Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries.

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target-focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase-focused library by chemically modifying helix-loop-helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip-3 retained the α-helical structure and bound to AurA with a KD value of 13.7 µM. Bip-3 and the adenosine-tethered peptide Bip-3-Adc provided IC50 values of 103 µM and 7.7 µM, respectively, suggesting that Bip-3-Adc bivalently inhibited AurA. In addition, the selectivity of Bip-3-Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase-focused library of phage-displayed HLH peptides.

Addressing the Analytical Challenges for the Detection of Ciguatoxins Using an Electrochemical Biosensor.

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 µg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

Highly Sensitive and Practical Fluorescent Sandwich ELISA for Ciguatoxins.

Ciguatera fish poisoning (CFP) caused by the consumption of fish that have accumulated ciguatoxins (CTXs) affects more than 50000 people annually. The spread of CFP causes enormous damage to public health, fishery resources, and the economies of tropical and subtropical endemic regions. The difficulty in avoiding CFP arises from the lack of sensitive and reliable analytical methods for the detection and quantification of CTXs in contaminated fish, along with the normal appearance, smell, and taste of fish contaminated with the causative toxins. Thus, an accurate, sensitive, routine, and portable detection method for CTXs is urgently required. We have successfully developed a highly sensitive fluorescent sandwich ELISA, which can detect, differentiate, and quantify four major CTX congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B) with a detection limit of less than 1 pg/mL. The ELISA protocol, using one microtiter plate coated with two mAbs (10C9 and 3G8), and ALP-linked 8H4, can detect any of the four CTX congeners in a single operation. CTX1B spiked into fish at the FDA guidance level of 0.01 ppb CTX1B equivalent toxicity in fish from Pacific regions was also proven to be reliably detected by this ELISA. Furthermore, the efficiency of extraction/purification procedures and the matrix effect of contaminants in fish were evaluated in detail, since pretreatment and matrix effects are critical for ELISA analysis.

Structural basis of the broad substrate tolerance of the antibody 7B9-catalyzed hydrolysis of p-nitrobenzyl esters.

Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (AsnH33, SerH95, and ArgL96) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (AsnH33Ala, SerH95Ala, and ArgL96Ala) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified the structural basis of the observed broad substrate tolerance of antibody 7B9-catalyzed hydrolyses. Our findings provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates, aiding their practical application in synthetic organic chemistry.

Preparation of anti-ciguatoxin monoclonal antibodies using synthetic haptens: sandwich ELISA detection of ciguatoxins.

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

Humanization and characterization of an anti-ciguatoxin CTX3C monoclonal antibody.

Ciguatera poisoning (CP), caused by ciguatoxins (CTXs), is one of the most common food-borne diseases, affecting more than 50,000 people each year. In most cases, CP are managed with symptomatic and supportive remedies, and no specific treatment has been devised. In this study, toward the development of therapeutic antibodies for CP, we examined to humanize mouse anti-CTX3C antibody 10C9 (m10C9), which exhibited neutralizing activity against ciguatoxin in vitro and in vivo. The complementarity determining regions were grafted onto a human germline sequence with high sequence identity to m10C9, and the backmutations were examined to maintain the binding affinity. The optimized humanized antibody, Opt.h10C9Fab, showed a strong binding affinity to CTX3C with a high affinity (KD = 19.0 nM), and only two backmutations of ArgL46 and CysH94 in the framework regions were involved in determining the antigen binding affinity.

Sensitive Detection of Ciguatoxins Using a Neuroblastoma Cell-Based Assay with Voltage-Gated Potassium Channel Inhibitors.

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 µg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.

Antibody-catalyzed decarboxylation and aldol reactions using a primary amine molecule as a functionalized small nonprotein component.

Catalytic antibody 27C1 bears binding sites for both a substrate- and a functionalized small nonprotein component in the active site. We investigated the possibility of exploiting imine and enamine intermediates using a primary amine molecule into the active site of antibody 27C1. The antibody catalyzed ß-keto acid decarboxylation with a rate enhancement (kcat/Km/kuncat) of 140,000, as well as highly regioselective cross-aldol reactions of ketones and p-nitrobenzaldehyde. These studies provide new strategies for the generation of catalytic antibodies possessing binding sites for functionalized components.

Evaluation of relative potency of calibrated ciguatoxin congeners by near-infrared fluorescent receptor binding and neuroblastoma cell-based assays.

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.

A smartphone-controlled amperometric immunosensor for the detection of Pacific ciguatoxins in fish.

Ciguatoxins (CTXs) are marine neurotoxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. CTXs may reach humans through food webs and cause ciguatera fish poisoning (CFP). An immunosensor for the detection of Pacific CTXs in fish was developed using multiwalled carbon nanotube (MWCNT)-modified carbon electrodes and a smartphone-controlled potentiostat. The biosensor attained a limit of detection (LOD) and a limit of quantification (LOQ) of 6 and 27 pg/mL of CTX1B, respectively, which were 0.001 and 0.005 µg/kg in fish flesh. In the analysis of fish samples from Japan and Fiji, excellent correlations were found with sandwich enzyme-linked immunosorbent assays (ELISAs), a cell-based assay (CBA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Stability of at least 3 months at -20 °C was predicted. In just over 2 h, the biosensor provides reliable, accurate and precise Pacific CTX contents in fish extracts, being suitable for monitoring and research programs.

Cell immunolocalization of ciguatoxin-like compounds in the benthic dinoflagellate Gambierdiscus australes M. Chinain & M.A. Faust by confocal microscopy.

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are able to produce potent neurotoxins like ciguatoxins (CTXs), which, after biooxidation in fish, are responsible for ciguatera intoxication. An isolate of G. australes from the Canary Islands, that revealed the presence of CTX-like compounds by immunosensing tools, was studied by immunocytochemistry to localize intracellular CTX-like compounds, using 8H4 monoclonal antibody that specifically recognizes the right wing of CTX1B and CTX3C analogues. Confocal microscopy observations of immunostained whole cells revealed a strong positive reaction on cell surface and all along the cell outline, while no reaction was detected inside the cells, probably because the antibody was not able to pass through thecal plates. Cell sections showed a positive antibody staining not only on thecal plates, but also inside cytoplasm, with numerous small dots and larger tubule-like reticulate structures. Small fluorescent dots were detected also on the nuclear surface. These observations indicate that CTX-like compounds are present in G. australes cytoplasm, and then are, at least in part, released to cover the cell surface.

Electrochemical biosensor for the dual detection of Gambierdiscus australes and Gambierdiscus excentricus in field samples. First report of G. excentricus in the Balearic Islands.

Several genera of marine dinoflagellates are known to produce bioactive compounds that affect human health. Among them, Gambierdiscus and Fukuyoa stand out for their ability to produce several toxins, including the potent neurotoxic ciguatoxins (CTXs), which accumulate through the food web. Once fishes contaminated with CTXs are ingested by humans, it can result in an intoxication named ciguatera. Within the two genera, only some species are able to produce toxins, and G. australes and G. excentricus have been highlighted to be the most abundant and toxic. Although the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, their presence in subtropical and temperate regions has been recently recorded. In this work, the combined use of species-specific PCR primers for G. australes and G. excentricus modified with short oligonucleotide tails allowed the development of a multiplex detection system for these two toxin-producing species. Simultaneous detection was achieved using capture probes specific for G. australes and G. excentricus immobilized on maleimide-coated magnetic beads (MBs), separately placed on the working electrodes of a dual electrode array. Additionally, a rapid DNA extraction technique based on a portable bead beater system and MBs was developed, significantly reducing the extraction time (from several hours to 30 min). The developed technique was able to detect as low as 10 cells of both Gambierdiscus species and allowed the first detection of G. excentricus in the Balearic Islands in 8 out of the 12 samples analyzed. Finally, field samples were screened for CTXs with an immunosensor, successfully reporting 13.35 ± 0.5 pg CTX1B equiv. cell-1 in one sample and traces of toxins in 3 out of the 9 samples analyzed. These developments provide rapid and cost-effective strategies for ciguatera risk assessment, with the aim of guaranteeing seafood safety.

Contribution of the trifluoroacetyl group in the thermodynamics of antigen-antibody binding.

We analyzed the binding of the 7C8 antibody to the chloramphenicol phosphonate antigens-one containing a trifluoroacetyl group (CP-F) and the other containing an acetyl group (CP-H)-by using isothermal titration calorimetry (ITC). The thermodynamic difference due to the substitution of F by H was evaluated using free energy calculations based on molecular dynamics (MD) simulations. We have previously shown that another antibody, namely, 6D9, binds more weakly to CP-H than to CP-F, mainly due to the different hydration free energies of the dissociated state and not due to the unfavorable hydrophobic interactions with the antibody in the bound state. Unlike in the binding of the trifluoroacetyl group with 6D9, in its binding with 7C8, it is exposed to the solvent, as seen in the crystal structure of the complex of 7C8 with CP-F. The thermodynamic analysis performed in this study showed that the binding affinity of 7C8 for CP-H is similar to that for CP-F, but this binding to CP-H is accompanied with less favorable enthalpy and more favorable entropy changes. The free energy calculations indicated that, upon the substitution of F by H, enthalpy and entropy changes in the associated and dissociated states were decreased, but the magnitude of enthalpy and entropy changes in the dissociated state was larger than that in the associated state. The differences in binding free energy, enthalpy, and entropy changes determined by the free energy calculations for the substitution of F by H are in good agreement with the experimental results.

Selection of inhibitory peptides for Aurora-A kinase from a phage-displayed library of helix-loop-helix peptides.

Conformationally constrained peptide libraries have been made by grafting randomized amino acid sequences onto a rigid scaffold derived from natural proteins. Here, as a library scaffold, we propose a de novo designed helix-loop-helix motif. We constructed a peptide library of the loop region and screened against Aurora-A, which is a member of the Aurora family of serine/threonine protein kinases, to successfully isolate the inhibitory peptides. A semi-rational strategy, which combines phage-displayed libraries and de novo designed peptides, would provide a new way to generate selective peptide inhibitors for the protein kinase family.

Molecular design of small organic molecules based on structural information for a conformationally constrained peptide that binds to G-CSF receptor.

Based on structural information for a peptide (P8-2KAQ) that binds to granulocyte-colony stimulating factor receptor (G-CSFR), small ligands with a biaryl scaffold were designed and their binding affinities were evaluated.

Further advance of Gambierdiscus Species in the Canary Islands, with the First Report of Gambierdiscus belizeanus.

Ciguatera Poisoning (CP) is a human food-borne poisoning that has been known since ancient times to be found mainly in tropical and subtropical areas, which occurs when fish or very rarely invertebrates contaminated with ciguatoxins (CTXs) are consumed. The genus of marine benthic dinoflagellates Gambierdiscus produces CTX precursors. The presence of Gambierdiscus species in a region is one indicator of CP risk. The Canary Islands (North Eastern Atlantic Ocean) is an area where CP cases have been reported since 2004. In the present study, samplings for Gambierdiscus cells were conducted in this area during 2016 and 2017. Gambierdiscus cells were isolated and identified as G. australes, G. excentricus, G. caribaeus, and G. belizeanus by molecular analysis. In this study, G. belizeanus is reported for the first time in the Canary Islands. Gambierdiscus isolates were cultured, and the CTX-like toxicity of forty-one strains was evaluated with the neuroblastoma cell-based assay (neuro-2a CBA). G. excentricus exhibited the highest CTX-like toxicity (9.5-2566.7 fg CTX1B equiv. cell-1) followed by G. australes (1.7-452.6.2 fg CTX1B equiv. cell-1). By contrast, the toxicity of G. belizeanus was low (5.6 fg CTX1B equiv. cell-1), and G. caribaeus did not exhibit CTX-like toxicity. In addition, for the G. belizeanus strain, the production of CTXs was evaluated with a colorimetric immunoassay and an electrochemical immunosensor resulting in G. belizeanus producing two types of CTX congeners (CTX1B and CTX3C series congeners) and can contribute to CP in the Canary Islands.

A single antibody catalyzes multiple chemical transformations upon replacement of the functionalized small nonprotein components.

A single antibody catalyzes multiple chemical transformations upon replacement of the funcctionalized small nonportein components.

Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins.

"Ciguatera" fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb.

Selection of a carbohydrate-binding domain with a helix-loop-helix structure.

We obtained a novel carbohydrate-binding peptide having a helix-loop-helix scaffold from a random peptide library. The helix-loop-helix peptide library randomized at five amino acid residues was displayed on the major coat protein of a filamentous phage. Affinity selection with a ganglioside, Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1), gave positive phage clones. Surface plasmon resonance spectroscopy showed that a corresponding 35-mer synthetic peptide had high affinity for GM1 with a dissociation constant of 0.24 microM. This peptide preferentially binds to GM1 rather than asialo GM1 and GM2, suggesting that a terminal galactose and sialic acid are required for the binding as for cholera toxin. Circular dichroism spectroscopic studies indicated that a helical structure is important for the affinity and specificity. Furthermore, alanine scanning at randomized positions showed that arginine and phenylalanine play an especially important role in the recognition of carbohydrates. Such a de novo helix-loop-helix peptide would be available for the design of carbohydrate-binding proteins.

Thermodynamic and structural basis for transition-state stabilization in antibody-catalyzed hydrolysis.

Catalytic antibodies 6D9 and 9C10, which were induced by immunization with a haptenic transition-state analog (TSA), catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. These antibodies stabilize the transition state to catalyze the hydrolysis reaction, strictly according to the theoretical relationship: for 6D9, k(cat)/k(uncat)=895 and K(S)/K(TSA)=900, and for 9C10, k(cat)/k(uncat)=56 and K(S)/K(TSA)=60. To elucidate the molecular basis of the antibody-catalyzed reaction, the crystal structure of 6D9 was determined, and the binding thermodynamics of 6D9 and 9C10 with both the substrate and the TSA were analyzed using isothermal titration calorimetry. The crystal structure of the unliganded 6D9 Fab was determined at 2.25 A resolution and compared with that of the TSA-liganded 6D9 Fab reported previously, showing that the TSA is bound into the hydrophobic pocket of the antigen-combining site in an "induced fit" manner, especially at the L1 and H3 CDR loops. Thermodynamic analyses showed that 6D9 binds the substrate of the TSA with a positive DeltaS, differing from general thermodynamic characteristics of antigen-antibody interactions. This positive DeltaS could be due to the hydrophobic interactions between 6D9 and the substrate or the TSA mediated by Trp H100i. The difference in DeltaG between substrate and TSA-binding to 6D9 was larger than that to 9C10, which is in good correlation with the larger k(cat) value of 6D9. Interestingly, the DeltaDeltaG was mainly because of the DeltaDeltaH. The correlation between k(cat) and DeltaDeltaH is suggestive of "enthalpic strain" leading to destabilization of antibody-substrate complexes. Together with X-ray structural analyses, the thermodynamic analyses suggest that upon binding the substrate, the antibody alters the conformation of the ester moiety in the substrate from the planar Z form to a thermodynamically unstable twisted conformation, followed by conversion into the transition state. Enthalpic strain also contributes to the transition-state stabilization by destabilizing the ground state, and its degree is much larger for the more efficient catalytic antibody, 6D9.