Screening for protease substrate by polyvalent phage display

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.     

Identification of calpain substrates by ORF phage display

Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases.     

Substrate phage as a tool to identify novel substrate sequences of proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.     

A modular assembly strategy for improving the substrate specificity of small catalytic peptides

In contrast to large proteins, small peptide catalysts typically display limited specificity for small molecule substrates. This is presumably a result of the limited opportunities small peptides have to fold in a manner that provides for the formation of an isolated reaction vessel that effectively binds and sequesters substrates from bulk solvent while at the same time catalyzing their transformation. For the preparation of small peptide catalysts that possess improved substrate specificity, we have developed a modular assembly strategy that involves appending phage display-derived substrate binding-domain modules to catalytically active peptide domains. We demonstrate the potential of this strategy with the construction of a small 35-amino acid residue aldolase peptide with improved substrate specificity. The advantages of this approach are that it reduces the demand on the functionalization of the catalytic site and it is modular, therefore making its adaptation to a variety of specificities rapid. The modular assembly strategy studied here may present advantages over exhaustive searches of large random-sequence peptide libraries for peptides with singular function.     

Substrate specificity of prostate-specific antigen (PSA)

Background: The serine protease prostate-specific antigen (PSA) is a useful clinical marker for prostatic malignancy. PSA is a member of the kallikrein subgroup of the (chymo)trypsin serine protease family, but differs from the prototypical member of this subgroup, tissue kallikrein, in possessing a specificity more similar to that of chymotrypsin than trypsin. We report the use of two strategies, substrate phage display and iterative optimization of natural cleavage sites, to identify labile sequences for PSA cleavage.Results: Iterative optimization and substrate phage display converged on the amino-acid sequence SS(Y/F)YIS(G/S) as preferred subsite occupancy for PSA. These sequences were cleaved by PSA with catalytic efficiencies as high as 220–3100 M⁻¹ s⁻¹, compared with values of 2–46 M⁻¹ s⁻¹ for peptides containing likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsites have a critical role in defining labile substrates and can even cause otherwise disfavored amino acids to bind in the primary specificity (S1) pocket.Conclusions: The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.     

Use of intein-directed peptide biosynthesis to improve serum stability and bioactivity of a gelatinase inhibitory peptide

Screening of phage display libraries allows rapid identification of peptides binding to a target. However, functional analysis of the phage sequences and their reproduction as soluble and stable peptides are often the most time-consuming part in the screening. We have used here intein-based peptide biosynthesis to produce a phage-display derived gelatinase inhibitory peptide CTTHWGFTLC and to identify the critical residues for gelatinase inhibitory activity by performing alanine-scanning mutagenesis. By biosynthetic incorporation of 5-fluorotryptophan, we obtained an inhibitor of MMP-2 and MMP-9 gelatinases that showed a 6-fold enhancement in serum stability in comparison to the wild-type peptide. The new peptide also had an improved ability to inhibit tumor cell migration. These studies indicate the utility of intein methodology for synthesis and design of peptides obtained by phage display.     

Identifying substrates for endothelium-specific Tie-2 receptor tyrosine kinase from phage-displayed peptide libraries for high throughput screening

The peptide substrate specificity of Tie-2 was probed using the phage display method in order to identify efficient substrate for high throughput screening. Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructed, in which all twenty amino acid residues were represented at the X positions flanking the fixed tyrosine residue Y. A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylation. The phosphorylated phage particles were enriched by panning over immobilized anti-phosphotyrosine antibody pY20 for a total of 5 rounds. Four phage clones (3T61, 3T68, C1-90 and D1-15) that express a peptide sequence that can be phosphorylated by the recombinant catalytic domain of human Tie-2 were identified. Synthetic peptides made according to the sequences of the 4 selected clones from the two libraries, which had widely different sequences, were active substrates of Tie-2. Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a k(cat)/K(m) of 5.9x10(4) M(-1) s(-1). Three high throughput screening assay formats, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-resolved fluorescent resonance energy transfer (TR-FRET) were developed to assess the suitability of these phage display selected peptides in screening Tie-2 inhibitors. Three out of four peptides were functional in the DELFIA assay and D1-15 was functional in the TR-FRET assay.     

A method for rapid protease substrate evaluation and optimization

We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.     

Peptide microarrays for the determination of protease substrate specificity

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.     

From phage display to dendrimer display: insights into multivalent binding

Phage display is widely used for the selection of target-specific peptide sequences. Presentation of phage peptides on a multivalent platform can be used to (partially) restore the binding affinity. Here, we present a detailed analysis of the effects of valency, linker choice, and receptor density on binding affinity of a multivalent architecture, using streptavidin (SA) as model multivalent receptor. For surfaces with low receptor densities, the SA binding affinity of multivalent dendritic phage peptide constructs increases over 2 orders of magnitude over the monovalent species (e.g., K(d,mono) = 120 μM vs K(d,tetra) = 1 μM), consistent with previous work. However, the affinity of the SA-binding phage presenting the exact same peptides was 16 pM when dense receptor surfaces used for initial phage display were used in assays. The phage affinity for SA-coated surfaces weakens severely toward the nanomolar regime when surface density of SA is decreased. A similarly strong dependence in this respect was observed for dendritic phage analogues. When presented with a dense SA-coated surface, dendrimer display affords up to a 10(4)-fold gain in affinity over the monovalent peptide. The interplay between ligand valency and receptor density is a fundamental aspect of multivalent targeting strategies in biological systems. The perspective offered here suggests that in vivo targeting schemes might best be served to conduct ligand selection under physiologically relevant receptor density surfaces, either by controlling the receptor density placed at the selection surface or by using more biologically relevant intact cells and tissues.     

A high-complexity, multiplexed solution-phase assay for profiling protease activity on microarrays

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.     

N-Terminomics Strategies for Protease Substrates Profiling

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.     

Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

BACKGROUND: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits. RESULTS: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(methyl)-sulfone (AdaAhx(3)L(3)VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. CONCLUSIONS: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts.     

A comparison of cyclohexanone and tetrahydro-4H-thiopyran-4-one 1,1-dioxide as pharmacophores for the design of peptide-based inhibitors of the serine protease plasmin

The plasminogen system is important in the proteolytic cascade that facilitates angiogenesis, a process that is essential for tumor growth and metastasis. The serine protease plasmin has a central role in the plasminogen system. This protease acts by degrading several components of the basement membrane and by activating other proteases. Therefore, inhibition of plasmin may be an effective method for blocking angiogenesis and, as a result, inhibiting the growth of primary tumors and secondary metastases. Three pairs of plasmin inhibitors were synthesized to compare the relative potency of inhibitors that are based upon a cyclohexanone or a tetrahydro-4H-thiopyran-4-one 1,1-dioxide nucleus. Compounds 1, 3, and 5 were cyclohexanone-based inhibitors, whereas compounds 2, 4, and 6 were tetrahydro-4H-thiopyran-4-one 1,1-dioxide-based inhibitors. Compounds 5 and 6 are reasonable inhibitors with IC50 values of 25 and 5.5 microM, respectively. Comparisons of the IC50 values of the three pairs show that the electron-withdrawing sulfone functional group is a beneficial element for the design of plasmin inhibitors. The presence of the sulfone increases inhibitor potency by a factor of 3-5 when compared to inhibitors that are based upon a simple cyclohexanone core.     

Biological identification of peptides that specifically bind to poly(phenylene vinylene) surfaces: recognition of the branched or linear structure of the conjugated polymer

Peptides that bind to poly(phenylene vinylene) (PPV) were identified by the phage display method. Aromatic amino acids were enriched in these peptide sequences, suggesting that a π-π interaction is the key interaction between the peptides and PPV. The surface plasmon resonance (SPR) experiments using chemically synthesized peptides demonstrated that the Hyp01 peptide, with the sequence His-Thr-Asp-Trp-Arg-Leu-Gly-Thr-Trp-His-His-Ser, showed an affinity constant (7.7 × 10(5) M(-1)) for the target, hyperbranched PPV (hypPPV) film. This value is 15-fold greater than its affinity for linear PPV (linPPV). In contrast, the peptide screened for linPPV (Lin01) showed the reverse specificity for linPPV. These results suggested that the Hyp01 and Lin01 peptides selectively recognized the linear or branched structure of PPVs. The Ala-scanning experiment, circular dichroism (CD) spectrometry, and molecular modeling of the Hyp01 peptide indicated that adequate location of two Trp residues by forming the polyproline type II (P(II)) helical conformation allowed the peptide to specifically interact with hypPPV.     

An inhibitor of sequence-specific proteolysis that targets the substrate rather than the enzyme

BACKGROUND: Traditional protease inhibitors target the active site of the enzyme. However, since most proteases act on multiple substrates, even the most specific protease inhibitors will affect the levels of a number of different proteins. However, if substrate-targeted inhibitors could be developed, much higher levels of specificity could be achieved. In theory, compounds that bind the cleavage site of a particular substrate could block its interaction with a protease without having any effect on the processing of other substrates of that protease. RESULTS: A model system is presented that demonstrates the feasibility of substrate-targeted inhibition of proteolysis. A peptide selected genetically to bind a 14-residue epitope that encompasses the cleavage site of human pro-IL-1beta was shown to inhibit interleukin-converting enzyme (ICE)-mediated proteolysis of model substrates containing the 14-mer target sequence. However, the peptide had no effect on the cleavage of other ICE substrates with different amino acids flanking the minimal cleavage site. CONCLUSIONS: This study demonstrates the feasibility of substrate-targeted inhibition of proteolysis. More potent compounds must be developed before substrate-targeted inhibitors can be used routinely. Nonetheless, this novel strategy for protease inhibition seems promising for the development of extremely selective molecules with which to manipulate the maturation of many important pro-hormones, -cytokines and -proteins.     

Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds. RESULTS: We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity. CONCLUSIONS: Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation.     

Fluorous-based peptide microarrays for protease screening

As ever more protease sequences are uncovered through genome sequencing projects, efficient parallel methods to discover the potential substrates of these proteases becomes crucial. Herein we describe the first use of fluorous-based microarrays to probe peptide sequences and begin to define the scope and limitations of fluorous microarray technologies for the screening of proteases. Comparison of a series of serine proteases showed that their ability to cleave peptide substrates in solution was maintained upon immobilization of these substrates onto fluorous-coated glass slides. The fluorous surface did not serve to significantly inactivate the enzymes. However, addition of hydrophilic components to the peptide sequences could induce lower rates of substrate cleavage with enzymes such as chymotrypsin with affinities to hydrophobic moieties. This work represents the first step to creating robust protease screening platforms using noncovalent microarray interface that can easily incorporate a range of compounds on the same slide.     

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

In biomedical research and clinical diagnostics, it is a major challenge to measure disease‐related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time‐consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge‐changing fluorescent peptide substrates. Charge‐changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic α‐chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both α‐chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross‐reactivity with the trypsin‐like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin‐specific), a detection limit of about 10–20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point‐of‐care diagnostics.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.