Intravenous phage display identifies peptide sequences that target the burn-injured intestine

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10¹² phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1 × 10¹² copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4–1: SGHQLLLNKMP, 4–5: ILANDLTAPGPR, 4–11: SFKPSGLPAQSL). Sequence 4–5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9 × 10⁵vs. 3.1 × 10⁴ particles per mg tissue). Sequences 4–1 and 4–11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4–11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics.     

Identification of PSA peptide mimotopes using phage display peptide library

Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression.     

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.     

Peptide sequences mediating tropism to intact blood–brain barrier: An in vivo biodistribution study using phage display

Peptide motifs that demonstrate tropism for the blood brain barrier (BBB) are of real translational value in developing innovative delivery strategies for biological brain targeted therapies. In vivo peptide-phage display affords peptide selection against the full complement of biological markers within the correct cellular macro- and micro-environments. Here a stringent in vivo biopanning protocol was employed in the rat aimed at identifying cyclic 7-mer peptide motifs that mediate tropism to brain microvasculature. Five rounds of biopanning identified 349 unique peptide motifs in the brain tissue gray matter compartment (microvasculature and parenchyma). While in general no consensus was evident linking peptide physico-chemical properties and brain tropism, peptides bearing c-SxTSSTx-c or c-xxxSSTx-c motifs were found to be present in high abundance. Based on amino acid frequency distribution of the 349 unique peptides sequences a theoretical ‘idealized’ peptide pattern, c-PP(S/P)SSST-c, could be derived. For the most abundant experimental peptide sequence found in brain tissue, c-SYTSSTM-c, an in vivo pharmacokinetic and whole body tissue biodistribution study was performed. Based upon tissue exposure data (i.e. tissue AUC_{(0–infinity)}) the sequence c-SYTSSTM-c efficiently retargeted phage virions to the brain providing an approximate 5-fold greater (P < 0.05) accumulation in brain over control phage; in all other organs no significant (P > 0.05) difference in tissue tropism between c-SYTSSTM-c and control phages were evident. This peptide and more generally the peptide motifs, -SxTSSTx- or -xxxSSTx-, warrant further investigation as agents mediating sequence-dependent tropism to brain microvasculature potentially able to deliver biologic cargo to the CNS.     

Identification of ligand-selective peptide antagonists of the mineralocorticoid receptor using phage display

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily. Pathological activation of the MR causes cardiac fibrosis and heart failure, but clinical use of MR antagonists is limited by the renal side effect of hyperkalemia. The glucocorticoid cortisol binds the MR with equivalent affinity to that of the mineralocorticoids aldosterone and deoxycorticosterone. In nonepithelial tissues, including the myocardium, which do not express the cortisol-inactivating enzyme 11β hydroxysteroid dehydrogenase 2, cortisol has been implicated in the activation of MR. The mechanisms for ligand- and tissue-specific actions of the MR are undefined. Over the past decade, it has become clear that coregulator proteins are critical for nuclear receptor-mediated gene expression. A subset of these coregulators may confer specificity to MR-mediated responses. To evaluate whether different physiological ligands can induce distinct MR conformations that underlie differential coregulator recruitment and ligand-specific gene regulation, we utilized phage display technology to screen 10(8) 19mer peptides for their interaction with the MR in the presence of agonist ligands. We identified ligand-selective MR-interacting peptides that acted as potent antagonists of MR-mediated transactivation. This represents a novel mechanism of MR antagonism that may be manipulated in the rational design of a ligand- or tissue-selective MR modulator to treat diseases like heart failure without side effects such as hyperkalemia.     

Identification of peptide inhibitors of penicillinase using a phage display library

There is a constant need to identify novel inhibitors to combat β-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM⁻¹) and binding affinity (K_D = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV–vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a K_i of 9.22 μM and a K_i′ of 33.12 μM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of β-lactamases and development of novel inhibitors of β-lactamases.     

Search for peptide sequences involved in human antisperm antibody-mediated male immunoinfertility by using phage display technology

The aim of the present study was to delineate peptide sequences against which antisperm antibodies (ASA) are raised in immunoinfertile men. Using the phage display technology, seven unique and novel dodecamer amino acid sequences were identified that reacted with the sera of immunoinfertile men. The peptides were synthesized based upon these amino acid sequences and examined for their immunoreactivity with sera from ASA-positive immunofertile men (n = 15) and ASA-negative fertile men (n = 18) for IgM, IgG, and IgA class of antibody in the enzyme-linked immunosorbent assay (ELISA). All the seven synthetic peptides showed a significantly (P < 0.001) higher mean absorbance values for IgG and/or IgA class of antibody with the immunoinfertile sera compared to fertile control sera. Three of the seven peptides demonstrated a stronger reaction (>2 SD units) with 27%-40% of immunoinfertile sera compared to fertile controls. These peptide sequences may find applications in the specific diagnosis and treatment of immunoinfertility and in contraceptive vaccine development. The phage display technique provides an exciting and novel technology to delineate sperm epitopes involved in immunoinfertility.     

Borrelia burgdorferi adhesins identified using in vivo phage display

Borrelia burgdorferi, the agent of Lyme disease, disseminates from the site of deposition by Ixodes ticks to cause systemic infection. Dissemination occurs through the circulation and through tissue matrices, but the B. burgdorferi molecules that mediate interactions with the endothelium in vivo have not yet been identified. In vivo selection of filamentous phage expressing B. burgdorferi protein fragments on the phage surface identified several new candidate adhesins, and verified the activity of one adhesin that had been previously characterized in vitro. P66, a B. burgdorferi ligand for beta(3)-chain integrins, OspC, a protein that is essential for the establishment of infection in mammals, and Vls, a protein that undergoes antigenic variation in the mammal, were all selected for binding to the murine endothelium in vivo. Additional B. burgdorferi proteins for which no functions have been identified, including all four members of the OspF family and BmpD, were identified as candidate adhesins. The use of in vivo phage display is one approach to the identification of adhesins in pathogenic bacteria that are not easily grown in the laboratory, or for which genetic manipulations are not straightforward.     

Identification of target ligands of CORYNE in Arabidopsis by phage display library

CORYNE (CRN) plays important roles in stem cell division and differentiation of shoot apical meristem (SAM) in Arabidopsis thaliana. The cytoplasmic kinase domain of CRN has been cloned and expressed in Escherichia coli, and further purified by two consecutive steps of affinity chromatography. By using this purified CRN as a ligand, a 12-mer random-peptide library was used to determine the specific amino acid sequences binding with the recombinant CRN molecule. After four rounds of biopanning, positive phage clones were isolated and sequenced, and further tested by enzyme linked immunosorbent assay for their binding ability and specificity. Two positive clones that specifically bind to the intracellular protein kinase domain of CRN have been identified. Alignment of these peptides and the kinase-associated protein phosphatase (KAPP) shows high similarity, indicating that KAPP might interact with the cytoplasmic kinase domain of CRN and negatively regulate the CLV signal. Our current study would be helpful to better understand the CLV3 signal pathway.     

Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display

The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.     

Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display

The pFc' fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc' fragments were separated from F(ab')2 fragments by affinity chromatography. The pFc' fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable-length peptide libraries. Interacting phage-displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc' binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography.     

Identification of extracytoplasmic proteins in Bradyrhizobium japonicum using phage display

A novel gene bank of Bradyrhizobium japonicum USDA110spc4 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. In this phagemid, the phage protein III lacks its indigenous signal peptide required for protein secretion, thus recombinant fusion proteins are displayed on the phage surface only if a functional signal peptide is provided by an inserted DNA fragment. In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein. The expression library described here, therefore, provides a powerful means to affinity select for B. japonicum genes encoding extracytoplasmic proteins. In total, 182 DNA sequences were analyzed, among which 132 different putative extracytoplasmic proteins could be identified. The function of most proteins could be predicted and support an extracytoplasmic localization. In addition, genes encoding novel extracytoplasmic proteins were found. In particular, a novel family of small proteins has been identified that is characterized by a conserved pattern of four cysteine residues.     

Identification of a novel calpain inhibitor using phage display

Calpains are calcium- and thiol-dependent proteases that cleave a variety of intracellular substrates. Overactivation of the calpains has been implicated in a number of diseases and conditions such as ischemic stroke indicating a need for the development of calpain inhibitors. A major problem with current calpain inhibitors has been specific targeting to calpain. To identify highly specific calpain interacting peptides, we developed a peptide-phage library screening method based on the calcium-dependent conformation change associated with calpain activation. A phage-peptide library representing greater than 2 billion expressed 12-mers was incubated with calpain I in the presence of calcium. The calcium-dependent bound phage was then eluted by addition of EGTA. After four rounds of selection we found a conserved 5-mer sequence represented by LSEAL. Synthetic LSEAL inhibited tau-calpain interaction and in vitro proteolysis of tau- and alpha-synuclein by calpains. Deletion of the portion of the tau protein containing a homologous sequence to LSEAL resulted in decreased calpain-mediated tau degradation. These data suggest that these peptides may represent novel calpastatin mimetics.     

Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology

ActRIIB (activin receptor type-2B) is an activin receptor subtype constitutively expressed in the whole body, playing a role in cellular proliferation, differentiation, and metabolism. For its various physiological activities, ActRIIB interacts with activin and multiple other ligands including myostatin (MSTN), growth differentiation factor 11 (GDF11), and bone morphogenetic protein 9 (BMP9). Notably, the protein-protein interaction (PPI) between ActRIIB and MSTN negatively controls muscular development. Therefore, this PPI has been targeted for effective treatment of muscle degenerative diseases such as muscular dystrophy and sarcopenia. Here, we report the identification of ligand-selective peptidic ActRIIB-antagonists by phage display technology. Our peptides bound to the extracellular domain of ActRIIB, inhibited PPIs between ActRIIB expressed on the cell surface and its ligands, and subsequently suppressed activation of Smad that serves as the downstream signal of the ActRIIB pathway. Interestingly, these peptidic antagonists displayed different ligand selectivities; the AR2mini peptide inhibited multiple ligands (activin A, MSTN, GDF11, and BMP9), AR9 inhibited MSTN and GDF11, while AR8 selectively inhibited MSTN. This is the first report of artificial peptidic ActRIIB-antagonists possessing ligand-selectivity.     

Identification of a methotrexate-binding peptide from a T7 phage display screen using a QCM device

An 11-mer unique peptide sequence SIFPLCNSGAL was identified as a methotrexate (MTX)-binding peptide from a T7 phage display screen using a quartz-crystal microbalance (QCM) biosensor. The synthetic peptide displayed weak interaction with MTX (K(D) 2.23x10(-5) M) using surface plasmon resonance (SPR). Interestingly, analysis of the primary amino acid sequence of the peptide identified similarities to the MTX-binding site of dihydrofolate reductase (DHFR). Our results highlight the importance of this primary sequence for the recognition of the MTX molecule.     

A broad-spectrum peptide inhibitor of beta-lactamase identified using phage display and peptide arrays

Hydrolysis of beta-lactam antibiotics by beta-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Several small-molecule, mechanism-based inhibitors of beta-lactamases such as clavulanic acid are clinically available although resistance to these inhibitors has been increasing in bacterial populations. In addition, these inhibitors act only on class A beta-lactamases. Here we utilized phage display to identify peptides that bind to the class A beta-lactamase, TEM-1. The binding affinity of one of these peptides was further optimized by the synthesis of peptide arrays using SPOT synthesis technology. After two rounds of optimization, a linear 6-mer peptide with the sequence RRGHYY was obtained. A soluble version of this peptide was synthesized and found to inhibit TEM-1 beta-lactamase with a K(i) of 136 micro M. Surprisingly, the peptide inhibits the class A Bacillus anthracis Bla1 beta-lactamase with a K(i) of 42 micro M and the class C beta-lactamase, P99, with a K(i) of 140 micro M, despite the fact that it was not optimized to bind these enzymes. This peptide may be a useful starting point for the design of non-beta-lactam, broad-spectrum peptidomimetic inhibitors of beta-lactamases.     

Identification of inorganic crystal-specific sequences using phage display combinatorial library of short peptides: A feasibility study

The feasibility of using a combinatorial phage display library of decapeptides to identify ligands which can interact with the surface of a crystal was assessed using geological calcium carbonate as a model. Two relatively strong binding clones were identified by ELISA, sequenced and the encoded oligopeptides were prepared by solid phase synthesis and their properties compared with those of casein hydrolysate.