(Received for publication, May 12, 1997)
From the Department of Vascular Biology, The Scripps
Research Institute, La Jolla, California 92037 and the
§ Department of Pharmacology and Howard Hughes Medical
Institute, University of Texas Southwestern Medical Center,
Dallas, Texas 75235
Human urokinase type plasminogen activator (u-PA)
is a member of the chymotrypsin family of serine proteases that can
play important roles in both health and disease. We have used substrate phage display techniques to characterize the specificity of this enzyme
in detail and to identify peptides that are cleaved 840-5300 times
more efficiently by u-PA than peptides containing the physiological target sequence of the enzyme. In addition, unlike peptides containing the physiological target sequence, the peptide substrates selected in
this study were cleaved as much as 120 times more efficiently by u-PA
than by tissue type plasminogen activator (t-PA), an intimately related
enzyme. Analysis of the selected peptide substrates strongly suggested
that the primary sequence SGRSA, from position P3 to P2, represents
optimal subsite occupancy for substrates of u-PA. Insights gained in
these investigations were used to design a variant of plasminogen
activator inhibitor type 1, the primary physiological inhibitor of both
u-PA and t-PA, that inhibited u-PA approximately 70 times more rapidly
than it inhibited t-PA. These observations provide a solid foundation
for the design of highly selective, high affinity inhibitors of u-PA
and, consequently, may facilitate the development of novel therapeutic
agents to inhibit the initiation and/or progression of selected human
tumors.
Local activation and aggregation of platelets, followed by initiation of the blood coagulation cascade, assure that a fibrin clot will form rapidly in response to vascular injury (1). The presence of this thrombus, however, must be transient if the damaged tissue is to be remodeled and normal blood flow restored. The fibrinolytic system, which accomplishes the enzymatic degradation of fibrin, is therefore an essential component of the hemostatic system (1). The ultimate product of the fibrinolytic system is plasmin, a chymotrypsin family enzyme with relatively broad, trypsin-like primary specificity that is directly responsible for the efficient degradation of a fibrin clot (2). Production of this mature proteolytic enzyme from the inactive precursor, or zymogen, plasminogen is the rate-limiting step in the fibrinolytic cascade (2, 3). Catalysis of this key, regulatory reaction is tightly controlled in vivo and is mediated by two enzymes present in human plasma, u-PA1 and t-PA (3-6).
u-PA and t-PA are very closely related members of the chymotrypsin gene family. These two proteases possess extremely high structural similarity (7, 8), share the same primary physiological substrate (plasminogen) and inhibitor (plasminogen activator inhibitor, type 1) (3), and, unlike plasmin, exhibit remarkably stringent substrate specificity (9-11). Despite their striking similarities, the physiological roles of t-PA and u-PA are distinct (5, 6), and many studies (5, 6, 12-18) suggest selective inhibition of either enzyme might have beneficial therapeutic effects. Mice lacking t-PA, for example, are resistant to specific excitotoxins that cause extensive neurodegeneration in wild type mice (13), and mice lacking u-PA exhibit defects in the proliferation and/or migration of smooth muscle cells in a model of restenosis following vascular injury (5, 6).
A large body of experimental evidence from studies involving both model systems and human patients suggests that u-PA may play an important role in tumor biology and provides a compelling rationale to pursue the development of u-PA inhibitors. For example, anti-u-PA antibodies inhibit metastasis of HEp3 human carcinoma cells to chick embryo lymph nodes, heart, and lung (19), and similar studies demonstrated that these antibodies inhibit lung metastasis in mice following injection of B16 melanoma cells into the tail vein (20). Anti-u-PA antibodies also inhibit both local invasiveness and lung metastasis in nude mice bearing subcutaneous MDA-MB-231 breast carcinoma tumors (21). In addition, a recent study indicated that u-PA-deficient mice are resistant to the induction and/or progression of several tumor types in a two-stage, chemical carcinogenesis model (18). Finally, high levels of tumor-associated u-PA correlate strongly with both a shortened disease-free interval and poor survival in several different human cancers (22-24).
Because mice lacking either u-PA or t-PA do not develop thrombotic disorders, selective inhibition of either of these two enzymes seems unlikely to create thrombotic complications in vivo. On the other hand, mice lacking both u-PA and t-PA suffer severe thrombosis in many organs and tissues, resulting in a significantly reduced life expectancy (5, 6). Nonselective inhibition of these two enzymes, therefore, seems almost certain to produce catastrophic consequences in the clinical setting. Consequently, significant interest exists in the development of inhibitors that are stringently specific for either t-PA or u-PA, which are expected to facilitate a detailed investigation of the precise roles of the two enzymes in several important pathological processes and may aid the development of novel therapeutic agents to combat these processes. Rational design of these selective inhibitors is greatly complicated, however, by the absence of obvious "lead compounds"; both their primary physiological substrate and inhibitors fail to discriminate between the two closely related proteases.
We have used substrate phage display (25, 26) to elucidate optimal subsite occupancy of u-PA. Peptide substrates that match the consensus sequence for substrates of u-PA derived from these studies are cleaved by u-PA 840-5300 times more efficiently than control peptides containing the physiological target sequence present in plasminogen. In addition, unlike the plasminogen-derived control peptides, the selected peptides exhibit substantial selectivity for cleavage by u-PA versus t-PA. Information gained in these investigations was used to augment the u-PA/t-PA selectivity of PAI-1, the physiological inhibitor of both t-PA and u-PA (27, 28); suggests potential lead compounds for the design of selective, small molecule inhibitors of u-PA; and provides new insights into the divergent evolution of molecular recognition by intimately related enzymes.
Competent MC1061 (F) Escherichia coli
and nitrocellulose were purchased from Bio-Rad Laboratories. Pansorbin
(Protein A-bearing Staphylococcus aureus) cells were
obtained from Calbiochem. K91 (F+) and MC1061
(F) strains of E. coli were provided by Steve
Cwirla (Affymax). mAb 3-E7 was purchased from Gramsch
Laboratories (Schwabhausen, FRG). u-PA was obtained from Jack
Henkin (Abbott Laboratories).
Construction of the phage vector fAFF1-tether C (fTC) and the random
hexapeptide library fAFF-TC-LIB has been previously described (26).
Control substrate phage fTC-PL, which contained the physiological target sequence for u-PA and t-PA, was constructed by
hybridizing the single-stranded oligonucleotides
5-TCGAGCGGTGGATCCGGTACTGGTCGTACTGGTCATGCTCTGGTAC-3
and
5
-CGCCACCTAGGCCAGGACCAGCACAACAACCACGAGAC-3
and then ligating the annealed, double-stranded products into the
XhoI/KpnI-cut vector fTC. All constructs were
first transformed into MC1061 by electroporation and then transferred
into K91.
Concentrations of functional t-PA and u-PA were measured by active site titration with 4-methylumbelliferyl p-guanidinobenzoate (29) using a Perkin-Elmer LS 50B Luminescence Fluorometer as described previously (9, 30). In addition, the enzymes were titrated with a standard PAI-1 preparation that had been previously titrated against a trypsin primary standard. Total enzyme concentrations were measured by enzyme-linked immunosorbent assay.
Phage Selection Using u-PASubstrate phage display was originally developed by Matthews and Wells (25) using monovalent phage, and an alternative method that used multivalent phage was reported later by Smith and Navre (26). Multivalent substrate phage were screened with u-PA using reaction conditions identical to those previously reported for t-PA (31) except that digestion of the phage was performed using enzyme concentrations varying from 2 to 10 µg/ml and incubation times varying from 0.5 to 10 h.
Dot Blot Assay of Phage ProteolysisPhage precipitation and dot blot analysis were performed as described previously (26, 31). Individual phage stocks were prepared and digested with no enzyme, t-PA, u-PA, or u-PA in the presence of 1 mM amiloride, a specific inhibitor of u-PA, for periods of time varying from 15 min to 10 h. Individual reaction mixtures were spotted onto a nitrocellulose filter using a dot blotter apparatus (Bio-Rad). The filter was probed with mAb 3E-7 and developed using the Amersham Western ECL kit. Loss of positive staining indicates loss of antibody epitopes from the phage due to proteolytic cleavage of the randomized hexamer region.
Preparation and Sequencing of DNA from Phage ClonesDNA samples were prepared from interesting phage clones as described previously (31). Briefly, phage were precipitated from a 1-ml overnight culture by adding 200 µl of 20% polyethylene glycol in 2.5 M NaCl. The mixture was incubated on ice for 30 min, and the phage pellet was collected by microcentrifugation for 5 min. The phage were resuspended in 40 µl of lysis buffer (10 mM Tris-HCl, pH 7.6, 0.1 mM EDTA, 0.5% Triton X-100) and heated at 80 °C for 15 min. Single-stranded DNA was purified by phenol extraction and ethanol precipitation and sequenced by the dideoxy method.
Kinetics of Cleavage of Synthetic Peptides by t-PA and u-PAPeptides were synthesized and purified as described (9). Kinetic data were obtained by incubating various concentrations of peptide with a constant enzyme concentration to achieve between 5 and 20% cleavage of the peptide in each reaction. For assays with u-PA, enzyme concentration was either 815 or 635 nM. For assays with t-PA enzyme, concentration was 700 nM. Peptide concentrations were chosen where possible to surround Km and in all cases were between 0.5 and 32 mM. The buffer used in these assays has been described (9). Reactions were stopped by the addition of trifluoroacetic acid to 0.33% or by freezing on dry ice. Cleavage of the 13- and 14-residue peptides was monitored by reverse phase HPLC as described (9). The 4-6-residue peptides were acylated at their amino termini and amidated at their carboxyl termini. Cleavage of the 4-6-residue peptides was monitored by hydrophilic interaction HPLC chromatography (32) using a polyhydroxyaspartamine column from PolyLC (Columbia, MD). Buffer A was 50 mM triethylamine phosphate in 10% acetonitrile, and buffer B was 10 mM triethylamine phosphate in 80% acetonitrile. Peptides were eluted by a gradient that was varied from 100% buffer B to 100% buffer A during a 13-min interval. The percentage of cleaved peptide was calculated by dividing the area under the product peaks by the total area under substrate and product peaks. For all peptides containing multiple basic residues, mass spectral analysis of products confirmed that cleavage occurred at a single site and identified the scissile bond. Data were interpreted by Eadie-Hofstee analysis. Errors were determined as described (33) and were <25%.
Site-directed Mutagenesis and Construction of an Expression Vector Encoding a Recombinant Variant of PAI-1The expression
vector pPAIST7HS was derived from the plasmid pBR322 and contained a
full-length cDNA encoding human PAI-1 that was transcribed from a
T7 gene 10 promoter (34). The 300-base pair
SalI/BamHI fragment of human PAI-1 was subcloned
from pPAIST7HS into bacteriophage M13mp18. Single-stranded DNA produced
by the recombinant M13mp18 constructs was used as a template for
site-specific mutagenesis according to the method of Zoller and Smith
(35) as modified by Kunkel (36). The mutagenic oligonucleotide
had the sequence
5-CCACAGCTGTCATAGGCAGCGGCAAAAGCGCCCCCGAGGAGATC-3
.
Following mutagenesis, single-stranded DNA corresponding to the entire
300-base pair SalI-BamHI fragment was fully
sequenced to ensure the presence of the desired mutations and the
absence of any additional mutation. The 300-base pair
SalI-BamHI double-stranded DNA fragment from the
mutated, replicative form DNA was used to replace the corresponding
fragment in pPAIST7HS to yield a full-length cDNA encoding
PAI-1/UK1, which contained the amino acid sequence GSGKSA from the P4
to P2 position of the reactive center loop.
Expression of wild type and the mutated
variant of PAI-1 was accomplished in the E. coli strain
BL21[DE3]pLyss (Novagen), which synthesizes T7 RNA
polymerase in the presence of
isopropyl-1-thio--D-galactopyranoside. Bacterial
cultures were grown at 37 °C with vigorous shaking to an
A595 of 0.9-1.1, and
isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 1 mM to induce the synthesis of T7
RNA polymerase and the production of PAI-1 proteins. Cultures were
grown for an additional 1-2 h at 37 °C and then shifted to 30 °C
for 2-6 h.
Cells were pelleted by centrifugation at 8000 × g for 20 min at 4 °C and resuspended in 40 ml of cold start buffer (20 mM sodium acetate, 200 mM NaCl and 0.01% Tween 20, pH 5.6). The cell suspension was disrupted in a French pressure cell (Aminco), and cellular debris was removed by ultracentrifugation for 25 min at 32,000 × g.
Purification of soluble, active PAI-1 was performed as described previously (37). PAI-1 containing supernatants were injected onto a XK-26 column (Pharmacia Biotech Inc.) packed with CM-50 Sephadex (Pharmacia). The column was washed with 5 column volumes of start buffer (20 mM sodium acetate, 200 mM NaCl, and 0.01% Tween 20, pH 5.6), and PAI-1 proteins were eluted using a 0.2-1.8 M linear gradient of NaCl in the same buffer. Peak fractions were collected, pooled, and concentrated using a Centriplus 30 concentrator (Amicon). Purified preparations were analyzed by activity measurements using standard, direct assays of t-PA, SDS-polyacrylamide gel electrophoresis, and measurement of optical density at 280 nm.
Measurement of Active PAI-1 in Purified PreparationsA primary standard of trypsin was prepared by active site titration using p-nitrophenyl guanidinobenzoate HCl as described previously (38). Concentrations of active molecules in purified preparations of wild type or mutated PAI-1s were determined by titration of standardized trypsin as described by Olson et al. (39) and by titration of standardized t-PA preparations.
Kinetic Analysis of the Inhibition of t-PA and u-PA by Recombinant PAI-1 and PAI-1/UK1Second order rate constants (ki) for inhibition of t-PA or u-PA were determined using pseudo-first order (ki < 2 × 106) or second order (ki > 2 × 106) conditions. For each reaction, the concentrations of enzyme and inhibitor were chosen to yield several data points for which the residual enzymatic activity varied between 20 and 80% of the initial activity. Reaction conditions and data analysis for pseudo-first order reactions were as described previously (40-43).
For second order reactions, equimolar concentrations of u-PA and PAI-1 were mixed directly in microtiter plate wells and preincubated at room temperature for periods of time varying from 0 to 30 min. Following preincubation, the mixtures were quenched with an excess of neutralizing anti-PAI-1 antibody (generously provided by Dr. David Loskutoff), and residual enzymatic activity was measured using a standard, indirect chromogenic assay. These indirect, chromogenic assays were compared with control reactions containing no PAI-1 or to which PAI-1 was added after preincubation and the addition of anti-PAI-1 antibody, plasminogen, and Spec PL to the reaction mixture. Data were analyzed by plotting the reciprocal of the residual enzyme concentration versus the time of preincubation.
A polyvalent fd phage library that displayed random hexapeptide sequences and contained 2 × 108 independent recombinants was prepared (25, 31). Each member of this library displayed an N-terminal extension from phage coat protein III that contained a randomized region of six amino acids, a six-residue linker sequence (SSGGSG), and the epitopes for mAbs 179 and 3-E7. Because u-PA did not digest the phage coat protein III sequence, the antibody epitopes, or the flexible linker sequence, the loss of antibody epitopes from the phage surface upon incubation with u-PA required cleavage of the randomized peptide insert. Incubation of the library with u-PA, followed by removal of phage retaining the antibody epitopes, therefore, accomplished a large enrichment of phage clones whose random hexamer sequence could be cleaved by u-PA.
Analysis of Selected Phage Clones and Identification of a Consensus SequenceFollowing five rounds of selection to enrich and amplify
phage that display sequences that are readily cleaved by u-PA, 100 phage clones were identified as u-PA substrates. DNA sequencing of
these clones revealed the presence of 91 distinct hexamer sequences among the selected phage (Table I). As
expected from the trypsin-like primary specificity of u-PA, each
hexamer contained at least one basic residue, and 89 of the 91 hexamer
sequences contained at least one arginine residue. 35 of the 91 substrate phage contained a single basic residue, and in 33 of these 35 cases the single basic residue was an arginine. An additional 22 phage
contained two basic residues but only a single arginine. Alignment and
analysis of these hexamer sequences suggested that the consensus
sequence for optimal subsite occupancy for substrates of u-PA, from P3 to P2, was SGR(S > R,K,A)X, where X
represents a variety of amino acid residues but was most often alanine,
glycine, serine, valine, or arginine.
|
Analysis of these data was complicated by the fact that approximately 72% of the selected substrate phage contained an arginine in the first position of the randomized hexamer and therefore utilized the amino-terminal flanking residues, Ser-Gly, to occupy the P3 and P2 subsites. While these results left no doubt that the P3-P1 SGR sequence created by the fusion was a very favorable recognition site for u-PA, this use of flanking residues necessitated a particularly careful examination of the P3 and P2 preferences of u-PA. Consequently, we altered our experimental protocol in two ways to address this issue. First, we isolated an unusually large collection of substrate phage (91 distinct substrates) to ensure that a reasonable number of these (23) would not utilize the flanking Ser-Gly to fill the P3 and P2 subsites. This allowed a meaningful comparison of the consensus sequence derived from the entire library with that derived from the non-fusion phage and the demonstration of good agreement between the two consensus sequences. Second, we performed a previously described dot blot analysis (26, 31) of the digestion of all 100 substrate phage by u-PA using a wide variety of stringencies of digestion. Although this semiquantitative assay cannot provide kinetic constants, it can provide an accurate rank ordering of the lability of the substrate phage clones.
Under the most stringent conditions examined, 11 of the 100 substrate
phage, containing eight distinct randomized hexamer sequences, proved
to be particularly labile u-PA substrates (Table II). All eight of the most labile
substrate phage contained the P3-P1 SGR motif, demonstrating that this
sequence is, in fact, a more labile u-PA site than related, selected
sequences present in the library such as SSR, TAR, TSR, TTR, etc. This
dot blot analysis also yielded additional information regarding the
preferences of u-PA for the unprimed subsites. While analysis of the
entire substrate phage library failed to reveal a clear consensus at P1 and P2
, the most labile substrate phage displayed an obvious preference at both of these positions. Five of the eight most labile
phage contained a serine residue at P1
, and seven of these eight phage
contained an alanine residue at P2
. These observations strongly
suggest that the primary sequence SGRSA, from P3 to P2
, represents
optimal subsite occupancy for substrates of u-PA.
|
Four peptides containing
amino acid sequences present in the randomized hexamer region of the
most labile phage were chosen for detailed kinetic analysis (Table
III) and compared with the hydrolysis of
a control peptide (I) containing the P3-P4 sequence of plasminogen, a
series of residues that fall within a disulfide-linked loop in the
native protein. All four of the selected peptides were substantially
improved substrates for u-PA, by factors of 840-5300, compared with
the control, plasminogen peptide (Table III). These increases in
catalytic efficiency were mediated primarily by increases in
kcat, suggesting that optimized subsite
interactions served to lower the energy of the transition state rather
than the ground state. For example, compared with that of control
peptide (I), the Km for cleavage of the most labile
selected peptide (II) was reduced by a factor of 5.6; however, the
kcat was increased by a factor of more than 940. In addition, peptide substrates that interacted optimally with the
primary subsites of u-PA were selective for cleavage by u-PA relative
to t-PA. The four selected peptides (II-V), for example, were cleaved
16-89 times more efficiently by u-PA than by t-PA, and improvements in
both Km and kcat contributed
to the preferential hydrolysis by u-PA.
|
The kinetic
analysis described above was performed using substrate peptides that
were 14 amino acids in length. To confirm that the specificity we
observed was inherent in the selected hexapeptide sequences, we
examined the kinetics of cleavage of short peptides containing only
sequences found within selected hexapeptide sequences. Pentapeptide
VII, for example, was cleaved by u-PA with a catalytic efficiency of
1200 M1 s
1 and exhibited a
u-PA/t-PA selectivity of 20. The behavior of pentamer VII in these
assays, therefore, was very similar to that of peptide IV, a 14-mer
that contains the same P3-P2
sequence as the pentamer. These
observations indicate that appropriate occupancy of the P3-P2
subsites alone can create selective substrates for u-PA.
Differences at position 190 (chymotrypsin numbering system) between u-PA and t-PA suggest that u-PA may exhibit decreased discrimination between arginine and lysine at the P1 position of a substrate compared with t-PA (44). Consistent with this hypothesis and in contrast to the selected t-PA substrate library, the u-PA library did include members that contained a P1 lysine. This observation suggested that the u-PA/t-PA selectivity of a peptide substrate might be enhanced by placement of lysine in the P1 position, although this increased selectivity was likely to be accompanied by decreased reactivity toward u-PA. To test this hypothesis, we analyzed hydrolysis of a variant of u-PA selective peptide (VI) that contained a P1 lysine (peptide VIII). The P1 lysine mutation decreased the catalytic efficiency for cleavage of this peptide by a factor of 49 for t-PA and by a factor of 7 for u-PA. As predicted, then, the P1 lysine mutation did enhance the u-PA/t-PA selectivity of the peptide substrate by a factor of approximately 7. It is not surprising, therefore, that the most selective u-PA substrate, peptide IX, which is cleaved approximately 121 times more efficiently by u-PA than by t-PA, is derived from the randomized hexamer region of a substrate phage that contained a P1 lysine.
Importance of P3 and P4 for Discrimination between u-PA and t-PARecent investigations that explored optimal subsite occupancy for substrates of t-PA suggested that the P3 residue was the primary determinant of the ability of a substrate to discriminate between t-PA and u-PA and that this selectivity could be enhanced modestly by appropriate occupancy of P4 (11). These suggestions were based on evidence obtained from a statistical analysis of phage selected using a substrate subtraction protocol rather than by a kinetic analysis of peptide substrates. Consequently, to test these hypotheses, we synthesized variants of the most labile u-PA-selective substrate (peptide II) that contained mutations in the P3 and/or P4 positions and analyzed the hydrolysis of these peptides by u-PA and t-PA. In peptide X the P3 serine of peptide II was replaced by a tyrosine, and in peptide XI the P3 serine was replaced by arginine. As expected, these mutations substantially decreased the u-PA/t-PA selectivity of the peptide by a factor of 330 or 360, respectively, and actually converted the peptide into a t-PA-selective substrate. Moreover, mutation of both the P3 serine and P4 glycine of the most labile u-PA substrate to arginine and glutamine, respectively (peptide XII), decreased the u-PA/t-PA selectivity by a factor of 1200. These data confirm the proposed status of the P3 and P4 residues as specificity determinants for substrates of t-PA and u-PA and suggest a particularly prominent role of the P3 residue in this capacity.
Design and Characterization of a Variant of PAI-1 That Is Selective for u-PATo test the prediction that information gained from the
study of peptide substrates could facilitate the design of selective, high affinity inhibitors of urokinase, we sought to augment the u-PA/t-PA selectivity of the serpin PAI-1, the primary physiological inhibitor of both t-PA and u-PA. We used oligonucleotide-directed, site-specific mutagenesis to construct a variant of PAI-1 that contained the primary sequence found in the peptide substrate that was
most selective for u-PA, GSGKS, from the P4-P1 position of the
reactive center loop. Kinetic analysis indicated that the PAI-1 variant
inhibited u-PA approximately 70 times more rapidly than it inhibited
t-PA with second order rate constants for inhibition of u-PA and t-PA
of 6.2 × 106 M
1
s
1 and 9 × 104
M
1 s
1, respectively (Table IV).
In contrast, wild type PAI-1 inhibits u-PA and t-PA with second order
rate constants of 1.9 × 107
M
1 s
1 and 1.8 × 106 M
1 s
1,
respectively. As anticipated, therefore, the mutated serpin possessed a
u-PA/t-PA selectivity that was approximately 7-fold greater than that
of wild type PAI-1. Moreover, the 70-fold selectivity of the
PAI-1 variant is consistent with the value of 120 observed for
hydrolysis of the corresponding peptide substrate by the two enzymes
(Tables III and IV).
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u-PA and t-PA possess distinct but overlapping physiological and pathological roles, and the ability to selectively inhibit either enzyme with small molecules would allow these roles to be examined comprehensively both in vitro and in vivo. Normally, such inhibitor design would be based on knowledge of the sequences of endogenous protein substrates or inhibitors. This approach, however, is not possible with u-PA and t-PA because these enzymes share the same physiological substrate, plasminogen, and inhibitor, PAI-1. Furthermore, this similarity calls into question the hypothesis that highly selective inhibitors can be generated, since the specificities of the two enzymes appear so similar. We find, however, both in this study and in a previous study aimed at the design of t-PA selective substrates (11), that there are subtle but significant differences in optimal subsite occupancy between the two enzymes, and these distinctions can be elucidated by substrate phage display protocols.
Sequences Selected for Optimal Cleavage Do Not Resemble the Physiological Target SequenceA key observation of this study is
that the primary sequence SGRSA, from the P3-P2 positions of a
peptide substrate, affords highly labile subsite occupancy for
urokinase. This sequence differs at P3, P1
, and P2
from the target
sequence found in plasminogen (PGRVV) and is cleaved by u-PA greater
than 5300 times more efficiently. This major discrepancy, in both
primary sequence and lability, of the physiological target sequence and
the consensus sequence derived using substrate phage display protocols
suggests that a physiological target sequence is not necessarily a
reasonable lead compound for the design of specific, small molecule
substrates or inhibitors of highly selective serine proteases.
A major contribution to the discrepancy between the physiological and
consensus target sequences of u-PA almost certainly arises from the
highly conserved mechanism of zymogen activation of chymotrypsin family
enzymes (45). Following activation cleavage of a chymotrypsinogen-like
zymogen, the P1 and P2
residues insert into the activation pocket,
where they form a number of conserved hydrophobic interactions as well
as a new, buried salt bridge with the aspartic acid residue adjacent to
the active site serine (45-47). Because these interactions
substantially stabilize the active conformation of the mature enzyme,
this key role after activation cleavage places severe functional
constraints on the P1
and P2
residues of a chymotrypsinogen-like
zymogen and consequently prevents the two residues from evolving simply
to interact optimally with the activating enzyme. Consistent with this
hypothesis, the consensus and physiological target sequences for u-PA
agree well on the unprimed side of the scissile bond; however, the two
target sequences diverge dramatically at the P1
and P2
subsites.
Additional factors are also likely to contribute to the observed
discrepancy between the consensus and physiological target sequences
for u-PA. For example, modeling studies reported by Lamba, Huber, Bode
and co-workers (8) suggest the S1 and/or the S2
pockets utilized by
u-PA when hydrolyzing plasminogen may actually differ from those used
when hydrolyzing peptide substrates. Moreover, as the enzyme diverged
from a trypsin-like precursor, u-PA may have evolved a strong
dependence for efficient catalysis upon productive interactions with
substrates at secondary sites that diminished the contribution of
optimal interactions with primary subsites in the active site cleft.
Although the location, role, and even the existence of such secondary
contacts between u-PA and plasminogen remain obscure at the present
time, previous studies of the interaction of u-PA and t-PA with PAI-1
have demonstrated very clearly that these two enzymes are capable of
using specific, secondary contacts efficiently both to enhance
selectivity and to dampen the influence of optimal primary subsite
interactions (42, 48-50). Although the reactive center loop of PAI-1
has evolved to match optimal subsite occupancy for urokinase very
closely, in the absence of productive contact with a single, strong
secondary site of interaction between the two proteins, PAI-1 becomes a poor inhibitor of u-PA (50).
The identification of synthetic peptides that are cleaved up to 120 times more efficiently by u-PA than by t-PA raises the possibility that similar u-PA-selective (or t-PA-selective) physiological substrates may exist that are currently not appreciated. Differences in the phenotypes exhibited by mice lacking either of the two enzymes are consistent with this possibility (5, 6). This issue remains uncertain, however, because selective expression of t-PA or u-PA in particular microenvironments could also account for these distinct phenotypes.
Importance of the P3 Residue in Discriminating between u-PA and t-PABy demonstrating that mutation of the P3 residue alone could alter the relative u-PA/t-PA selectivity of a peptide substrate by a factor of greater than 300 (Table III), this study provided strong support for the hypothesis that the P3 residue was the primary determinant of the ability of a substrate to discriminate between u-PA and t-PA. We have previously reported that occupancy of P3 by arginine or large aromatic or hydrophobic residues favored cleavage by t-PA (11), and this investigation showed that a P3 serine residue favored cleavage by u-PA. In addition, this study demonstrated that more modest alterations of specificity could be achieved by selective occupancy of the P4 and P1 subsites. These data indicated that PAI-1, which contains a P3 serine, has evolved to match optimal subsite occupancy of u-PA more closely than that of t-PA. This observation may explain why PAI-1 inhibits u-PA more rapidly than it inhibits t-PA (Table IV) and suggests that, during the evolution of the fibrinolytic system, there may have been a greater need to suppress the activity of u-PA in the circulation than to regulate t-PA activity. Consistent with this hypothesis, the circulating, single chain form of u-PA is a true zymogen, while t-PA is secreted into the circulation as an active, single chain enzyme.
Substrate Phage Display Can Aid Inhibitor DesignAnother
implication of these studies is that information gained from the
application of substrate phage display libraries can lead directly to
the design of specific inhibitors. Although hydrolysis of the
selective, small peptide substrates by u-PA is characterized by
Km values in the 0.6-3 mM range, it has
been routinely observed that the introduction of a transition state
bond geometry adjacent to the P1 residue of a protease substrate can
create either a reversible inhibitor whose affinity for the target
protease is enhanced by 3-6 orders of magnitude or an irreversible inhibitor with an impressive second order rate constant for inhibition of the target protease (>105 M1
s
1) (for a review, see Ref. 51). Similar results using
the substrates identified in this study would create highly selective,
small molecule u-PA inhibitors, with affinities in the low nanomolar range, that might be further improved by subsequent, systematic chemical modification.
The ability to identify subtle but significant specificity differences between enzymes that share the same physiological substrates and inhibitors, as demonstrated in this study, is a fundamental challenge both for basic enzymology and rational drug design. Advances in this area will significantly enhance understanding of the molecular determinants and mechanisms of specific catalysis and may facilitate the design of highly selective and therapeutically valuable new enzymes.
We thank Steven Madden, Bikash C. Pramanik, and Dr. Clive Slaughter for peptide synthesis and mass spectral analysis.