Peptide ligand screening of α-synuclein aggregation modulators by in silico panning

<p>Abstract</p> <p>Background</p> <p>α-Synuclein is a Parkinson's-disease-related protein. It forms aggregates <it>in vivo</it>, and these aggregates cause cell cytotoxicity. Aggregation inhibitors are expected to reduce α-synuclein cytotoxicity, and an aggregation accelerator has recently been reported to reduce α-synuclein cytotoxicity. Therefore, amyloid aggregation modulating ligands are expected to serve as therapeutic medicines.</p> <p>Results</p> <p>We screened peptide ligands against α-synuclein by <it>in silico </it>panning, a method which we have proposed previously. In this study, we selected as the target a very hydrophobic region known as the amyloid-core-forming region. Since this region cannot be dissolved in water, it is difficult to carry out the <it>in vitro </it>screening of its peptide ligand. We carried out 6 rounds of <it>in silico </it>panning using a genetic algorithm and a docking simulation. After the <it>in silico </it>panning, we evaluated the top peptides screened <it>in silico </it>by <it>in vitro </it>assay. These peptides were capable of binding to α-synuclein.</p> <p>Conclusion</p> <p>We demonstrated that it is possible to screen α-synuclein-binding peptides by <it>in silico </it>panning. The screened peptides bind to α-synuclein, thus affecting the aggregation of α-synuclein.</p

Control of Aptamer Function Using Radiofrequency Magnetic Field

Remote control of aptamer function has allowed us to control protein function in space and time. Here, we propose a novel control system for aptamer function by radiofrequency magnetic field- (RFMF-) induced local heating of a gold nanoparticle conjugated with an aptamer. In this study, we used a 31-mer thrombin-binding aptamer (TBA), which can inhibit thrombin activity, as a model aptamer. We evaluated the RFMF control of the inhibitory activity of a gold nanoparticle-conjugated TBA. To evaluate the effect of RFMF on enzymatic activity, we utilized a complementary DNA strand that maintains the broken structure during the activity assay. We observed a decrease in the inhibitory activity of TBA after RFMF irradiation. It indicates that RFMF is capable of controlling the TBA structure. Because RFMF allows noninvasive control of aptamer function, this strategy is expected to be novel way of controlling aptamer drug activity

In silico panning for a non-competitive peptide inhibitor

BACKGROUND: Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs). In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH). RESULTS: The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs), which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with K(i )value of 20 μM. PQQGDH activity, in terms of the V(max )value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (K(D)) value was calculated as 60 μM by surface plasmon resonance (SPR) analysis. CONCLUSION: We demonstrate an effective methodology of in silico panning for the selection of a non-competitive peptide inhibitor from small virtual peptide library. This study is the first to demonstrate the usefulness of in silico evolution using experimental data. Our study highlights the usefulness of this strategy for structure-based screening of enzyme inhibitors

Development of engineered chromatic acclimation sensor with strict and reverse response to light signal, and application to optogenetic control in cyanobacteria

Genetic regulation and metabolic engineering enabled cyanobacteria to produce renewable chemical compounds from carbon dioxide via photosynthesis. Optogenetic control enables to precisely regulate the timing and level of gene expression without chemical inducer which is environment-hazardous. We recently developed a green-light regulated gene expression system in a model cyanobacterial strain Synechocystis sp. PCC6803 (hereafter PCC6803) [1] and a fast-growing marine cyanobacterial strain Synechococcus sp. NKBG15041c (hereafter NKBG15041c) [2] using a PCC6803-derived chromatic acclimation sensor, CcaS/CcaR two-component system [3]. However, the regulation of gene expression by CcaS is not strictly controllable and the background expression level under non-inductive condition is not negligible. Furthermore, altering the direction of gene expression, that is induction under red-light and repression under green-light, may expand its flexibility as one of the genetic tools. To obtain stricter and versatile system, we fabricated engineered CcaSs focusing on its domain structure using Escherichia coli expression system. One of the engineered CcaSs, CcaS#11, showed reverse response to light signal, i.e. inducible under red-light and strictly repressible under green-light [4]. To investigate the potential application and versatility of CcaS#11 as the red-light regulated gene expression system in cyanobacteria, we next introduced CcaS#11/CcaR two-component system and GFPuv as a probe of gene expression into PCC6803 after knocking out genomic CcaS/CcaR two-component system to exclude the interference. In this strain, the gene expression was induced under red-light and strictly repressed under green-light as we expected. Then, we applied this system to NKBG15041c. Similarly, red-light inducible gene expression with 2-fold higher ON/OFF ratio compared with the original system was successfully observed in NKBG15041c. Remarkably, there was no leaky expression under green-light, indicating that this system enables strict regulation of gene expression by light signal. In conclusion, we successfully constructed the engineered CcaS, CcaS#11, with strict and reverse response to light signal. Then we also confirmed its versatility and applicability as the red-light regulated gene expression system with strict regulation in cyanobacteria. Further development of the light regulated bioprocess will be expected using cyanobacterial hosts with this system, as a cell factory for the renewable chemical compounds production. [1] K. Abe et al., ‘Engineering of a Green-light Inducible Gene Expression System in Synechocystis sp. PCC 6803’, Microb. Biotechnol. 7 (2014) 177-183 [2] A. Badary et al., ‘The Development and Characterization of an Exogenous Green-light-regulated Gene Expression System in Marine Cyanobacteria’, Mar. Biotechnol. 17 (2015) 245-251 [3] Y. Hirose et al., ‘Cyanobacteriochrome CcaS is the Green Light Receptor That Induces the Expression of Phycobilisome Linker Protein’, Proc. Natl. Acad. Sci. USA 105 (2008) 9528-9533 [4] M. Nakajima et al., ‘Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal’, Sci. Rep. 6 (2016) 3759

An Amine-Reactive Phenazine Ethosulfate (arPES)—A Novel Redox Probe for Electrochemical Aptamer-Based Sensor

Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at −0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB

Development of an Anti-Idiotype Aptamer-Based Electrochemical Sensor for a Humanized Therapeutic Antibody Monitoring

Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1–100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands

Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method