Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.

Engineering potent and selective analogues of GpTx-1, a tarantula venom peptide antagonist of the Na(V)1.7 sodium channel.

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.

Position-specific scoring matrix and hidden Markov model complement each other for the prediction of conopeptide superfamilies.

Classified into 16 superfamilies, conopeptides are the main component of cone snail venoms that attract growing interest in pharmacology and drug discovery. The conventional approach to assigning a conopeptide to a superfamily is based on a consensus signal peptide of the precursor sequence. While this information is available at the genomic or transcriptomic levels, it is not present in amino acid sequences of mature bioactives generated by proteomic studies. As the number of conopeptide sequences is increasing exponentially with the improvement in sequencing techniques, there is a growing need for automating superfamily elucidation. To face this challenge we have defined distinct models of the signal sequence, propeptide region and mature peptides for each of the superfamilies containing more than 5 members (14 out of 16). These models rely on two robust techniques namely, Position-Specific Scoring Matrices (PSSM, also named generalized profiles) and hidden Markov models (HMM). A total of 50 PSSMs and 47 HMM profiles were generated. We confirm that propeptide and mature regions can be used to efficiently classify conopeptides lacking a signal sequence. Furthermore, the combination of all three-region models demonstrated improvement in the classification rates and results emphasise how PSSM and HMM approaches complement each other for superfamily determination. The 97 models were validated and offer a straightforward method applicable to large sequence datasets.

Peak capacity optimisation for high resolution peptide profiling in complex mixtures by liquid chromatography coupled to time-of-flight mass spectrometry: application to the Conus consors cone snail venom.

The high resolution profiling of complex mixtures is indispensable for obtaining online structural information on the highest possible number of the analytes present. This is particularly relevant for natural extracts, as for the venom of the predatory marine snail Conus consors, which contains numerous bioactive peptides with molecular masses ranging between 1000 and 5000 Da. The goal of the present work was to maximise peak capacity of peptides separations by LC-MS while maintaining a reasonable analysis time. The best gradient performance using the C. consors venom as a real sample was obtained with a mobile phase flow rate as high as possible to maximise performance in the gradient mode, and gradient time comprised between 75 and 350 min when using a 150 mm column length. The present study also confirmed that an elevated temperature (up to 90 °C) improves performance under ultra-high pressure liquid chromatography (UHPLC) conditions. However, the thermal stability of the analytes had to be critically evaluated. For the profiling of C. consors, analyte degradation was not clearly observable at 90 °C with analysis times of approximately 100 min. Finally, the MS source was found to cause significant additional band broadening in the UHPLC mode (σ(ext)(2) was 10-24 times higher using TOF-MS vs. UV detection). Thus, if the MS contributes strongly to the peak capacity loss, classical 2.1mm I.D. columns can be replaced by 3.0mm I.D. to mitigate this problem. Based on these considerations, the optimal generic profiling conditions applied to the C. consors venom provided a peak capacity higher than 1100 for a gradient time of around 100 min, doubling the values reached by classical HPLC separation. UHPLC-QTOF-MS/MS experiments carried out in these conditions provided exploitable data that matched with peptides present in the C. consors venom. These optimal LC conditions are thus compatible with online peptide deconvolution and matching against transcriptomic data and, to some extent, de novo sequencing in such complex mixtures.

Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach.

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.

A novel µ-conopeptide, CnIIIC, exerts potent and preferential inhibition of NaV1.2/1.4 channels and blocks neuronal nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH: µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS: Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC(50) = 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked Na(V)1.4 (IC(50) = 1.3 nM) and Na(V)1.2 channels in a long-lasting manner. Cardiac Na(V)1.5 and DRG-specific Na(V)1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3ß2 nAChR subtype (IC(50) = 450 nM) and, to a lesser extent, on the α7 and α4ß2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS: µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

High-resolution picture of a venom gland transcriptome: case study with the marine snail Conus consors.

Although cone snail venoms have been intensively investigated in the past few decades, little is known about the whole conopeptide and protein content in venom ducts, especially at the transcriptomic level. If most of the previous studies focusing on a limited number of sequences have contributed to a better understanding of conopeptide superfamilies, they did not give access to a complete panorama of a whole venom duct. Additionally, rare transcripts were usually not identified due to sampling effect. This work presents the data and analysis of a large number of sequences obtained from high throughput 454 sequencing technology using venom ducts of Conus consors, an Indo-Pacific living piscivorous cone snail. A total of 213,561 Expressed Sequence Tags (ESTs) with an average read length of 218 base pairs (bp) have been obtained. These reads were assembled into 65,536 contiguous DNA sequences (contigs) then into 5039 clusters. The data revealed 11 conopeptide superfamilies representing a total of 53 new isoforms (full length or nearly full-length sequences). Considerable isoform diversity and major differences in transcription level could be noted between superfamilies. A, O and M superfamilies are the most diverse. The A family isoforms account for more than 70% of the conopeptide cocktail (considering all ESTs before clustering step). In addition to traditional superfamilies and families, minor transcripts including both cysteine free and cysteine-rich peptides could be detected, some of them figuring new clades of conopeptides. Finally, several sets of transcripts corresponding to proteins commonly recruited in venom function could be identified for the first time in cone snail venom duct. This work provides one of the first large-scale EST project for a cone snail venom duct using next-generation sequencing, allowing a detailed overview of the venom duct transcripts. This leads to an expanded definition of the overall cone snail venom duct transcriptomic activity, which goes beyond the cysteine-rich conopeptides. For instance, this study enabled to detect proteins involved in common post-translational maturation and folding, and to reveal compounds classically involved in hemolysis and mechanical penetration of the venom into the prey. Further comparison with proteomic and genomic data will lead to a better understanding of conopeptides diversity and the underlying mechanisms involved in conopeptide evolution.

Structural identification by mass spectrometry of a novel antimicrobial peptide from the venom of the solitary bee Osmia rufa (Hymenoptera: Megachilidae).

The venom from the solitary bee Osmia rufa (Hymenoptera: Megachilidae) was analyzed using mass spectrometry (MS)-based techniques. Sensitive proteomic methods such as on-line LC-ESI-MS and nanoESI-MS analyses revealed more than 50 different compounds with molecular masses ranging from 400 to 4000Da. The major component has a monoisotopic molecular mass of 1924.20Da and its amino acid sequence was elucidated by de novo sequencing using tandem mass spectrometry and Edman degradation. This 17-residue cysteine-free peptide, named osmin, shows some similarities with the mast cell degranulation (MCD) peptide family. Free acid and C-terminally amidated osmins were chemically synthesized and tested for antimicrobial and haemolytic activities. The synthetic C-amidated peptide (native osmin) was found to be about three times more haemolytic than its free acid counterpart, but both peptides are much less lytic than melittin from social bee venom. Preliminary antimicrobial and antifungal tests indicate that both peptides are able to inhibit bacterial and fungal growth at micromolar concentrations.

Comparative proteomic study of the venom of the piscivorous cone snail Conus consors.

In the context of an exhaustive study of the piscivorous cone snail Conus consors, we performed an in-depth analysis of the intact molecular masses that can be detected in the animal's venom, using MALDI and ESI mass spectrometry. We clearly demonstrated that, for the venom of this species at least, it is essential to use both techniques in order to obtain the broadest data set of molecular masses. Only 20% of the total number of molecules detected were found in both mass lists. The two data sets were also compared in terms of mass range and relative hydrophobicity of the components detected in each. With a view to an extensive analysis of this venom's proteome, we further performed a comparative study by ESI-MS between venom obtained after classical dissection of the venom duct versus venom obtained by milking live animals. Surprisingly, although many fewer components were found in the milked venom than in the dissected venom, approximately 50% of those found had not been seen in the dissected venom. Several questions raised by these observations are discussed. With regards to the current knowledge of the cone snail venom composition, our results emphasize the complementary nature of the mass spectrometry methods and of the two techniques used in venom collection.

High throughput screening of bradykinin-potentiating peptides in Bothrops moojeni snake venom using precursor ion mass spectrometry.

Snake venoms are known to be an extensive source of bioactive peptides. Bradykinin-potentiating peptides (BPPs) are inhibitors of the angiotensin-converting enzyme that have already been identified in the venom of many snake, scorpion, spider and batrachian species. Their most characteristic structural features are an invariable N-terminal pyroglutamate residue (pGlu or Z) and two consecutive proline residues at the C-terminus. Fragmentation of BPPs by collision-induced dissociation during electrospray tandem mass spectrometry analysis (ESI-MS/MS) generates a predominant signal at m/z 213.1 corresponding to the y-ion of the terminal Pro-Pro fragment. In addition, signals at m/z 226.1 and 240.1 that correspond to the b ions of the N-terminus pGlu-Asn and pGlu-Lys, respectively, can often be observed. Based on these structural determinants, the present work describes an original methodology for the discovery of BPPs in natural extracts using liquid chromatography coupled to ESI-MS/MS operated in precursor ion-scan mode. The venom of the Bothrops moojeni snake was used as a model and the methodology was applied for subsequent structural analysis of the identified precursors by tandem mass spectrometry on quadrupole-time-of-flight (Q-TOF) and matrix-assisted laser-desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) instruments. More than 40 peptides below 2500 Da could be detected, among them 20 were shown to belong to the BPP-like family including the related tripeptides pGlu-Lys-Trp and pGlu-Asn-Trp. A total of 15 new sequences have been identified using this approach.

The venom of the snake genus Atheris contains a new class of peptides with clusters of histidine and glycine residues.

We investigated venoms from members of the genus Atheris (Serpentes, Viperidae), namely the rough scale bush viper (Atheris squamigera), the green bush viper (A. chlorechis) and the great lakes bush viper (A. nitschei), using mass spectrometry-based strategies, relying on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with de novo peptide sequencing. We discovered a set of novel peptides with masses in the 2-3 kDa range and containing poly-His and poly-Gly segments (pHpG). Complete primary structural elucidation and confirmation of two sequences by Edman degradation indicated the consensus sequence EDDH(9)GVG(10). Bioinformatic investigations in protein sequence databanks did not show relevant homology with known peptides or proteins. However, a more extensive investigation of data in nucleic acid databases revealed some similarities to the precursor sequences of bradykinin potentiating peptides (BPP) and C-type natriuretic peptides (CNP), agents that are known to affect the cardiovascular system by acting on specific metalloproteases and receptors. The novel pHpG peptides found in Atheris venoms might also act on the cardiovascular system by inhibiting particular metalloproteases, which however remain to be identified.

A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles.

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.

Profiling and in vivo quantification of proteins by high resolution mass spectrometry: the example of goserelin, an analogue of luteinizing hormone-releasing hormone.

Proteins are essential biomolecules which are frequently involved in major pathological syndromes and are widely used as diagnostic markers or therapeutic agents. The emergence of proteomics will doubtless further increase the significance of proteins both in the clinic and in the life sciences in general. Our main objective is to offer innovative solutions to what we like to call the "post-proteomics era". To achieve our goal, we intend to develop novel approaches and technologies for in vivo metabolic studies of proteins using mass spectrometry (MS), focusing on pharmacokinetics and pharmacodynamics. Using goserelin as a model, we have successfully developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of an intact analogue of luteinizing hormone-releasing hormone (LHRH) in small volumes of rat plasma samples at concentrations ranging from 0.3 to 405.0 microg/l. To this end, a microbore reversed-phase-HPLC system was coupled on-line to a tandem high resolution quadrupole time-of-flight (Q-TOF) instrument fitted with an electrospray ion source and operated in LC-MS/MS mode. External calibration was used and the high resolution was crucial to discard contaminating signals, which would not have been possible with the more conventional triple quadrupole mass spectrometers operated in a static mode. For low sample amounts, calibration curves were constructed corresponding to rat plasma levels of 0.3 to 16.4 microg/l and found to be of third order with a coefficient of determination greater than 0.999. The relative standard deviation was found to be lower than 15%. A lower limit of detection (LLOD) of 0.17 microg/l and a lower limit of quantification (LLOQ) of 0.3 microg/l were determined.