the second group using computationally-derived conformations. The peptide conformations, either experimentally available or predicted, were clustered in an “all vs. all” fashion and the most populated clusters were then analyzed. It was confirmed that these peptides tend to assume an amphipathic conformation regardless of the environment. It was also observed that positively-charged amino acid residues can often be found next to aromatic residues. Finally, a protocol was evaluated for the investigation of the behavior of short cationic peptides in the presence of a membrane-like environment such as dodecylphosphocholine (DPC) micelles. The results presented herein introduce a promising approach to inform the design of novel short peptides with a potential antimicrobial activity.Peer reviewedFinal Published versio

Structural and functional characterization of venom pore forming proteins

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular, leída el 11-12-2020.Las proteínas formadoras de poros (PFP) son una familia de toxinas capaces de matar células por choque osmótico, precisamente porque forman poros en sus membranas. En disolución acuosa, estas proteínas permanecen plegadas y estables. Al interactuar con un receptor específico de la membrana (proteína, lípido o azúcar), se unen a ella, oligomerizan y forman un poro a través del núcleo hidrófobo de la membrana. Las actinoporinas son α-PFPs producidas por anémonas marinas como parte de su cóctel venenoso. Son pequeñas (≈ 20 kDa) y suelen tener un punto isoeléctrico básico ( ≈ 9). Comparten un motivo conformacional común, caracterizado por un sándwich β flanqueado por dos hélices α. Su receptor de membrana específico es la esfingomielina. Cuando las actinoporinas se unen a una membrana que contiene este esfingolípido, extienden su segmento helicoidal N-terminal, oligomerizan y forman un poro selectivo de cationes, insertando sus hélices α del extremo N-terminal a través del núcleo de la membrana. Sin embargo, el orden específico de las etapas que conducen a la formación del poro, así como la naturaleza de posibles estados intermedios, o su estequiometría final, todavía son objeto de debate. Estas actinoporinas constituyen además familias multigénicas: una sola especie produce una variedad de toxinas similares que no necesariamente muestran una actividad lítica o una especificidad idénticas. Debido a su simplicidad, son un modelo apropiado para estudiar la todavía no bien comprendida transición de una proteína soluble en agua a un estado en el que se integra en membrana...Pore forming proteins (PFPs) are a family of toxins that form pores in cell membranes leading to cell death by osmotic shock. These proteins remain stably folded and soluble in aqueous solution. Upon interaction with a specific receptor in the membrane (protein, lipid or sugar), they bind, oligomerize and form a pore through the membrane hydrophobic core.Actinoporins are α-PFPs produced by sea anemones as part of their venomous cocktail. They are small (≈ 20 kDa) and usually have a basic isoelectric point ( ≈ 9). They share a common fold characterized by a β-sandwich flanked by two α-helices. Their specific membrane receptor is sphingomyelin. When actinoporins bind to a membrane containing this sphingolipid, they extend their N-terminal α-helical segment, oligomerize and form a cation selective pore by inserting the α-helices through the membrane core. However, the specific step order leading to a final pore, the necessity of a pre-pore and the final stoichiometry are still debated. Actinoporins constitute multigene families: A single species produces a variety of similar toxins which not necessarily display identical lytic activity or specificity. Because of their simplicity, they are an appropriate model to study the biophysical challenging transition from a water-soluble protein to a membrane bound state...Fac. de Ciencias QuímicasTRUEunpu