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

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

Selective inhibition of miR-21 by phage display screened peptide

miRNAs are nodal regulators of gene expression and deregulation of miRNAs is causally associated with different diseases, including cancer. Modulation of miRNA expression is thus of therapeutic importance. Small molecules are currently being explored for their potential to downregulate miRNAs. Peptides have shown to have better potency and selectivity toward their targets but their potential in targeting and modulating miRNAs remain unexplored. Herein, using phage display we found a very selective peptide against pre-miR-21. Interestingly, the peptide has the potential to downregulate miR-21, by binding to pre-miR-21 and hindering Dicer processing. It is selective towards miR-21 inside the cell. By antagonising miR-21 function, the peptide is able to increase the expression of its target proteins and thereby increase apoptosis and suppress cell proliferation, invasion and migration. This peptide can further be explored for its anti-cancer activity in vivo and may be even extended to clinical studies

Development of G9a-targeted BioPROTACs based on peptides and nanobodies

This project arose motivated by the high incidence that neoplastic pathologies have nowadays, and therefore by the need that our society has to find a solution against them. The main objective of this research was to identify specific peptides and nanobodies against G9a methyltransferase, whose role in cancer is to introduce epigenetic modifications to silence tumor suppressor genes. The strategy followed focuses on degrading this enzyme through the Biological Proteolysis Targeting Chimera (BioPROTAC) system, made up of either the aforementioned peptides or nanobodies. A search for these specific molecules was carried out using phage display, to find the most specific candidates against the previously produced and purified G9a. Later, the binding affinity of the selected clones was evaluated, the BioPROTAC construct was designed, and immunocytochemistry (ICQ) was performed to check their cellular internalization. In short, it was possible to select 3 peptide clones and 16 nanobodies with a good affinity for G9a, in addition to designing a system capable of being introduced into the tumor cell. This represents a project of Targeted Protein Degradation (TPD) for drug discovery with its main objectives met. Furthermore, this study will facilitate future investigations aimed at confirming the efficacy of the system to degrade the enzyme, in order to conduct additional testing of its therapeutic effect

Novel applications of shotgun phage display

In a shotgun phage display library, theoretically, the entire proteome of a bacterium is represented. Phages displaying specific polypeptides can be isolated by affinity selection, while the corresponding gene remains physically linked to the gene product. The overall objective of the study in this thesis was to explore the shotgun phage display technique in new areas. Initially, it was used to study interactions between Staphylococcus aureus and an in vivo coated biomaterial. It was shown to be well suited for the identification of bacterial proteins that bind to ex vivo central venous catheters. Several known interactions were detected, but it was also found that β2-glycoprotein I (β2-GPI) is deposited on this type of biomaterial – a finding that is of interest both for the adherence of S. aureus, but perhaps also in view of the occurrence of autoantibodies in certain autoimmune diseases. Further, it is of interest to identify the subset of extracellular proteins in a bacterium since they are involved in important functions like pathogenesis and symbiosis. A method that allows for the rapid and general isolation of extracellular proteins is desirable, and may prove particularly useful when applied to bacteria for which the genome sequences are not known. For this purpose, a specialised phage display method was developed to isolate extracellular proteins by virtue of the presence of signal peptides (SS phage display). It was successfully applied to S. aureus and, on a larger scale, to the symbiotic bacterium Bradyrhizobium japonicum. In elaboration of the SS phage display method, an inducible antisense RNA system was incorporated to enable gene silencing of the isolated genes. A tetracycline-regulated promoter was inserted in such a way, that an antisense RNA covering the cloned gene could be expressed. The new element was shown to be compatible with the properties of SS phage display, and to promote gene expression upon induction on both the transcriptional and translational level. However, screening for clones affected by the induction of antisense RNA transcription was unsuccessful, and further developments of the system are required to improve the efficiency of this attractive application

Computational Evolution Protocol for Peptide Design

Computational peptide design is useful for therapeutics, diagnostics, and vaccine development. To select the most promising peptide candidates, the key is describing accurately the peptide-target interactions at the molecular level. We here review a computational peptide design protocol whose key feature is the use of all-atom explicit solvent molecular dynamics for describing the different peptide-target complexes explored during the optimization. We describe the milestones behind the development of this protocol, which is now implemented in an open-source code called PARCE. We provide a basic tutorial to run the code for an antibody fragment design example. Finally, we describe three additional applications of the method to design peptides for different targets, illustrating the broad scope of the proposed approach

Phage Display-Based Nanotechnology Applications in Cancer Immunotherapy

Phage display is a nanotechnology with limitless potential, first developed in 1985 and still awaiting to reach its peak. Awarded in 2018 with the Nobel Prize for Chemistry, the method allows the isolation of high-affinity ligands for diverse substrates, ranging from recombinant proteins to cells, organs, even whole organisms. Personalized therapeutic approaches, particularly in oncology, depend on the identification of new, unique, and functional targets that phage display, through its various declinations, can certainly provide. A fast-evolving branch in cancer research, immunotherapy is now experiencing a second youth after being overlooked for years; indeed, many reports support the concept of immunotherapy as the only non-surgical cure for cancer, at least in some settings. In this review, we describe literature reports on the application of peptide phage display to cancer immunotherapy. In particular, we discuss three main outcomes of this procedure: (i) phage display-derived peptides that mimic cancer antigens (mimotopes) and (ii) antigen-carrying phage particles, both as prophylactic and/or therapeutic vaccines, and (iii) phage display-derived peptides as small-molecule effectors of immune cell functions. Preclinical studies demonstrate the efficacy and vast potential of these nanosized tools, and their clinical application is on the way

Studies on the immunosuppressive effects and detection of naturally-occuring toxins

Episodes of toxin-producing phytoplankton occur worldwide, causing both animal and human fatalities. Toxicity occurs through consumption of phycotoxins, including azaspiracid, which accumulate in filter-feeding shellfish. Microcystins are hepatotoxins, produced mainly by freshwater cyanobacteria. Aflatoxins are potent, fungal hepatocarcinogens, which occur mainly in food and feed products. The purpose of this research was to examine the cytotoxic and immunosuppressive effects of aflatoxins (B1, B2 and G1), azaspiracid-1 and microcystin-LR in vitro, using the murine macrophage cell line, J774A.1. The results clearly demonstrated that azaspiracid and microcystin had a significant effect on host defence functions, through deregulation of IL-6, IL-10, IL12p40 and TNF-α cytokine expression. Microcystin exposure significantly decreased IL-1β expression. ‘Toll-like’ receptor (TLR2 and CD14) expression was altered following aflatoxin exposure, while apoptotic marker (caspase-1) expression was affected following microcystin exposure. This knowledge should be taken into consideration in the implementation of detection limits, aimed at minimising risks to human health through toxin exposure. Increased awareness of the hazards presented by toxins led to the requirement for recombinant antibodies for these targets, for incorporation into sensitive detection immunoassays. This thesis describes the production of leprine and avian immune libraries for azaspiracid and microcystin, respectively. Attempts were made to isolate azaspiracid-specific antibodies with little success. Phage display was utilised to successfully isolate two single chain antibody fragments (scFvs) to microcystin from the avian library. Error-prone PCR resulted in the isolation of a mutant clone which displayed a 2.3-fold improvement in sensitivity by ELISA, with an LOD of 1.4 ng/mL. The mutant scFv displayed an altered cross-reactivity profile to the microcystin variants tested using Biacore™ inhibition analysis. The recombinant antibodies were successfully applied to the development of fluorescence-based immunoassay formats. The biotinylated mutant scFv was incorporated into a slide-based assay format on a functionalised glass substrate (IC50 ~ 1 µg/L). This assay had the potential to accurately detect microcystin and its variants, below the regulatory limit of 1 µg/L. The application of these highly-sensitive recombinant antibodies into rapid and inexpensive fluorescence detection systems could aid in the development of an early warning system for toxin outbreaks

Phage display and experimental brain therapeutics

Phage display, a powerful polypeptide display technology, affords the rapid identification of peptides and proteins that interact with a target of interest The aims of the project were the phage display identification of peptides that interact with a druggable target in a brain disorder (glioblastoma multiforme) and the identification of peptides that serve as targeting vectors for brain delivery. Validation studies were undertaken to qualify the use of a cyclic 7-mer peptide phage library against targets including streptavidin and paracetamol chosen as examples of a large complex and small simple molecule, respectively. With the aim of identifying peptide phages that bind to the luminal surface of brain micro vasculature, a primary in-vitro porcine model of the blood-brain barrier (BBB) comprising primary brain capillary endothelial cells was established and characterised. An in-vivo phage display was undertaken in the rat with the aim of identifying peptide sequences that mediated translocation across the BBB into brain grey matter. A 7-mer cyclic peptide was identified with sequence AC-SYTSSTM-CGGGS that enhanced the uptake of phages into brain grey matter by 4-fold compared to control wild-type phages. This peptide may serve as a novel targeting vector for the delivery of a therapeutic cargo to the brain. Caveolin-1 was identified as a potential new therapeutic target in in-vitro models of grade IV astrocytomas (glioblastoma multiforme), with siRNA knockdown of caveolin-1 associated with reduced glioma cell proliferation and invasiveness. With the caveolin-1 scaffolding domain (aa 81-101 in the caveolin-1 protein) as a target, an in-vitro peptide phage selection was undertaken and identified a series of peptides that bind the scaffolding domain with high affinity. These peptides will serve as a template for the development of low molecular weight peptidomimetics that inhibit caveolin-1 function. In conclusion, the studies in this thesis have demonstrated the utility of phage display in experimental therapeutics of brain disorders.EThOS - Electronic Theses Online ServiceGBUnited Kingdo