Synthesis and evaluation of aminobenzothiazoles as blockers of N- and T-type calcium channels.

Both N- and T-type calcium ion channels have been implicated in pain transmission and the N-type channel is a well-validated target for the treatment of neuropathic pain. An SAR investigation of a series of substituted aminobenzothiazoles identified a subset of five compounds with comparable activity to the positive control Z160 in a FLIPR-based intracellular calcium response assay measuring potency at both CaV2.2 and CaV3.2 channels. These compounds may form the basis for the development of drug leads and tool compounds for assessing in vivo effects of variable modulation of CaV2.2 and CaV3.2 channels.

Isolation and characterization of a structurally unique ß-hairpin venom peptide from the predatory ant Anochetus emarginatus.

BACKGROUND: Most ant venoms consist predominantly of small linear peptides, although some contain disulfide-linked peptides as minor components. However, in striking contrast to other ant species, some Anochetus venoms are composed primarily of disulfide-rich peptides. In this study, we investigated the venom of the ant Anochetus emarginatus with the aim of exploring these novel disulfide-rich peptides. METHODS: The venom peptidome was initially investigated using a combination of reversed-phase HPLC and mass spectrometry, then the amino acid sequences of the major peptides were determined using a combination of Edman degradation and de novo MS/MS sequencing. We focused on one of these peptides, U1-PONTX-Ae1a (Ae1a), because of its novel sequence, which we predicted would form a novel 3D fold. Ae1a was chemically synthesized using Fmoc chemistry and its 3D structure was elucidated using NMR spectroscopy. The peptide was then tested for insecticidal activity and its effect on a range of human ion channels. RESULTS: Seven peptides named poneritoxins (PONTXs) were isolated and sequenced. The three-dimensional structure of synthetic Ae1a revealed a novel, compact scaffold in which a C-terminal ß-hairpin is connected to the N-terminal region via two disulfide bonds. Synthetic Ae1a reversibly paralyzed blowflies and inhibited human L-type voltage-gated calcium channels (CaV1). CONCLUSIONS: Poneritoxins from Anochetus emarginatus venom are a novel class of toxins that are structurally unique among animal venoms. GENERAL SIGNIFICANCE: This study demonstrates that Anochetus ant venoms are a rich source of novel ion channel modulating peptides, some of which might be useful leads for the development of biopesticides.

Spider Knottin Pharmacology at Voltage-Gated Sodium Channels and Their Potential to Modulate Pain Pathways.

Voltage-gated sodium channels (NaVs) are a key determinant of neuronal signalling. Neurotoxins from diverse taxa that selectively activate or inhibit NaV channels have helped unravel the role of NaV channels in diseases, including chronic pain. Spider venoms contain the most diverse array of inhibitor cystine knot (ICK) toxins (knottins). This review provides an overview on how spider knottins modulate NaV channels and describes the structural features and molecular determinants that influence their affinity and subtype selectivity. Genetic and functional evidence support a major involvement of NaV subtypes in various chronic pain conditions. The exquisite inhibitory properties of spider knottins over key NaV subtypes make them the best lead molecules for the development of novel analgesics to treat chronic pain.

Venomic, Transcriptomic, and Bioactivity Analyses of Pamphobeteus verdolaga Venom Reveal Complex Disulfide-Rich Peptides That Modulate Calcium Channels.

Pamphobeteus verdolaga is a recently described Theraphosidae spider from the Andean region of Colombia. Previous reports partially characterized its venom profile. In this study, we conducted a detailed analysis that includes reversed-phase high-performance liquid chromatography (rp-HPLC), calcium influx assays, tandem mass spectrometry analysis (tMS/MS), and venom-gland transcriptome. rp-HPLC fractions of P. verdolaga venom showed activity on CaV2.2, CaV3.2, and NaV1.7 ion channels. Active fractions contained several peptides with molecular masses ranging from 3399.4 to 3839.6 Da. The tMS/MS analysis of active fraction displaying the strongest activity to inhibit calcium channels showed sequence fragments similar to one of the translated transcripts detected in the venom-gland transcriptome. The putative peptide of this translated transcript corresponded to a toxin, here named ω-theraphositoxin-Pv3a, a potential ion channel modulator toxin that is, in addition, very similar to other theraphositoxins affecting calcium channels (i.e., ω-theraphotoxin-Asp1a). Additionally, using this holistic approach, we found that P. verdolaga venom is an important source of disulfide-rich proteins expressing at least eight superfamilies.

Protein classification with Extended-Sequence Coding by sliding window.

A large number of unclassified sequences is still found in public databases, which suggests that there is still need for new investigations in the area. In this contribution, we present a methodology based on Artificial Neural Networks for protein functional classification. A new protein coding scheme, called here Extended-Sequence Coding by Sliding Windows, is presented with the goal of overcoming some of the difficulties of the well method Sequence Coding by Sliding Window. The new protein coding scheme uses more than one sliding window length with a weight factor that is proportional to the window length, avoiding the ambiguity problem without ignoring the identity of small subsequences Accuracy for Sequence Coding by Sliding Windows ranged from 60.1 to 77.7 percent for the first bacterium protein set and from 61.9 to 76.7 percent for the second one, whereas the accuracy for the proposed Extended-Sequence Coding by Sliding Windows scheme ranged from 70.7 to 97.1 percent for the first bacterium protein set and from 61.1 to 93.3 percent for the second one. Additionally, protein sequences classified inconsistently by the Artificial Neural Networks were analyzed by CD-Search revealing that there are some disagreement in public repositories, calling the attention for the relevant issue of error propagation in annotated databases due the incorrect transferred annotations.