©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation of a High Affinity Inhibitor of Urokinase-type Plasminogen Activator by Phage Display of Ecotin (*)

Cheng-I Wang, Qing Yang, and Charles S. Craik (§)

From the (1) Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
REFERENCES

ABSTRACT

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unique in its ability and mechanism of inhibiting serine proteases of a broad range of substrate specificity. However, although the catalytic domain of human urokinase-type plasminogen activator (uPA) has 40% identity to bovine trypsin and the substrate specificities of these two proteases are virtually identical, ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin. Ecotin was expressed on the surface of filamentous bacteriophage (ecotin phage) to allow the isolation of more potent inhibitors of uPA from a library of ecotin variants. The 142-amino acid inhibitor was fused to the C-terminal domain of the M13 minor coat protein, pIII, through a Gly-Gly-Gly linker and assembled into phage particles. The ecotin phage were shown to react with anti-ecotin antibodies, revealing a stoichiometry of approximately one ecotin per bacteriophage. The ecotin displayed on the surface of phage inhibited trypsin with an equilibrium dissociation constant of 6.7 nM, in close approximation to that of free ecotin, indicating that phage-associated ecotin is correctly folded and functionally active. Reactive-site amino acids 84 and 85 of ecotin were then randomized and a library of 400 unique ecotin phage was created. Three hundred thousand members of the library were screened with immobilized uPA and subjected to three rounds of binding and in vitro selection. DNA sequence analysis of the selected ecotin phage showed that ecotin M84R/M85R predominated while ecotin M84R, M84K, and M84R/M85K were present at a lower frequency. The four ecotin variants were overexpressed and purified and their affinities toward uPA were determined. Each of the selected ecotin variants exhibited increased affinity for uPA when compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold increase in binding affinity.


INTRODUCTION

Urokinase-type plasminogen activator (uPA)() and tissue-type plasminogen activator (tPA) are two serine proteases that catalyze the conversion of the inactive precursor plasminogen, to plasmin, a serine protease of broad substrate specificity (1, 2) . uPA has been found to be involved in the activation of pericellular proteolysis during cell migration and tissue remodeling, while the function of tPA is primarily connected to intravascular clot dissolution (3, 4) . Although no three-dimensional structure currently exists for human uPA, it is thought to be composed of three domains: an NH-terminal domain (residues 1-45), which has partial homology to EGF (5, 6) followed by a kringle domain (residues 50-131) (5) , and a COOH-terminal protease domain (residues 159-411) with 40% identity to trypsin (7) . The EGF-like domain of uPA binds the membrane-bound urokinase receptor (8) , localizing the proteolytic activity of uPA to the cell surface. Receptor-bound uPA has been shown to cleave a 66-kDa extracellular matrix protein (9) as well as fibronectin (10) . The presence of uPA on the cell surface also allows the formation of cell-surface plasmin, which is capable of degrading most components of the extracellular matrix, either directly or through activation of procollagenases (3) . Cell surface uPA has been implicated in mediating processes such as tumor growth, cell invasion, metastasis, cell migration, and tissue remodeling (3, 11) , all of which require extracellular proteolytic activity.

High levels of receptor-bound uPA are found on the surface of many cancer cells (12, 13, 14) . The role of uPA and its receptor in tumor invasion and metastasis suggests two possible approaches for chemotherapeutic intervention: one by blocking specific interactions between the EGF-like domain of uPA and the uPA receptor, and the other by specific inhibition of the proteolytic activity of uPA. A truncated, soluble form of the uPA receptor was produced genetically and shown to reduce the amount of uPA that bound to cells expressing wild-type uPA receptor (15) . By acting as a scavenger for uPA, the soluble uPA receptor also inhibited the proliferation and invasion of human cancer cells (16) . Alternatively, a uPA mutant which lacked proteolytic activity while retaining full receptor binding affinity was shown to compete for cell surface receptors and, in turn, inhibit metastasis (17). Finally, high-affinity urokinase receptor antagonists were identified from a pentadecamer random peptide library and were shown to compete with the EGF-like domain of uPA for binding to the uPA receptor (18). Although a certain extent of success in anti-invasion and anti-metastasis has been achieved in vitro using this approach, rapid clearance of uPA, uPA receptor derivatives, or natural peptides may pose a problem if they are used as therapeutic agents.

Less effort has been made toward developing specific inhibitors for uPA, presumably because of the difficulty of discriminating uPA from other serine proteases. Of the synthetic uPA inhibitors that have been described to date, the 4-substituted benzo[b]thiophene-2-carboxamidines were the most potent and were shown to inhibit cell surface uPA as well as cell surface uPA-mediated fibronectin degradation (19) . The natural macromolecular inhibitor of the plasminogen activators is the type 1 plasminogen activator inhibitor (PAI-1), a single-chain glycoprotein with a molecular mass of approximately 50 kDa (20) . However, PAI-1 does not discriminate between the plasminogen activators, inactivating tPA and uPA with nearly identical secondary rate constants (21) . Furthermore, high PAI-1 levels have recently been found to associate with malignancy in a number of cancers (22, 23, 24, 25) . These findings suggest that, in addition to functioning as a uPA inhibitor in normal cells, PAI-1 or its complex may play a role in promoting growth or spreading of cancers. These attributes disfavor the use of PAI-1 as a therapeutic agent.

Ecotin is a dimeric serine protease inhibitor found in the periplasm of Escherichia coli, where each unit of the dimer contains 142 amino acids (26, 27) . Ecotin has been found to inhibit pancreatic serine proteases of a broad range of specificity but not any known proteases from E. coli(26) . Recently, ecotin has also been found to be a highly potent anticoagulant and a reversible tight-binding inhibitor of human factor Xa (28) . Ecotin belongs to the ``substrate-like'' class of inhibitors (29) with Met-84 at the reactive-site (the P1 site) (27) . A crystal structure of ecotin complexed with trypsin showed that two trypsin molecules bind to an ecotin dimer in a 2-fold symmetry (30) . In addition to the interactions through a primary site that includes the reactive-site loop, ecotin makes a total of 9 hydrogen bonds to trypsin through a secondary binding site located at the distal end of ecotin relative to the reactive site. Modeling studies with ecotin and other proteases including chymotrypsin and elastase indicates that similar interactions could occur, along with other unique contacts. The chelation of a target protease through the two binding sites is a unique feature of ecotin since most serine protease inhibitors interact with their target proteases predominately through their reactive-site loop (31) . The bidentate binding scheme utilized by ecotin may allow fine tuning of protease inhibition toward specific targets through protein engineering efforts. Although the catalytic domain of uPA and trypsin are homologous (7) and their substrate specificities are virtually identical, ecotin is a poor inhibitor of uPA proteolytic activity. We attempted to convert ecotin into a potent uPA inhibitor using phage display to aid our understanding of protease-inhibitor recognition and uPA function.

It was previously shown that a bovine pancreatic trypsin inhibitor variant with altered specificity and high affinity toward human neutrophil elastase could be isolated from a library of bovine pancreatic trypsin inhibitor variants by phage display technology (32) . This demonstrated the feasibility of studying protease-inhibitor interactions using this technique. Phage display allows the expression of a diverse library of peptides or protein variants on the surface of filamentous M13 bacteriophage (33, 34) . This in turn allows the isolation of individual phage particles that display desired binding properties by an in vitro selection process. Since the phenotype of each phage is directly linked to its genotype, specific mutations in the displayed peptide or protein that confer a desired function can be readily identified. Using this technology, a number of antigen-antibody interactions have been studied (34, 35) as well as hormone-receptor interactions (36, 37) , protein-nucleic acid interactions (38, 39) , and inhibitor-protease interactions (32, 40) .

We have shown that the phage display approach can be broadly generalized to other protease inhibitors by displaying ecotin on the surface of phagemid-derived bacteriophage (ecotin phage). Ecotin was chosen because of its unusual ability to inhibit trypsin, chymotrypsin, and elastase (26) , and its unique mechanism of inhibition (30) . Its broad specificity suggests a structural flexibility which would allow modifications via protein engineering to confer novel properties. The crystal structure of ecotin complexed with a variant of rat trypsin was solved recently (30) . Combined with information from the three-dimensional structure, phage display can be used to search designed libraries of ecotin mutants that affect interactions at the inhibitor/protease interface. Variants with high affinity toward a particular target protease can then be readily isolated and characterized. Herein, we report the display of ecotin on the surface of phagemid-derived bacteriophage, and the isolation of mutants with high affinity toward uPA.


EXPERIMENTAL PROCEDURES

Materials

Enzymes and reagents for molecular cloning were purchased from New England Biolabs and were used following the manufacturer's instructions. The E. coli strain JM101 and the VCSM13 helper phage were from Stratagene. Low molecular weight uPA (LMuPA) was obtained from American Diagnostica. Bovine trypsin was from Sigma. The chromogenic substrate Z-Gly-Pro-Arg-p-nitroanilide used for trypsin kinetics analysis was from Bachem, and the chromogenic substrate Z--Glu(-t-butoxy)-Gly-Arg-p-nitroanilide (Spectrozyme UK) used for LMuPA kinetics analysis was from American Diagnostica. 4-Methylumbelliferyl p-guanidinobenzoate was from Sigma. -S-dATP was from DuPont NEN. Sequenase Version 2.0 sequencing kit was from U. S. Biochemical Corp. Oligonucleotides were synthesized with an Applied Biosystems 391 DNA synthesizer.

Plasmid and Library Constructions

The phagemid pBSeco-gIII was constructed to produce ecotin on the surface of the surface of filamentous phage. These phage are referred to as ecotin phage. The ecotin expression plasmid pTacTacEcotin (41) was digested with the restriction endonucleases BamHI and HindIII. The resulting DNA fragment encoding the ecotin gene and its signal sequence was ligated to the large fragment of BamHI/HindIII-digested pBluescript to produce pBSecotin. The DNA sequence coding for amino acids 198-406 of gene III of M13 was generated from M13 mp18 DNA using polymerase chain reaction; the forward primer was 5`-GTC ACG AAG CTT CCA TTC GTT TGT GAA TAT CAA GG-3`, and the reverse primer was 5`-GCA CGA AGC TTA AGA CTC CTT ATT ACG CAG TAT G-3`. After HindIII digestion, the polymerase chain reaction product was inserted into a HindIII site at the 3` end of the ecotin gene of pBSecotin. The stop codon at the COOH terminus of the ecotin gene was then removed, and a Gly-Gly-Gly tether was introduced at the junction of the fusion gene. This was achieved by site-directed mutagenesis (42) using the primer 5`-CG GTA GTT CGC GGC GGC GGA GCT GAA AGC GTC CAG-3`. The resulting plasmid construct was named pBSeco-gIII. A pBSeco-gIII mutant in which codons 84 and 85 and the third base pair of codon 83 of the ecotin gene were deleted was constructed to provide control phage that did not express the ecotin gene. This mutant, pBSeco-gIII, was made by site-directed mutagenesis using the primer 5`-C AGT TCC CCG GTT AGT AC GCC TGC CCG GAT GG-3`. A pBSeco-gIII library with random mutations at codons 84 and 85 of the ``reactive-site'' loop of ecotin was created by oligonucleotide-directed mutagenesis using the oligonucleotide 5`-C AGT TCC CCG GTT AGT ACT NNS NNS GCC TGC CCG GAT GG-3` (N = A/C/G/T; S = G/C) as the primer and the uracilated, single-stranded pBSeco-gIII as the template. Also introduced by this primer was a ScaI site, which enabled facile differentiation between native templates and mutant templates. The library of ecotin phage had 1024 possible DNA sequences that resulted in 400 possible protein sequences.

Phage Preparations

For the preparation of pBluescript, pBSeco-gIII, and pBSeco-gIII bacteriophage, plasmid DNAs were transformed into a male strain (F`) of JM101. A single colony selected on ampicillin plates was grown in 3 ml of 2YT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl/liter) containing 60 µg/ml ampicillin at 37 °C for 7 h. The culture was diluted into 30-100 ml of 2YT/ampicillin, grown to A = 0.25, and infected with the helper phage VCSM13 at a multiplicity of infection of approximately 100 helper phage per cell. The infected culture was allowed to grow at 37 °C with shaking for approximately 12 h. Phage particles were harvested by precipitation with 5% polyethylene glycol and resuspended in 1 ml of TBS buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4). Phage titers typically ranged from 5 10 to 2 10 cfu/ml culture. For library phage preparation, the mutagenesis reaction mixture was ethanol-precipitated, redissolved in water, electroporated into F` JM101, and plated on 150-mm LB/ampicillin plates. Cells from the plates were recovered in 5 ml of LB/ampicillin and diluted in 50 ml of 2YT/ampicillin to an A = 0.25, and then infected with VCSM13 helper phage. The infected culture was grown for 12 h at 37 °C with shaking, and the phage were harvested as described above.

Immunoblotting Analysis of Ecotin Fusion Phage

Approximately 5 10 cfu were loaded in duplicate onto a single 1% agarose gel with 25 mM Tris, 250 mM glycine (pH 8.6) as the running buffer (43) . The gel was electrophoresed at 6 mA constant current for 16 h. One set of samples was transblotted onto a nitrocellulose filter. The filter was immunostained for ecotin by allowing it to react with rabbit anti-ecotin antibodies followed by reaction with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies. The other set of samples were denatured by soaking the gel in 0.5 N NaOH for 4 h, washed by soaking in water for 4-8 h, and stained with ethidium bromide.

Inhibition of Trypsin Activity by Ecotin-pIII Fusion Phage

pBSeco-gIII and pBSeco-gIII phage were suspended in trypsin assay buffer (50 mM Tris-HCl, 100 mM NaCl, 20 mM CaCl, pH 8.0) and adjusted to 1.5 10 cfu/ml. Various volumes of phage solution were incubated with 0.5 nM trypsin in a total volume of 125 µl of trypsin assay buffer, 0.01% Tween 80 in a 96-well microtiter plate at room temperature for 20 min. After adding 125 µl of 0.1 mM substrate Z-Gly-Pro-Arg-p-nitroanilide, the residual trypsin activity was measured by monitoring the increase of optical density at 405 nm.

Binding Enrichments

Polystyrene Petri dishes (35 mm, Falcon) were coated with 1 ml of 10 µg/ml bovine trypsin or LMuPA in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO, pH 7.5) overnight, and excess binding sites were blocked with 5% non-fat dry milk solution for 2 h. For control experiments, Petri dishes were coated with 5% non-fat dry milk solution. Phage were added to the dishes in buffer containing 1 ml of phosphate-buffered saline, 0.5% Tween 20 and incubated overnight with gentle agitation at room temperature. Solutions containing the phagemid were then removed and the dishes were washed 9 times with 5 ml of phosphate-buffered saline/Tween 20. Each wash was approximately 1 min. Bound phage were serially eluted by incubation with 1 ml of 0.1 N HCl/glycine solution (pH 2.2) with gentle shaking for 15 min at room temperature. Three elutions were performed. The eluates were neutralized with 185 µl of 1 M Tris-HCl (pH 8.8). For biopanning against immobilized trypsin, a mixture of pBSeco-gIII phage (1.1 10 cfu) and pBluescript phage (2.8 10 cfu) was used. An aliquot of the appropriately diluted solution of each wash and elution was used to infect 100 µl of saturated JM101 cells. After incubation for 15 min at 37 °C, the infected cells were plated on LB/ampicillin plates containing IPTG and 5-bromo-4-chloro-3-indolyl -D-galactoside. The cfu ratio of pBSeco-gIII phage to pBluescript phage was calculated by the number of white and blue colonies, respectively. For biopanning against immobilized LMuPA, a mixture of 2.5 10 cfu library phage were used. Phage from the third elution were amplified for the next cycle of panning.

Expression and Purification of Recombinant Ecotin and Ecotin Mutants

Ecotin and ecotin mutants were produced in bacteria from the expression vector pTacTacEcotin (41) . The expression and purification procedures were as follows. JM101 cells were freshly transformed with expression plasmid DNA. A single colony selected from ampicillin plates was used to inoculate 3 ml of LB containing 60 µg/ml ampicillin. The cultures were grown at 37 °C for 9 h and diluted to 1 liter of LB/ampicillin. Following growth at 37 °C for 1 h, IPTG was added to the cultures to a final concentration of 0.2 mM, and continued to grow for 12 h at for 37 °C. Cells were harvested and treated with lysozyme in a solution containing 25% sucrose, 10 mM Tris-HCl (pH 8.0). The periplasmic fraction was dialyzed against 10 mM sodium citrate (pH 2.8). Following the dialysis, the supernatant was adjusted to pH 7.4 with 1 M Tris-HCl (pH 8.0), and to 0.3 M NaCl. The solution was heated in boiling water for 10 min, and then cooled to room temperature. The precipitate was removed by centrifugation, and the supernatant was dialyzed against water overnight at 4 °C. The solution containing the ecotin was loaded onto a Vydac C4 reverse-phase high performance liquid chromatography column (2.2 25 cm) which was equilibrated with 0.1% trifluoroacetic acid. The column was washed and then eluted with a linear gradient of 34-37% acetonitrile, 0.1% trifluoroacetic acid at a flow rate of 10 ml/min over 30 min. Fractions were analyzed with SDS-polyacrylamide gel electrophoresis (44) , and the ones containing pure ecotin were pooled and lyophilized. Purified ecotin was redissolved in buffer containing 10 mM Tris-HCl (pH 7.4) and stored at 4 °C. The concentrations of ecotin and ecotin mutants were determined using a calculated molar extinction coefficient (45) of 2.2 10 cmM and were in good agreement with that from the Bradford assay (data not shown) (46) .

Determination of Equilibrium Dissociation Constants

LMuPA was titrated with 4-methylumbelliferyl p-guanidinobenzoate to obtain an accurate concentration of enzyme active sites. Various concentrations of ecotin or ecotin mutants were incubated with human LMuPA in a total volume of 990 µl of buffer containing 50 mM NaCl, 50 mM Tris-HCl (pH 8.7), 0.01% Tween 80. The final concentrations of LMuPA used for the determination of equilibrium dissociation constants, K, of ecotin or ecotin mutants were as follows: 0.5 nM (M84R/M85R); 1.0 nM (M84R, M84K, and M84R/M85K); 7.2 nM (wild-type). The final concentrations of ecotin mutants ranged from 0.6 to 40 nM, and the concentrations of wild-type ecotin ranged from 1.25 to 50 µM. Following a 30-min incubation at room temperature to reach equilibrium, 10 µl of 10 mM substrate Z--Glu (-t-butoxy)-Gly-Arg-p-nitroanilide was added and the rate of p-nitroaniline formation was measured by monitoring the change of absorption at 410 nm in a 10-min period. The data were fit to the equation derived for kinetics of reversible tight-binding inhibitors (47, 48) by nonlinear regression analysis, and the values for apparent Kwere determined.

RESULTS

Design of the Phage Display System

Ecotin was expressed on the surface of phagemid-derived bacteriophage to establish an in vitro selection for ecotin variants. The pBluescript vector was chosen for ecotin phagemid construction due to its high copy number, small size, and stability. The presence of the M13 origin of replication permits efficient packaging of phage particles when bacteria carrying the pBluescript phagemid are infected with helper phage. The presence of the -complementation factor permits monitoring of the phagemid by -galactosidase activity, and the -lactamase gene permits quantitation of phagemid titer by cfu.

The ecotin gene for this construction was isolated from the expression plasmid pTacTacEcotin (41) and was inserted into the multiple cloning site of pBluescript to generate pBSecotin. The inserted DNA contained a Shine-Dalgarno sequence at the 5` end, followed by the sequence encoding a 20-amino acid ecotin signal peptide from the genomic clone (27), and the mature form of ecotin. The DNA sequence coding for the COOH-terminal domain (residues 198-406) of the gene III protein, which is embedded in the phage coat and is essential for proper phage assembly (49) , was generated by polymerase chain reaction. This DNA fragment was fused to the end of the ecotin gene of pBSecotin through a Gly-Gly-Gly linker (Fig. 1). It should be noted that the ecotin gene and the ecotin-gene III fusion were inserted in pBluescript in the same orientation as the lacZ gene and disrupted the lacZ reading frame. Ecotin and ecotin-pIII fusion were expressed from pBSecotin and pBSeco-gIII, respectively, and the protein expression level was increased approximately 5-fold by the addition of IPTG in the cultures (data not shown). These results verified that the lac promoter of pBluescript was utilized for transcription and the Shine-Dalgarno sequence upstream of the ecotin gene was recognized for initiating translation. However, phagemid propagation in the presence of IPTG resulted in very low phage titer, perhaps due to the increased levels of ecotin-pIII fusion which may be toxic to the cells. Since ecotin-pIII fusion was expressed at a significant level in the absence of IPTG without affecting phage titer, IPTG was not used in the phage preparations. Under these conditions, the phage titer for pBSeco-gIII was approximately 2 to 8 10 cfu/ml of culture.


Figure 1: Schematic representation of the DNA fragment encoding the ecotin-pIII fusion and the lac regulatory sequence of the phagemid pBluescript. Ecotin was fused to the carboxyl-terminal domain (residues 198-406) of M13 pIII through a Gly-Gly-Gly linker. The amino acid and nucleotide sequences between BamHI restriction site and the start of the ecotin signal peptide (S.P.), at the junction of the gene fusion, and at the end of gene III are shown. Transcription of the fusion protein is under control of the lac promoter/operator sequence of the phagemid pBluescript, and secretion is directed by a 20-amino acid signal peptide from the ecotin genomic clone. The fusion construct was inserted between BamHI and HindIII restriction sites of pBluescript in the same direction as lacZ gene and disrupted the lacZ reading frame. pBluescript contains the -lactamase gene which provides ampicillin resistance and allows quantification of the ecotin phage by colony forming units.



Characterization of the Ecotin-pIII Fusion

pBSeco-gIII phage were fractionated by electrophoresis in a 1% agarose gel and analyzed for their interaction with anti-ecotin antibodies. Fig. 2shows that ecotin co-migrated with intact phage particles (lane 1), indicating that the ecotin-pIII fusion was expressed and incorporated into the phage particles. In contrast, phagemids lacking the ecotin-pIII fusion (pBluescript, lane 2) or having a deletion/frameshift mutation within ecotin-pIII fusion (pBSeco-gIII, lane 3) did not interact with anti-ecotin antibodies. These results established that ecotin was displayed on phage only in the context of a correct ecotin-pIII fusion. To determine whether the ecotin molecules displayed on the surface of phage were active, the ability of ecotin phage to inhibit the proteolytic activity of trypsin was determined. Ecotin phage were incubated with rat trypsin for 20 min and the residual proteolytic activity was measured. Fig. 3shows that trypsin activity decreased as the concentration of ecotin phage increased, whereas trypsin activity remained essentially unchanged when incubated with pBSeco-gIII phage. Based on the observation that approximately 10% of the phagemid particles were infectious and that, on average, each virion contained one copy of the ecotin-pIII fusion protein as judged by its immunochemical activity compared to a known amount of free ecotin protein (data not shown), a measure of the affinity of ecotin phage for trypsin could be calculated. An apparent equilibrium dissociation constant, K, of 6.7 nM for ecotin phage and trypsin was calculated using reversible tight-binding kinetics. These results suggested that ecotin was correctly folded and active as a fusion with pIII.


Figure 2: Characterization of ecotin-pIII fusion phage. Phage samples were loaded in duplicate and fractionated on a single 1% agarose gel. One set of samples was stained with ethidium bromide (panel A). The other set of samples was transblotted onto a nitrocellulose filter, which was immunostained for ecotin (panel B) (see ``Experimental Procedures'' for details). Lane 1, pBSeco-gIII; lane 2, pBluescript; lane 3, pBSeco-gIII; lane 4, pBSeco-gIII P1/P1` library. See ``Experimental Procedures'' for details.




Figure 3: Inhibition of rat trypsin activity by pBSeco-gIII phage. Various concentrations of phage were mixed with rat trypsin in a 96-well microtiter plate. Following a 20-min incubation at room temperature, the substrate, Z-Gly-Pro-Arg-p-nitroanilide, was added to the mixture. Residual trypsin activity in percentage is expressed as the ratio of the rate of Z-Gly-Pro-Arg-p-nitroanilide hydrolysis in the presence of phage to that in the absence of phage at various phage concentrations. Data were average of two independent experiments and agreed within 12%. Circle, pBSeco-gIII; triangle, pBSeco-gIII.



Binding Enrichments on Trypsin-coated Dishes

Biopanning against immobilized trypsin was carried out to determine whether ecotin phage could be selectively enriched from a background phage population. Bovine trypsin was used to coat the bottom of polystyrene Petri dishes by incubating a 10 µg/ml trypsin solution in the polystyrene dishes. After extensive washing, the presence of immobilized trypsin on the dish was verified by its ability to hydrolyze the chromogenic substrate Z-Gly-Pro-Arg-p-nitroanilide (data not shown). A mixture of ecotin phage and pBluescript phage at an approximately 1:2800 ratio was incubated with the trypsin-coated dishes overnight at room temperature. After extensive washing, the bound phage were eluted with low pH buffer (0.1 N HCl/glycine, pH 2.2). These conditions are known to dissociate the trypsin-ecotin complex (27) . Because the insertion of the ecotin-gIII fusion gene in pBluescript disrupted the gene coding for lacZ, F` JM101 cells infected with pBSeco-gIII phage yielded white colonies on ampicillin plates containing IPTG and 5-bromo-4-chloro-3-indolyl -D-galactoside. This permitted discrimination between pBluescript phage (blue colonies) and ecotin phage (white colonies). The results in show that, starting with a ratio of ecotin phage to pBluescript phage of 1:2800, the ratio of ecotin phage to pBluescript phage exceeded 4:1 after one cycle of binding and elution on trypsin-coated dishes. Thus, a single step of selection yielded greater than 10-fold enrichment. In contrast, only a slight enrichment (10-fold) was found when the experiment was performed with dishes coated with 5% milk. Subsequent elutions also showed greater than 10-fold enrichments on trypsin-coated dishes.

Library Construction and the Search for a High Affinity Urokinase Inhibitor

Oligonucleotide-directed mutagenesis was used to introduce random mutations at the P1 (84) and P1` (85) sites of ecotin. These amino acids are in the reactive-site loop of the inhibitor and flank the scissile peptide bond. The sequence NNS, where n = A/C/G/T, and S = G/C, was incorporated into each codon to create a mutant library with all possible amino acid substitutions, 1024 possible DNA sequences and 400 possible protein sequences. After the mutagenesis reaction, the DNA mixture was ethanol-precipitated and electroporated into JM101 cells. Approximately 5 10 individual clones were obtained in a single electroporation. A ScaI restriction digest of DNA from 20 individual clones revealed that approximately 60% of the transformants were derived from mutagenesis and 40% were background resulting from the single-stranded DNA template. Based on the nucleotide sequences of 64 mutated codons from the library, the frequencies of nucleotide occurrence were: n = 23% A, 19% C, 31% G, and 27% T; S = 30% C, 70% G. Although the occurrence of nucleotides in this library is not evenly distributed, especially at the third position of the codon, the large number of individual transformants (3 10) greatly exceeds the 4.8 10 transformants necessary for a 99% confidence level that each member of the library is represented.

Our specific target for binding selection is human uPA. It has been shown that receptor binding of uPA through the EGF-like domain does not shield uPA from the action of its endogenous inhibitors, PAI-1 (50, 51) and PAI-2 (52, 53) , nor does it modulate the proteolytic activity of uPA (54) . Since intact uPA is unstable and readily degrades to its low molecular weight form (LMuPA) which contains the protease domain (7) , our binding selections were carried out using LMuPA. Library phage displaying ecotin variants were cycled through rounds of binding selection to isolate mutants with high affinity. A strong consensus sequence was observed after only one cycle of binding selection (). Of the 15 clones sequenced, 13 had Arg at the P1 position, while Met predominated at the P1` position (10) , along with a minor population of Arg (3) , Lys (1) , and Thr (1) . It should be noted that only two wild-type clones were observed from 15 sequenced clones, in spite of the fact that wild-type ecotin phage accounted for approximately 40% of the original library. This suggested that the panning process was not a random selection. As the binding selection was carried through subsequent cycles, a dramatic change in ecotin variant phage population was observed. Wild-type ecotin phage were not detected after the second cycle of selection. The observed population of the ecotin M84R phage decreased from 53% in the first cycle to 20% in the second cycle and to 10% in the third cycle, while the population of ecotin M84R/M85R phage that was initially less abundant increased from 20% in the first cycle to 60% in the second cycle and to 70% in the third cycle.

The wild-type ecotin and ecotin mutants derived from the third cycle of selection were cloned into the expression vector pTacTac. Proteins were expressed in E. coli, and purified to >95% homogeneity (data not shown). The equilibrium dissociation constants, K, of the mutants toward LMuPA were determined using tight-binding kinetics (47, 48) and are shown in I. All mutants exhibited significantly higher affinity than that of wild-type ecotin. The double mutant, ecotin M84R/M85R, was the most potent, with a 2800-fold increase in affinity toward uPA.

DISCUSSION

There is an extensive array of naturally evolved macromolecular inhibitors that regulate the proteolytic activities of serine proteases. Although the three-dimensional structure of the various scaffolds may differ among these inhibitors, usually a reactive-site loop complementary to the substrate specificity of the proteases is presented to the target protease in such a way to cripple the active-site machinery of the enzyme. A classic example of this type of interaction can be found in the bovine pancreatic trypsin inhibitor-trypsin complex. It was previously shown that phage display can be used to alter the inhibitory profile of bovine pancreatic trypsin inhibitor against the serine protease, human neutrophil elastase (32) . It had yet to be shown that this approach could be applied to other protease inhibitors. Ecotin is unique among the serine protease inhibitors in having a dyad-related secondary binding site that permits recognition of a wide range of proteases through a network-linked array of interactions. By showing that ecotin can be displayed on the surface of filamentous phage and can be remodeled to inhibit uPA, the application has been broadened to show that protein engineering methods can be used to design an even greater array of potent macromolecular inhibitors.

Ecotin was shown to be associated with the filamentous phage by localizing ecotin antibodies to phage particles. Ecotin phage also inhibited trypsin activity, suggesting that ecotin is folded and active even when produced as a fusion protein. In contrast, phagemids lacking ecotin-pIII on their surface failed to inhibit trypsin activity, suggesting that inhibition occurred through a specific interaction between trypsin and ecotin rather than a nonspecific interaction between trypsin and phage particles. The equilibrium dissociation constant for ecotin phage and trypsin was estimated to be 6.7 nM. This value is within 3-fold of the Kof free ecotin (2.3 nM). The lowered affinity of the ecotin-pIII fusion may be due to blocked access for trypsin by the phage particle, or may simply be experimental error due to the inherent difficulty of direct measurement of the number of phage particles and ecotin molecules incorporated. Nevertheless, this result suggested that the phage-associated ecotin was highly active and virtually unhindered by the presence of the associated bacteriophage.

Ecotin phage were enriched from a pool of background phage when biopanning against immobilized trypsin was carried out; an enrichment of greater than 10-fold was achieved after the first elution of the bound phage in the first cycle of binding selection. This enrichment was comparable to those reported for antigen-antibody (33, 34) and hormone-hormone receptor (36) interactions. Subsequent elutions (elution 2 and elution 3) yielded lower numbers of phage, but the enrichment ratio essentially remained unchanged. In contrast, almost no enrichment was observed when biopanning was carried out in the absence of a target protease. These results showed that the presence of trypsin on the plate was required for enrichment, suggesting that binding occurred by specific interaction between ecotin and trypsin.

Having established conditions for binding selection, a phage library was constructed with random mutations at the P1 (Met-84) and P1` (Met-85) sites of ecotin in an attempt to search for high affinity inhibitors for uPA. These two sites were chosen for randomization because they flanked the scissile peptide bond. Furthermore, the side chains of Met-84 and Met-85 have been shown to interact with the S1 and S1` sites of trypsin in the three-dimensional structure of the trypsin-ecotin complex. Like most substrate-like inhibitors, these sites are major determinants for inhibition specificity. A strong consensus sequence developed after one cycle of selection of 3 10 phage; all except two wild-type ecotin phage had Arg at the P1 site, with ecotin M84R/M85 predominating. Arg and Lys were the only residues found at the P1 site in the subsequent binding selection cycles, and Arg was also the favored residue at the P1` site. Examination of the equilibrium dissociation constant, K, using tight-binding kinetics showed that all ecotin mutants from the third cycle of selection exhibited high affinity toward uPA and represented an 800-2,800-fold increase in potency relative to wild-type ecotin. The engineered protease inhibitors selected here are the most potent uPA inhibitors described to date; using comparable assay conditions the affinity of ecotin M84R/M85R for uPA (K= 1 nM) was 160-fold higher than that of a synthetic inhibitor, 4-benzodioxolanylethenly benzo[b]thiophene-2-carboxamidine (K= 160 nM), which was the most potent synthetic uPA inhibitor previously described (19) .

Previous studies on the roles of the P1-P1` residues of PAI-1 found that residues other than Arg or Lys at P1 displayed little or drastically reduced affinity for tPA or uPA (55) . Although ecotin and PAI-1 belong to different inhibitor families and their mechanisms of action are quite different (31, 56) , our selection results are consistent with that of a mutagenesis study of PAI-1 in that Arg and Lys were the only residues found at the P1 site of ecotin. The P1 residue of ecotin, Met-84, did not appear to interact strongly with the substrate binding pocket of trypsin (30) , suggesting that interactions between ecotin and trypsin at sites other than the reactive center have a significant contribution to the observed affinity. We hypothesize that these interaction sites fail to interact with uPA properly so that a basic residue at P1 that forms a strong electrostatic interaction with Asp in the substrate binding pocket of uPA is required to confer significant inhibition. A comparison of the crystal structures of ecotin-trypsin (30) and ecotin-collagenase() complexes revealed a significant displacement at the binding loops of ecotin, suggesting that these loops adopted different conformations when encountering different proteases in order to attain an optimal complementary fit. This property is unique among the known protease inhibitors, in which the reactive loops are less affected by complex formation and their canonical conformations remain essentially unchanged when complexed with different proteases (31) . Based on these observations, mutations can be introduced at the inhibitor-protease interface to remodel ecotin to attain improved binding affinity and selectivity. Phage display can then provide an efficient means of screening a conformationally diverse library from which an inhibitor with the desired specificity can be selected.

The exact structural role of the P1` residue in binding with uPA is not known, due to the unavailability of the uPA structure. The fact that basic residues (Arg and Lys) were preferred at the P1` site suggests an electrostatic interaction at the interface with uPA. Sequence alignment of bovine trypsin and uPA (7) revealed that an Asp is inserted in a surface loop that, in trypsin, interacts with the prime sites of the inhibitor (30, 57) and, in the case of ecotin, may form an electrostatic interaction with Arg or Lys at the P1` site. Further evidence supporting this hypothesis came from a previous study of the PAI-1-uPA interaction in which a Glu at the P1` site was found to significantly reduce the second order rate constant of inhibition (55) , presumably by charge repulsion.

The specific inhibition of proteases associated with various disease states holds great promise as an alternative to current therapies. Highly specific inhibitors of the proteases involved in cancer may prove to be a less toxic alternative to the chemotherapies currently in use. Particular stages of cancer are marked by an imbalance of proteolytic activity. Elevated levels of proteases such as cathepsins B and D, collagenase IV, and uPA are found in tumor tissues and correlate with tumor cell invasion and metastasis (3, 58, 59) . Specific inhibition of a given protease by a macromolecular inhibitor can serve as a starting point for subsequent development of small molecule inhibitors. We have successfully displayed ecotin on the surface of filamentous bacteriophage and demonstrated the use of this system to engineer potent inhibitors for uPA. Owing to the unique structural property of ecotin and the power of the in vitro selection, the system described here could find a general application in engineering specific ecotin-based inhibitors for target proteases, particularly for those of clinical importance.

  
Table: Binding enrichments of ecotin phage on trypsin- or milk-coated plates


  
Table: Identity of ecotin mutants from the P1/P1` library after various cycles of binding selection to immobilized LMuPA


  
Table: Apparent equilibrium dissociation constants for ecotin and ecotin mutants with LMuPA



FOOTNOTES

*
This work was supported by National Science Foundation Grant MCB-9219806 and National Institutes of Health Pharmaceutical Chemistry, Pharmacology, Toxicology Training Grant GM07175 (to Q. Y.).

§
To whom correspondence should be addressed. Tel.: 415-476-8146; Fax: 415-476-0688; E-mail: craik@cgl.ucsf.edu.

The abbreviations used here are: uPA, urokinase-type plasminogen activator; cfu, ampicillin-resistant colony forming unit; EGF, epidermal growth factor; IPTG, isopropylthio--D-galactoside; LMuPA, low molecular weight urokinase-type plasminogen activator; pIII, gene III product of M13 bacteriophage; PAI-1, type 1 plasminogen activator inhibitor; tPA, tissue-type plasminogen activator; Z, carbobenzoxy.

J. Peron, C. Tsu, C. Craik, and R. Fletterick, unpublished data.


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