Computational Design of α-Conotoxins to Target Specific Nicotinic Acetylcholine Receptor Subtypes.

Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α-conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high-resolution experimental structures, which are not widely available for α-conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α-conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α-conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α-conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype-specific nAChR ligands and potentially accelerated drug development.

Mutations within the selectivity filter reveal that Kv1 channels have distinct propensities to slow inactivate.

Voltage-activated potassium (Kv) channels open in response to membrane depolarization and subsequently inactivate through distinct mechanisms. For the model Shaker Kv channel from Drosophila, fast N-type inactivation is thought to occur by a mechanism involving blockade of the internal pore by the N-terminus, whereas slow C-type inactivation results from conformational changes in the ion selectivity filter in the external pore. Kv channel inactivation plays critical roles in shaping the action potential and regulating firing frequency, and has been implicated in a range of diseases including episodic ataxia and arrhythmias. Although structures of the closely related Shaker and Kv1.2 channels containing mutations that promote slow inactivation both support a mechanism involving dilation of the outer selectivity filter, mutations in the outer pores of these two Kv channels have been reported to have markedly distinct effects on slow inactivation, raising questions about the extent to which slow inactivation is related in both channels. In this study, we characterized the influence of a series of mutations within the external pore of Shaker and Kv1.2 channels and observed many distinct mutant phenotypes. We find that mutations at four positions near the selectivity filter promote inactivation less dramatically in Kv1.2 when compared to Shaker, and they identify one key variable position (T449 in Shaker and V381 in Kv1.2) underlying the different phenotypes in the two channels. Collectively, our results suggest that Kv1.2 is less prone to inactivate compared to Shaker, yet support a common mechanism of inactivation in the two channels.

ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (CaV 2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABAB receptor activation.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABAB receptors (GABAB R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAB R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAB R1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAB R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K+ currents mediated by human GIRK1/2 channels co-expressed with GABAB R in HEK293T cells. This study highlights GABAB R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.

Interactions of Globular and Ribbon [γ4E]GID with α4ß2 Neuronal Nicotinic Acetylcholine Receptor.

The α4ß2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson's and Alzheimer's diseases. Designing α4ß2 nAChR selective inhibitors could help define the role of the α4ß2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4ß2 nAChR through competitive inhibition of the α4(+)ß2(-) or α4(+)α4(-) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3ß2, α4ß2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4ß2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4ß2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)3(ß2)2 nAChR was deduced using the information from the cryo-electron structure of (α4)3(ß2)2 nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(-) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)3(ß2)2 nAChR.

Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation - Suggestion of a Binding Mode at the α7 nAChR.

Peptides derived from animal venoms provide important research tools for biochemical and pharmacological characterization of receptors, ion channels, and transporters. Some venom peptides have been developed into drugs (such as the synthetic ω-conotoxin MVIIA, ziconotide) and several are currently undergoing clinical trials for various clinical indications. Challenges in the development of peptides include their usually limited supply from natural sources, cost-intensive chemical synthesis, and potentially complicated stereoselective disulfide-bond formation in the case of disulfide-rich peptides. In particular, if extended structure-function analysis is performed or incorporation of stable isotopes for NMR studies is required, the comparatively low yields and high costs of synthesized peptides might constitute a limiting factor. Here we investigated the expression of the 4/7 α-conotoxin TxIA, a potent blocker at α3ß2 and α7 nicotinic acetylcholine receptors (nAChRs), and three analogs in the form of maltose binding protein fusion proteins in Escherichia coli. Upon purification via nickel affinity chromatography and release of the toxins by protease cleavage, HPLC analysis revealed one major peak with the correct mass for all peptides. The final yield was 1-2 mg of recombinant peptide per liter of bacterial culture. Two-electrode voltage clamp analysis on oocyte-expressed nAChR subtypes demonstrated the functionality of these peptides but also revealed a 30 to 100-fold potency decrease of expressed TxIA compared to chemically synthesized TxIA. NMR spectroscopy analysis of TxIA and two of its analogs confirmed that the decreased activity was due to an alternative disulfide linkage rather than the missing C-terminal amidation, a post-translational modification that is common in α-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the α7 nAChR, but not the α3ß2 subtype, the loss of potency could be rescued by an R5D substitution. In conclusion, we demonstrate efficient expression of functional but alternatively folded ribbon TxIA variants in E. coli and provide the first structure-function analysis for a ribbon 4/7-α-conotoxin at α7 and α3ß2 nAChRs. Computational analysis based on these data provide evidence for a ribbon α-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.

Stoichiometry dependent inhibition of rat α3ß4 nicotinic acetylcholine receptor by the ribbon isomer of α-conotoxin AuIB.

The ribbon isomer of α-conotoxin AuIB has 10-fold greater potency than the wild-type globular isomer at inhibiting nicotinic acetylcholine receptors (nAChRs) in rat parasympathetic neurons, and unlike its globular isoform, ribbon AuIB only targets a specific stoichiometry of the α3ß4 nAChR subtype. Previous electrophysiological recordings of AuIB indicated that ribbon AuIB binds to the α3(+)α3(-) interface within the nAChR extracellular domain, which is displayed by the (α3)3(ß4)2 stoichiometry but not by (α3)2(ß4)3. This specificity for a particular stoichiometry is remarkable and suggests that ribbon isoforms of α-conotoxins might have great potential in drug design. In this study, we investigated the binding mode and structure-activity relationships of ribbon AuIB using a combination of molecular modeling and electrophysiology recording to determine the features that underpin its selectivity. An alanine scan showed that positions 4 and 9 of ribbon AuIB are the main determinants of the interaction with (α3)3(ß4)2 nAChR. Our computational models indicate that the first loop of ribbon AuIB binds in the "aromatic box" of the acetylcholine orthosteric binding site, similar to that of globular AuIB. In contrast, the second loop and the termini of the ribbon isomer have different orientations and interactions in the binding sites to those of the globular isomer. The structure-activity relationships reported herein should be useful to design peptides displaying a ribbon α-conotoxin scaffold for inhibition of nAChR subtypes that have hitherto been difficult to selectively target.

Backbone cyclization of analgesic conotoxin GeXIVA facilitates direct folding of the ribbon isomer.

Conotoxin GeXIVA inhibits the α9α10 nicotinic acetylcholine receptor (nAChR) and is analgesic in animal models of pain. α-Conotoxins have four cysteines that can have three possible disulfide connectivities: globular (CysI-CysIII and CysII-CysIV), ribbon (CysI-CysIV and CysII-CysIII), or bead (CysI-CysII and CysIII-CysIV). Native α-conotoxins preferably adopt the globular connectivity, and previous studies of α-conotoxins have focused on the globular isomers as the ribbon and bead isomers typically have lower potency at nAChRs than the globular form. A recent report showed that the bead and ribbon isomers of GeXIVA are more potent than the globular isomer, with low nanomolar half-maximal inhibitory concentrations (IC50). Despite this high potency, the therapeutic potential of GeXIVA is limited, because like most peptides, it is susceptible to proteolytic degradation and is challenging to synthesize in high yield. Here we used backbone cyclization as a strategy to improve the folding yield as well as increase the serum stability of ribbon GeXIVA while preserving activity at the α9α10 nAChR. Specifically, cyclization of ribbon GeXIVA with a two-residue linker maintained the biological activity at the human α9α10 nAChR and improved stability in human serum. Short linkers led to selective formation of the ribbon disulfide isomer without requiring orthogonal protection. Overall, this study highlights the value of backbone cyclization in directing folding, improving yields, and stabilizing conotoxins with therapeutic potential.

Comparative study on acid-soluble and pepsin-soluble collagens from skin and swim bladder of grass carp (Ctenopharyngodon idella).

BACKGROUND: Collagen has a wide range of applications in food, biomedical and pharmaceutical products. RESULTS: The collagens in grass carp (Ctenopharyngodon idella) skin and swim bladder were extracted using acetic acid and pepsin. Higher yield of pepsin-soluble collagen (PSC) was obtained from skin (178 g kg(-1)) than from swim bladder (114 g kg(-1)). Not surprisingly, yields of PSC from skin and swim bladder were also higher than those of acid-soluble collagen (ASC) from the same organs (89 and 51 g kg(-1)). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that ASC and PSC were type I collagens, with PSC containing a higher proportion of α components than ASC. Fourier transform infrared spectra revealed that ASC and PSC were very similar in their protein secondary structures. Scanning electron micrographs showed that the collagens had a spongy structure, with more pores being obtained in swim bladder than in skin collagens. The collagens showed high solubility in the acidic pH range. However, their solubility decreased in the presence of NaCl at concentrations above 20 g kg(-1). CONCLUSION: Collagens were successfully extracted from the skin and swim bladder of grass carp. These fish by-products could serve as an alternative source of collagens for a wide variety of applications in the food and nutraceutical industries.

The neuroprotective and antioxidant activities of protein hydrolysates from grass carp (Ctenopharyngodon idella) skin.

To observe the neuroprotective and antioxidant activities of the grass carp protein hydrolysates (GPH) obtained from grass carp (Ctenopharyngodon idella) skin by enzymatic hydrolysis. GPH prepared using Protamex, at different (5, 10, 15, 20 and 30 %) degrees of hydrolysis (DH) were investigated. The DPPH radial scavenging, reducing power and inhibition of linoleic acid oxidation activities of GPH were significantly improved by a low DH (5 %) compared with those of GPH with a higher DH (p < 0.05). A low degree of enzymatic hydrolysis was appropriate to obtain GPH with improved neuroprotective activities. These results suggest that the control of the DH may be an effective strategy to modify specific neuroprotective and antioxidant activities of GPH, and GPH has potential as a functional food ingredient for related functional and health benefits.

Physicochemical responses and quality changes of red sea bream (Pagrosomus major) to gum arabic coating enriched with ergothioneine treatment during refrigerated storage.

The combined effects of gum arabic coating (GA) and ergothioneine (ER) treatment on the sensory and physicochemical characteristics of red sea bream (Pagrosomus major) stored at 4 ± 1 °C for 16 days were investigated. Fish proximate composition, pH value, total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), K-value, TCA-soluble peptides, colour, texture profile analyses (TPA), microbiological properties and sensory quality were measured. The results indicate that treatment with gum arabic and ergothioneine (GAER) retarded nucleotide breakdown, lipid oxidation, protein degradation, and reduced microbial growth compare with the control. The efficiency was better than that of GA or ER treatment. Sensory evaluation proved the efficacy of GAER coating by maintaining the overall quality of red sea bream during the storage period. Furthermore, GAER maintained better colour and textural characteristics. Our study suggests that GAER treatment has potential to improve the quality of red sea bream and extend its storage life.

Influence of disulfide connectivity on structure and bioactivity of α-conotoxin TxIA.

Cone snails express a sophisticated arsenal of small bioactive peptides known as conopeptides or conotoxins (CTxs). Through evolutionary selection, these peptides have gained the ability to interact with a range of ion channels and receptors, such as nicotinic acetylcholine receptors (nAChRs). Here, we used reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization-mass spectrometry (ESI-MS) to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction of C. textile crude venom potently blocked α3ß2 nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to α-CTx TxIA, an antagonist of α3ß2 nAChRs. Then three disulfide isoforms of α-CTx TxIA were synthesized and their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for α-CTx TxIA potency. Although both globular and ribbon isomers showed similar retention times in RP-HPLC, globular TxIA potently inhibited α3ß2 nAChRs with an IC50 of 5.4 nM, while ribbon TxIA had an IC50 of 430 nM. In contrast, beads isomer had little activity towards α3ß2 nAChRs. Two-step oxidation synthesis produced the highest yield of α-CTx TxIA native globular isomer, while a one-step production process based on random oxidation folding was not suitable. In summary, this study demonstrated the relationship between conotoxin activity and disulfide connectivity on α-CTx TxIA.

Optimal cleavage and oxidative folding of α-conotoxin TxIB as a therapeutic candidate peptide.

Alpha6beta2 nicotinic acetylcholine receptors (nAChRs) are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including addiction and Parkinson's disease. Alpha-conotoxin (α-CTx) TxIB is a uniquely selective ligand, which blocks α6/α3ß2ß3 nAChRs only, but does not block the other subtypes. Therefore, α-CTx TxIB is a valuable therapeutic candidate peptide. Synthesizing enough α-CTx TxIB with high yield production is required for conducting wide-range testing of its potential medicinal applications. The current study optimized the cleavage of synthesized α-CTx TxIB resin-bounded peptide and folding of the cleaved linear peptide. Key parameters influencing cleavage and oxidative folding of α-CTx TxIB were examined, such as buffer, redox agents, pH, salt, co-solvent and temperature. Twelve conditions were used for cleavage optimization. Fifty-four kinds of one-step oxidative solution were used to assess their effects on each α-CTx TxIB isomers' yield. The result indicated that co-solvent choices were particularly important. Completely oxidative folding of globular isomer was achieved when the NH4HCO3 or Tris-HCl folding buffer at 4 °C contained 40% of co-solvent DMSO, and GSH:GSSG (2:1) or GSH only with pH 8~8.7.

Expression and secretion of functional recombinant µO-conotoxin MrVIB-His-tag in Escherichia coli.

µO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.