Characterization of the Native Disulfide Isomers of the Novel χ-Conotoxin PnID: Implications for Further Increasing Conotoxin Diversity.

χ-Conotoxins are known for their ability to selectively inhibit norepinephrine transporters, an ability that makes them potential leads for treating various neurological disorders, including neuropathic pain. PnID, a peptide isolated from the venom of Conus pennaceus, shares high sequence homology with previously characterized χ-conotoxins. Whereas previously reported χ-conotoxins seem to only have a single native disulfide bonding pattern, PnID has three native isomers due to the formation of different disulfide bond patterns during its maturation in the venom duct. In this study, the disulfide connectivity and three-dimensional structure of these disulfide isomers were explored using regioselective synthesis, chromatographic coelution, and solution-state nuclear magnetic resonance spectroscopy. Of the native isomers, only the isomer with a ribbon disulfide configuration showed pharmacological activity similar to other χ-conotoxins. This isomer inhibited the rat norepinephrine transporter (IC50 = 10 ± 2 µM) and has the most structural similarity to previously characterized χ-conotoxins. In contrast, the globular isoform of PnID showed more than ten times less activity against this transporter and the beaded isoform did not display any measurable biological activity. This study is the first report of the pharmacological and structural characterization of an χ-conotoxin from a species other than Conus marmoreus and is the first report of the existence of natively-formed conotoxin isomers.

Research into the Bioengineering of a Novel α-Conotoxin from the Milked Venom of Conus obscurus.

The marine cone snail produces one of the fastest prey strikes in the animal kingdom. It injects highly efficacious venom, often causing prey paralysis and death within seconds. Each snail has hundreds of conotoxins, which serve as a source for discovering and utilizing novel analgesic peptide therapeutics. In this study, we discovered, isolated, and synthesized a novel α3/5-conotoxins derived from the milked venom of Conus obscurus (α-conotoxin OI) and identified the presence of α-conotoxin SI-like sequence previously found in the venom of Conus striatus. Five synthetic analogs of the native α-conotoxin OI were generated. These analogs incorporated single residue or double residue mutations. Three synthetic post-translational modifications (PTMs) were synthetically incorporated into these analogs: N-terminal truncation, proline hydroxylation, and tryptophan bromination. The native α-conotoxin OI demonstrated nanomolar potency in Poecilia reticulata and Homosapiens muscle-type nicotinic acetylcholine receptor (nAChR) isoforms. Moreover, the synthetic α-[P9K] conotoxin OI displayed enhanced potency in both bioassays, ranging from a 2.85 (LD50) to 18.4 (IC50) fold increase in comparative bioactivity. The successful incorporation of PTMs, with retention of both potency and nAChR isoform selectivity, ultimately pushes new boundaries of peptide bioengineering and the generation of novel α-conotoxin-like sequences.

Mechanisms of Herb-Drug Interactions Involving Cinnamon and CYP2A6: Focus on Time-Dependent Inhibition by Cinnamaldehyde and 2-Methoxycinnamaldehyde.

Information is scarce regarding pharmacokinetic-based herb-drug interactions (HDI) with trans-cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), components of cinnamon. Given the presence of cinnamon in food and herbal treatments for various diseases, HDIs involving the CYP2A6 substrates nicotine and letrozole with MCA (KS = 1.58 µM; Hill slope = 1.16) and CA were investigated. The time-dependent inhibition (TDI) by MCA and CA of CYP2A6-mediated nicotine metabolism is a complex process involving multiple mechanisms. Molecular dynamic simulations showed that CYP2A6's active site accommodates two dynamic ligands. The preferred binding orientations for MCA and CA were consistent with the observed metabolism: epoxidation, O-demethylation, and aromatic hydroxylation of MCA and cinnamic acid formation from CA. The percent remaining activity plots for TDI by MCA and CA were curved, and they were analyzed with a numerical method using models of varying complexity. The best-fit models support multiple inactivator binding, inhibitor depletion, and partial inactivation. Deconvoluted mass spectra indicated that MCA and CA modified CYP2A6 apoprotein with mass additions of 156.79 (142.54-171.04) and 132.67 (123.37-141.98), respectively, and it was unaffected by glutathione. Heme degradation was observed in the presence of MCA (48.5% ± 13.4% loss; detected by liquid chromatography-tandem mass spectrometry). In the absence of clinical data, HDI predictions were made for nicotine and letrozole using inhibition parameters from the best-fit TDI models and parameters scaled from rats. Predicted area under the concentration-time curve fold changes were 4.29 (CA-nicotine), 4.92 (CA-letrozole), 4.35 (MCA-nicotine), and 5.00 (MCA-letrozole). These findings suggest that extensive exposure to cinnamon (corresponding to ≈ 275 mg CA) would lead to noteworthy interactions. SIGNIFICANCE STATEMENT: Human exposure to cinnamon is common because of its presence in food and cinnamon-based herbal treatments. Little is known about the risk for cinnamaldehyde and methoxycinnamaldehyde, two components of cinnamon, to interact with drugs that are eliminated by CYP2A6-mediated metabolism. The interactions with CYP2A6 are complex, involving multiple-ligand binding, time-dependent inhibition of nicotine metabolism, heme degradation, and apoprotein modification. An herb-drug interaction prediction suggests that extensive exposure to cinnamon would lead to noteworthy interactions with nicotine.

Incorporation of post-translational modified amino acids as an approach to increase both chemical and biological diversity of conotoxins and conopeptides.

Bioactive peptides from Conus venom contain a natural abundance of post-translational modifications that affect their chemical diversity, structural stability, and neuroactive properties. These modifications have continually presented hurdles in their identification and characterization. Early endeavors in their analysis relied on classical biochemical techniques that have led to the progressive development and use of novel proteomic-based approaches. The critical importance of these post-translationally modified amino acids and their specific assignment cannot be understated, having impact on their folding, pharmacological selectivity, and potency. Such modifications at an amino acid level may also provide additional insight into the advancement of conopeptide drugs in the quest for precise pharmacological targeting. To achieve this end, a concerted effort between the classical and novel approaches is needed to completely elucidate the role of post-translational modifications in conopeptide structure and dynamics. This paper provides a reflection in the advancements observed in dealing with numerous and multiple post-translationally modified amino acids within conotoxins and conopeptides and provides a summary of the current techniques used in their identification.

Training fellows for retinopathy of prematurity care: a Web-based survey.

PURPOSE: To characterize the training received by pediatric ophthalmology and retina fellows in retinopathy of prematurity (ROP) management. METHODS: Pediatric ophthalmology and retina fellowship programs were emailed a Web-based survey to assess fellowship training in ROP management. RESULTS: Of 140 programs contacted, 42 (30%) participated, resulting in 87 surveys for analysis. Of the 87 respondents, 25 (29%) reported that two-thirds or less of ROP examinations performed by fellows were also seen by an attending. When stratified by specialty, this trend was statistically different between pediatric ophthalmology and retina fellows (P = 0.03). Additionally, pediatric ophthalmology fellows performed fewer laser photocoagulation procedures than retina fellows (P < 0.001). Regarding fellows' perceived competency in ROP management, 3 of 51 (6%) felt competent at the start of their fellowship and 43 of 51 (84%) felt competent at the time of the survey. Only 7% of respondents reported the use of formal evaluations at their programs to assess fellow competence in ROP examination. CONCLUSIONS: Training programs for fellows in pediatric ophthalmology and retina vary greatly with respect to ROP training and the quality of clinical care. Many clinical ROP examinations are being performed by pediatric ophthalmology and retina fellows without involvement and/or direct supervision by attending ophthalmologists. Our findings have important implications for the development of a future workforce for ROP management.

Accuracy of retinopathy of prematurity image-based diagnosis by pediatric ophthalmology fellows: implications for training.

PURPOSE: To measure the accuracy of image-based retinopathy of prematurity (ROP) diagnosis by pediatric ophthalmology fellows. METHODS: This was a comparative case series of expert versus nonexpert clinicians in image-based ROP diagnosis. An atlas of 804 retinal images was captured from 248 eyes of 67 premature infants with a wide-angle camera (RetCam-II, Clarity Medical Systems, Pleasanton, CA). Images were uploaded to a study website from which an expert pediatric retinal specialist and five pediatric ophthalmology fellows independently provided a diagnosis (no ROP, mild ROP, type 2 ROP, or treatment-requiring ROP) for each eye. Two different retinal specialists experienced in ROP examination served as additional controls. Primary outcome measures were sensitivity and specificity of image-based ROP diagnosis by fellows compared to a reference standard of image-based interpretation by the expert pediatric retinal specialist. Secondary outcome measure was intraphysician reliability. RESULTS: For detection of mild or worse ROP, the mean (range) sensitivity among the five fellows was 0.850 (0.670-0.962) and specificity was 0.919 (0.832-0.964). For detection of type 2 or worse ROP by fellows, mean (range) sensitivity was 0.527 (0.356-0.709) and specificity was 0.938 (0.777-1.000). For detection of treatment-requiring ROP, mean (range) sensitivity was 0.515 (0.267-0.765) and specificity was 0.949 (0.805-1.00). CONCLUSIONS: Pediatric ophthalmology fellows in this study demonstrated high diagnostic specificity in image-based ROP diagnosis; however, sensitivity was lower, particularly for clinically significant disease.