Tetravalent Peptide Research Articles

A tetravalent peptide efficiently inhibits the intestinal toxicity of heat-labile enterotoxin by targeting the receptor-binding region of the B-subunit pentamer

Infection by enterotoxigenic Escherichia coli (ETEC) causes severe watery diarrhea and dehydration in humans. Heat-labile enterotoxin (LT) is a major virulence factor produced by ETEC. LT is one of AB5-type toxins, such as Shiga toxin (Stx) and cholera toxin (Ctx), and the B-subunit pentamer is responsible for high affinity binding to the LT-receptor, ganglioside GM1, through multivalent interaction. In this report, we found that Glu51 of the B-subunit plays an essential role in receptor binding compared with other amino acids, such as Glu11, Arg13, and Lys91, all of which were previously shown to be involved in the binding. By targeting Glu51, we identified four tetravalent peptides that specifically bind to the B-subunit pentamer with high affinity by screening tetravalent random-peptide libraries, which were tailored to bind to the B-subunit through multivalent interaction. One of these peptides, GGR-tet, efficiently inhibited the cell-elongation phenotype and the elevation of cellular cAMP levels, both induced by LT. Furthermore, GGR-tet markedly inhibited LT-induced fluid accumulation in the mouse ileum. Thus, GGR-tet represents a novel therapeutic agent against ETEC infection.

Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.

Structural basis to identify a target site in Shiga toxin for the inhibitor discovery against growth of Shiga toxin-producing E. coli

BackgroundCertain peptides that bind Shiga toxin 2 (Stx2) have been reported to treat Shiga toxin-producing Escherichia coli (STEC) infections. However, their mechanisms of action remain unknown. STEC infections lead to serious diseases, such as hemolytic uremic syndrome, in humans. Antibiotic therapy is usually not recommended because of the major challenges of antibiotic resistance and SOS repair. Currently, there is no human vaccine for STEC infection, leaving rehydration therapy as the recommended supportive therapy. Therefore, there is a need for targeted therapeutic intervention to inhibit STEC growth. The purpose of this study was to evaluate the interaction of five known peptides with Stx2 to identify a more suitable peptide based on structural changes. These peptides have been used to inhibit the growth of STEC.ResultsThe current study demonstrated that only tetravalent peptide (TVP) out of 5 common peptides interrupted the Y77-E259 interaction of Stx2, making it active by exposing active site, which ultimately leads to STEC cell death. We also demonstrated that amino acids R170 and F171 of Stx2 in the docked complex of Stx2 and TVP form a helix-loop-helix (HLH). This might lead to the differential expression of genes regulated by Stx2 and ultimately inhibit STEC growth. However, in the case of Stx2-ribosomal P-stalk, these residues did not form HLH. The 3D refined model of TVP showed a low MolProbity score and low energy zones in the ANOLEA profile compared to the original one. Moreover, the low radius of gyration of the refined TVP suggests that it is more compact than the original TVP. Therefore, TVP is a suitable drug candidate for the inhibition of STEC growth. However, the low antigenicity of TVP makes it unsuitable as a drug candidate. We also evaluated three antibiotics that have been used as active ingredients in FDA-approved peptides. Only Oritavancin diphosphate showed strong polar interactions with Y77-E259 and also had the highest binding affinity.ConclusionsPotential drug candidates that inhibit or interrupt the interaction between Y77-E259 and have high antigenicity, low toxicity, and no allergenicity should be explored against the growth of STEC.

A tailored tetravalent peptide displays dual functions to inhibit amyloid β production and aggregation

Inhibition of amyloid-β peptide (Aβ) accumulation in the brain is a promising approach for treatment of Alzheimer’s disease (AD). Aβ is produced by β-secretase and γ-secretase in endosomes via sequential proteolysis of amyloid precursor protein (APP). Aβ and APP have a common feature to readily cluster to form multimers. Here, using multivalent peptide library screens, we identified a tetravalent peptide, LME-tet, which binds APP and Aβ via multivalent interactions. In cells, LME-tet-bound APP in the plasma membrane is transported to endosomes, blocking Aβ production through specific inhibition of β-cleavage, but not γ-cleavage. LME-tet further suppresses Aβ aggregation by blocking formation of the β-sheet conformation. Inhibitory effects are not observed with a monomeric peptide, emphasizing the significance of multivalent interactions for mediating these activities. Critically, LME-tet efficiently reduces Aβ levels in the brain of AD model mice, suggesting it may hold promise for treatment of AD.

A tetravalent peptide that binds to the RANK-binding region of TRAF6 via a multivalent interaction efficiently inhibits osteoclast differentiation

Inhibition of osteoclast differentiation is a promising approach for the treatment of osteoporosis and rheumatoid arthritis. Receptor activator of nuclear factor kappa B (NF-κB) (RANK), which is an essential molecule for osteoclast differentiation, interacts with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to transduce downstream signals. Both RANK and TRAF6 have homo-trimeric structures, forming a multivalent interaction between the Pro-X-Glu-X-X-(aromatic/acidic) motif of RANK and the C-terminal domain of TRAF6 (TRAF-C), that markedly increases the binding affinity. Here, we designed a tetravalent peptide, RANK-tet, containing the TRAF-C-binding motif of RANK and found that RANK-tet binds to TRAF-C with high affinity. In contrast, a monomeric form of RANK-tet (RANK-mono) with the same TRAF-C-binding motif did not bind to TRAF-C, clearly indicating the multivalent interaction is strictly required for the high-affinity binding to TRAF-C. RANK-tet did not bind to a series of TRAF-C-mutants with an amino acid substitution in the RANK-binding region, indicating that RANK-tet specifically targets the RANK-binding region of TRAF-C. A cell-permeable form of RANK-tet that has poly-Arg residues at each C-terminal of the TRAF-C-binding motif efficiently inhibited the RANK ligand (RANKL)-induced differentiation of bone marrow cells to osteoclasts. Thus, this compound can be an effective anti-osteoclastogenic agent.

Development of a novel tetravalent peptide that absorbs subtilase cytotoxin by targeting the receptor-binding B-subunit

Subtilase cytotoxin (SubAB) is a major virulence factor produced by eae-negative Shiga-toxigenic Escherichia coli (STEC) that can cause fatal systemic complications. SubAB binds to target cells through multivalent interactions between its B-subunit pentamer and receptor molecules such as glycoproteins with a terminal N-glycolylneuraminic acid (Neu5Gc). We screened randomized multivalent peptide libraries synthesized on a cellulose membrane and identified a series of tetravalent peptides that efficiently bind to the receptor-binding region of the SubAB B-subunit pentamer. These peptides competitively inhibited the binding of the B-subunit to a receptor-mimic molecule containing clustered Neu5Gc (Neu5Gc-polymer). We selected the peptide with the highest inhibitory efficacy, FFP-tet, and covalently bound it to beads to synthesize FFP-tet-beads, a highly clustered SubAB absorber that displayed potency to absorb SubAB cytotoxicity through direct binding to the toxin. The efficacy of FFP-tet-beads to absorb SubAB cytotoxicity in solution was similar to that of Neu5Gc-polymer, suggesting that FFP-tet-beads might be an effective therapeutic agent against complications arising from eae-negative STEC infection.

Resolution of Eczema with Multivalent Peptides

The integrity of the skin is an important aspect of QOL. Whether caused by genetic deficiencies or environmental insults, disruption of the surface barrier allows irritants and allergens to penetrate the skin, which initiates inflammatory responses by immune cells that often lead to life-long allergies. In this study, eczema was induced on depilated mouse skin with topical lipopolysaccharide or a mixture of Staphylococcal enterotoxin B and an extract of house dust mites, which resulted in thickening of the epidermis, epidermal disruption, and abundant neutrophils in the dermis. Within 14 days of topical treatment with 1 μM svL4, a tetravalent peptide, neutrophils were absent, and the epidermis had returned to a normal morphology. The sequence of svL4 contains glutamine residues that serve as a cross-linking substrate for transglutaminase 2, which gains access to the skin surface where the epidermis becomes disrupted. In contrast, topical application of 1 μM svH1C, a peptide mimetic of sialic acid that lacks glutamine residues, or 1 μM dexamethasone was ineffective in restoring normal epidermal morphology. The data suggest that svL4 would be a powerful treatment for resolving severe eczema.

Peptide-Antibody Fusions Engineered by Phage Display Exhibit an Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants.

The spread of COVID-19 has been exacerbated by the emergence of variants of concern (VoC). Many VoC contain mutations in the spike protein (S-protein) and are implicated in infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but they are limited by low potency and vulnerability to RBD mutations in VoC. To address these issues, we used naïve phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate-affinity nAb to generate tetravalent peptide–IgG fusions, and we showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrated that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide–IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format, including the dominant B.1.1.529 (Omicron) variant that is resistant to most clinically approved therapeutic nAbs. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug and showed that it enabled the neutralization of a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. Here, we identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. A monomeric peptide with the same motif, however, did not bind to the B-subunit pentamer. Instead, the monomer inhibited cytotoxicity with remarkable potency by binding to the catalytic A-subunit. An X-ray crystal structure analysis to 1.6 Å resolution revealed that the monomeric peptide fully occupied the catalytic cavity, interacting with Glu167 and Arg170, both of which are essential for catalytic activity. Thus, the peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx.

Multivalent, Soluble Nano-Self Peptides Increase Phagocytosis of Antibody-Opsonized Targets while Suppressing "Self" Signaling.

Macrophages engulf "foreign" cells and particles, but phagocytosis of healthy cells and cancer cells is inhibited by expression of the ubiquitous membrane protein CD47 which binds SIRPα on macrophages to signal "self". Motivated by some clinical efficacy of anti-CD47 against liquid tumors and based on past studies of CD47-derived polypeptides on particles that inhibited phagocytosis of the particles, here we design soluble, multivalent peptides to bind and block SIRPα. Bivalent and tetravalent nano-Self peptides prove more potent (Keff ∼ 10 nM) than monovalent 8-mers as agonists for phagocytosis of antibody opsonized cells, including cancer cells. Multivalent peptides also outcompete soluble CD47 binding to human macrophages, consistent with SIRPα binding, and the peptides suppress phosphotyrosine in macrophages, consistent with inhibition of SIRPα's "self" signaling. Peptides exhibit minimal folding, but functionality suggests an induced fit into SIRPα's binding pocket. Pre-clinical studies in mice indicate safety, with no anemia that typifies clinical infusions of anti-CD47. Multivalent nano-Self peptides thus constitute an alternative approach to promoting phagocytosis of "self", including cancer cells targeted clinically.

Enhancement of Tetravalent Immune Responses to Highly Conserved Epitopes of a Dengue Peptide Vaccine Conjugated to Polystyrene Nanoparticles

Vaccination remains the major approach to the prevention of dengue. Since the only licensed live attenuated vaccine (LAV) lacked efficacy against all four serotypes, other vaccine platforms, such as synthetic peptide vaccines, should be explored. In this study, four multi-epitope peptides (P1–P4) were designed by linking a universal T-helper epitope (PADRE or TpD) to the highly conserved CD8 T cell epitope and B cell epitope (B1 or B2) against all four DENV serotypes. The multi-epitope peptides were conjugated to polystyrene nanoparticles (PSNPs) and four nanovaccines (NP1–NP4) were constructed. Mice immunized with NP1–NP4 elicited significantly higher titers of IgG and neutralizing antibodies when compared to immunization with naked P1–P4. The immune responses in mice immunized with peptide vaccines were compared with nanovaccines using ELISA, ELISPOT, and a neutralization test based on FRNT50. Among the four conjugated peptide nanovaccines, NP3 comprising the TpD T-helper epitope linked to the highly conserved B1 epitope derived from the E protein was able to elicit significant levels of IFN-γ and neutralizing antibodies to all four dengue serotypes. NP3 is a promising tetravalent synthetic peptide vaccine, but the selection of a more effective CD8+ T cell epitope and adjuvants to further improve the immunogenicity is warranted.

Effective Access to Multivalent Inhibitors of Carbonic Anhydrases Promoted by Peptide Bioconjugation.

Multivalency has impressive effects on (bio)molecular recognition, through the simultaneous presentation of multiple copies of a ligand, which can change a weak millimolar binder into a potent nanomolar one. The implementation of multivalency in enzyme inhibition is rather recent, being exemplified by few serendipitous discoveries, and hitherto relying on the random exploration of new multivalent structures as potential enzyme inhibitors. Here, a straightforward and versatile method is reported that enables the construction of multivalent systems for the inhibition of carbonic anhydrases (CA), widespread enzymes that catalyze a fundamental biochemical reaction. Oxime and hydrazone click-type bioconjugation techniques were successfully used for the preparation of tetravalent peptide conjugates tethered with sulfonamide CA inhibitors. The enzyme inhibition assays show that multivalent effects were present with these novel compounds, but also reveal various structural effects provided by the scaffolds. The versatility of this approach may facilitate the exploration of structure-activity relationships for other types of enzyme inhibitors.

Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC.

Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid

We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

Identification of a Wide Range of Motifs Inhibitory to Shiga Toxin by Affinity-Driven Screening of Customized Divalent Peptides Synthesized on a Membrane

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli, binds to target cells through a multivalent interaction between its B-subunit pentamer and the cell surface receptor globotriaosylceramide, resulting in a remarkable increase in its binding affinity. This phenomenon is referred to as the "clustering effect." Previously, we developed a multivalent peptide library that can exert the clustering effect and identified Stx neutralizers with tetravalent peptides by screening this library for high-affinity binding to the specific receptor-binding site of the B subunit. However, this technique yielded only a limited number of binding motifs, with some redundancy in amino acid selectivity. In this study, we established a novel technique to synthesize up to 384 divalent peptides whose structures were customized to exert the clustering effect on the B subunit on a single cellulose membrane. By targeting Stx1a, a major Stx subtype, the customized divalent peptides were screened to identify high-affinity binding motifs. The sequences of the peptides were designed based on information obtained from the multivalent peptide library technique. A total of 64 candidate motifs were successfully identified, and 11 of these were selected to synthesize tetravalent forms of the peptides. All of the synthesized tetravalent peptides bound to the B subunit with high affinities and effectively inhibited the cytotoxicity of Stx1a in Vero cells. Thus, the combination of the two techniques results in greatly improved efficiency in identifying biologically active neutralizers of Stx.

Synthetic Multivalent Antifungal Peptides Effective against Fungi

Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2–4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides.

Identification of a Peptide-Based Neutralizer That Potently Inhibits Both Shiga Toxins 1 and 2 by Targeting Specific Receptor-Binding Regions

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli that occasionally causes fatal systemic complications. We recently developed a tetravalent peptide (PPP-tet) that neutralizes the cytotoxicity of Stx2 using a multivalent peptide library approach. In this study, we used this technique to identify a series of tetravalent peptides that bound to Stx1, another major Stx family member, with high affinity by targeting one receptor-binding site of the B subunit. One peptide, MMA-tet, markedly inhibited Stx1 and Stx2 cytotoxicity with greater potency than PPP-tet. After forming a complex with Stx1 through its specific receptor-binding region, MMA-tet did not affect vesicular transport of the toxin to the endoplasmic reticulum but substantially rescued inhibition of the protein synthesis induced by Stx1. Oral application of MMA-tet protected mice from a fatal dose of an E. coli O157:H7 strain producing both toxins. MMA-tet may be a promising therapeutic agent against the infection.

Staphylococcus aureus resistance on titanium coated with multivalent PEGylated-peptides

Bacterial infections can have adverse effects on the efficacy, lifetime and safety of an implanted device and are the second most commonly attributed cause of orthopedic implant failure. We have previously shown the assembly of PEGylated titanium-binding peptides (TBPs) on Ti to obtain a bacteriophobic surface coating that can effectively resist protein adsorption and Staphylococcus aureus ( S. aureus) adhesion. In the present study, we examine the effect of multiple TBP repeats on coating performance in vitro. Mono, di, and tetravalent peptides were synthesized and assessed for binding affinity and serum stability. PEGylated analogs were prepared and evaluated for their effect on S. aureus attachment and biofilm formation. Coating performance improved with the number of TBP repeats, with the tetravalent coating, TBP 4 – PEG, showing the best performance in all assays. In particular, TBP 4 – PEG forms a serum-resistant surface coating capable of preventing S. aureus colonization and subsequent biofilm formation. These results further support the role that multivalency can play in the development of improved surface coatings with enhanced stabilities and efficacy for in vivo clinical use.

Reversible and Noncompetitive Inhibition of β‐Tryptase by Protein Surface Binding of Tetravalent Peptide Ligands Identified from a Combinatorial Split–Mix Library

Molecular plug: On-bead screening of a combinatorial library of 216 tetravalent oligopeptides reveals highly specific, noncompetitive inhibitors of the serine protease β-tryptase with nanomolar affinity. The ligands most likely bind to the protein surface and act as a molecular plug that blocks access to the active sites, which are buried inside a central cavity.

Reversible and Noncompetitive Inhibition of β-Tryptase by Protein Surface Binding of Tetravalent Peptide Ligands Identified from a Combinatorial Split-Mix Library

Reversible and Noncompetitive Inhibition of β-Tryptase by Protein Surface Binding of Tetravalent Peptide Ligands Identified from a Combinatorial Split-Mix Library