Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substrates Sylvain M.. Selected substrates were then transplanted into the reactive site
Trang 1Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substrates
Sylvain M Cloutier1,2, Christoph Ku¨ndig2, Loyse M Felber1, Omar M Fattah1, Jair R Chagas3,
Christian M Gygi1, Patrice Jichlinski1, Hans-Ju¨rg Leisinger1and David Deperthes1,2
1
Urology Research Unit, Department of Urology, CHUV, Epalinges, Switzerland;2Med Discovery SA, Epalinges, Switzerland;
3
Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes, Brazil
The reactive site loop of serpins undoubtedly defines in part
their ability to inhibit a particular enzyme Exchanges in
the reactive loop of serpins might reassign the targets and
modify the serpin–protease interaction kinetics Based on
this concept, we have developed a procedure to change the
specificity of known serpins First, reactive loops are very
good substrates for the target enzymes Therefore, we have
used the phage-display technology to select from a
penta-peptide phage library the best substrates for the human
prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach,
J.P., Gygi, C.M., Leisinger, H.J & Deperthes, D (2002)
Eur J Biochem 269, 2747–2754] Selected substrates were then transplanted into the reactive site loop of a1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease Thus, we have developed some highly specific a1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity These inhibitors might be useful to help elucidate the importance
of hK2 in prostate cancer progression
Keywords: phage-display; protease; human kallikrein; inhibitor; a1-antichymotrypsin
Prostate cancer is currently the most commonly diagnosed
cancer in American men This pathology is the second
leading cause of cancer death after lung cancer and the
majority of the patients with locally advanced prostate
cancer have an increased risk for disease progression In this
progression, proteases are believed to play a pivotal role
in the malignant behaviour of cancer cells, including rapid
tumor growth, invasion and metastasis Human glandular
kallikrein (hK2) protein is a trypsin-like serine protease
expressed predominantly in the prostate epithelium First
isolated from human seminal plasma [1], hK2 has emerged
recently as a diagnostic marker for prostate cancer When
tested in combination with assays for various forms of
prostate specific antigen (PSA), hK2 seemed to be better
suited to distinguish malignant from benign prostate disease
than the well established marker PSA (prostate specific
antigen or hK3) [2–4] In addition to its role as a marker, the
proteolytic activities suggest that hK2 could contribute to
cancer progression Several potential functions for this
enzyme have been proposed, including the activation of
urokinase-type plasminogen activator [5] and inactivation
of plasminogen activator inhibitor-1 [6], activation of
pro-PSA [7], degradation of fibronectin [8] and degradation of
insulin-like growth factor binding protein (IGF-BP) [9]
Taking into account its prostate tissue-specific expression and the involvement of all its potential substrates in cancer development, hK2 can be considered as a potential thera-peutic target
The serpins (serine protease inhibitors) are a large family
of proteins implicated in the regulation of complex physio-logical processes These proteins of about 45 kDa can be subdivided into two groups, one being inhibitory and the other noninhibitory Serpins contain an exposed flexible reactive-site loop (RSL), which is implicated in the inter-action with the putative target protease Following the binding to the enzyme and cleavage of the P1-P’1 scissile bond of the RSL, a covalent complex is formed [10] Formation of this complex induces a major conformational rearrangement and thereby traps irreversibly the target protease The inhibitory specificity of serpins is attributed largely to the nature of the residues at P1-P¢1 positions and the length of the RSL Changing the RSL domain or the reactive site of serpins is one approach to understand the inhibitory process between a serpin and an enzyme [11–13] and to develop specific inhibitors
Several serpins, such as protein C inhibitor, a2-antiplas-min, antithrombin-III, a1-antichymotrypsin (ACT), or protease inhibitor 6 [8,14,15] have been identified as hK2 inhibitors The relatively slow complex formation between hK2 and ACT [14] is attributed mainly to residues Leu358-Ser359 at P1-P¢1 positions of the RSL, an unfavourable peptide bond for this trypsin-like enzyme
Modifications of the RSL of a1-antichymotrypsin have been performed with the aim of changing the specificity of this serpin Peptide sequences, selected as substrates for the enzyme hK2 by phage-display technology [16], have been used to replace the scissile bond and neighbour amino acid residues of the RSL Recombinant inhibitors were produced
in bacteria and purified by affinity chromatography
Correspondence to D Deperthes, Urology Research Unit, Biopoˆle,
Ch Croisettes 22, CH-1066 Epalinges, Switzerland.
Fax: + 41 21 6547133, Tel.: + 41 21 6547130,
E-mail: david.deperthes@urology-research.ch
Abbreviations: ACT, a1-antichymotrypsin; Chtr, chymotrypsin; HNE,
human neutrophil elastase; PK, plasma kallikrein; PSA, prostate
specific antigen; uPA, urokinase plasminogen activator.
(Received 7 November 2003, revised 5 December 2003,
accepted 12 December 2003)
Trang 2Compared to wild-type rACT, which inhibited hK2 very
slowly (12–16 h), the modified rACTs formed a covalent
complex very quickly (within minutes) Three of the six rACT
variants were specific to hK2 with high association constants
Materials and methods
Materials
hK2 and hK3 (PSA) were purified from human semen as
described previously [14,17] Anti-hK2 and anti-PSA
monoclonal Igs were a gift from R R Tremblay (Laval
University, Canada) Human chymotrypsin (Chtr),
urokin-ase plasminogen activator (uPA), human kallikrein hK1,
human plasma kallikrein (PK), human neutrophil elastase
(HNE) and commercial ACT (human plasma
a1-antichy-motrypsin) were purchased from Calbiochem
Z-Phe-Arg-AMC, Suc-Ala-Ala-Pro-Phe-Z-Phe-Arg-AMC, Z-Gly-Gly-Arg-Z-Phe-Arg-AMC,
MeOSuc-Ala-Ala-Pro-Val-AMC were purchased from
Calbiochem CFP-TFRSA-YFP fluorescent substrate was
developed as described previously [16,18] The cDNA for
human a1-antichymotrypsin (ACT) was a generous gift
from H Rubin (University of Pennsylvania)
Site-directed mutagenesis
Following the subcloning of ACT cDNA into pQE-9
expression vector (Qiagen, Germany) and the introduction
of a His6tag at the N terminal of rACTWT, two restriction
sites SacII and MluI, were incorporated 18 bp upstream
and 18 bp downstream of the P1 codon in RSL domain,
respectively These sites were created by a silent mutation
using oligonucleotides 5¢-GTGATTTTGACCGCGGTGG
CAGCAG-3¢ for SacII and 5¢-GCACAATGGTACGCG
TCTCCACTAATG-3¢ for MluI site and following the
quickchange mutagenesis protocol supplied by Stratagene
Construction and expression of recombinant wild-type
ACT and its variants
Six variants, which correspond to a change in the reactive
site loop in positions between P3 and P3¢ (Table 1), were
generated by PCR extension of the template
oligonucleo-tides: rACT8.20, 5¢-TACCGCGGTCAAAATCACCCTCC
5¢-TACCGCGGTCAAAATCACCAGGAGGTCTATC GATGTGGAGACGCGTGA-3¢; rACT8.3, 5¢-TACCGCG GTCAAAATCAGGGGGAGATCTGAGTTAGTGGA GACGCGTGA-3¢; rACT6.7, 5¢-TACCGCGGTCAAAAT CAAGCTTAGAACAACATTAGTGGAGACCGCTG A-3¢; rACT6.1, 5¢-TACCGCGGTCAAAATCATGACAA
5¢-TACCGCGGTCAAAATCACCGAGCGTGTCTCG CCCGTGGAGACGCGTGA-3¢ (where underlined sequ-ences encode new cleavage sites in the reactive site loop), using primers corresponding to the flanking regions: 5¢-TACCGCGGTCAAAATC-3¢ and 5¢-TCACGCGTGT CCAC-3¢ PCR products were digested with SacII and MluI restriction enzymes and then subcloned into digested rACTWTconstruct Recombinant serpins were produced in TG1 Escherichia coli strain Cells were grown at 37C in 2· TY media (16 g tryptone, 10 g yeast extract, 5 g NaCl per L) containing 100 lgÆmL)1 ampicillin to A600¼ 0.5 Isopropyl thio-b-D-galactoside (IPTG) was then added to a final concentration of 0.5 mM allowing the expression of recombinant serpins for 16 h at 16C The cells from
100 mL of culture were harvested by centrifugation, resus-pended in cold NaCl/Piand then passed through a French press to recover the total soluble cytoplasmic proteins Cell debris were removed by centrifugation and Ni2+ -nitrilotri-acetic affinity agarose beads were added to the supernatant for 90 min at 4C to bind recombinant serpins The resin was washed subsequently with 50 mMTris, pH 8.0, 500 mM NaCl, 25 mMimidazole and the bound proteins were eluted for 10 min with 50 mM Tris, pH 8.0, 500 mM NaCl and
150 mMimidazole Once purification was completed, rACT were dialysed against 50 mMTris, pH 8.0, 500 mMNaCl, 0.05% Triton X-100 for 16 h at 4C The protein concentration was determined for each purification by Bradford assay and normalized by densitometry of Coo-massie Blue-stained SDS/PAGE gels [19]
Inhibition assays and stoichiometry of inhibition The stoichiometry of inhibition (SI) values were determined for the inhibition of rACTWTand its variants with hK2 and different other enzymes An initial test was made with a molar excess of rACT (100-fold) over hK2, PSA, hK1, chymotrypsin (Chtr), plasma kallikrein (PK), urokinase (uPA) and human neutrophile elastase (HNE) enzymes The
Table 1 Alignment of RSL (reactive serpin loop) of recombinant serpin a1-antichymotrypsin (ACT) and its variants Substrate peptides selected by kallikrein hK2 using a phage-displayed random pentapeptide library (12) Plain type residues are common to rACT WT , bold residues correspond to substrate peptides relocated in RSL of ACT variants The scissile bond by hK2 in substrate peptides is designated by fl and putative cleavage site in serpins is marked by asterisks between the P1-P1¢ residues.
Serpin
Selected
Trang 3reaction was performed for 30 min at 25C (90 min at
37C for PSA) in reaction buffer (50 mM Tris, pH 7.5,
150 mM NaCl, 0.05% Triton X-100, 0.01% BSA) and
residual enzyme activity was measured by adding fluorescent
substrates (Z-Phe-Arg-AMC for hK1, hK2 and PK,
Suc-Ala-Ala-Pro-Phe-AMC for Chtr, Z-Gly-Gly-Arg-AMC for
uPA, MeOSuc-Ala-Ala-Pro-Val-AMC for HNE, and
CFP-TFRSA-YFP for PSA) Activity of enzyme in presence
of inhibitors was compared to uninhibited reaction For
reactions where an inhibition was observed, SI was
deter-mined by incubating different concentrations of
recombin-ant serpins Using linear regression analysis of fractional
activity (velocity of inhibited enzyme reaction/velocity of
uninhibited enzyme reaction) vs the molar ratio of the
inhibitor to enzyme ([Io]/[Eo]), the stoichiometry of
inhibi-tion, corresponding to the abscissa intercept, was obtained
Kinetics
The association rate constants for interactions of hK2,
chymotrypsin, PK and HNE with different rACTs were
determined under pseudo-first order conditions using the
progress curve method [20] Under these conditions, a fixed
amount of enzyme (2 nM) was mixed with different
concentrations of inhibitor (0–800 nM) and an excess of
substrate (10 lM) Each reaction was made in reaction
buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% (v/v)
Triton X-100, 0.01% (w/v) BSA] at 25C for 45 min and
the rate of product formation was measured using a FLx800
fluorescence 96-well microplate reader (Biotek, USA) In
this model, inhibition is considered to be irreversible over
the course of reaction and the progress of enzyme activity is
expressed by product formation (P), beginning at a rate (vz)
and is inhibited over time (t) at a first-order rate (kobs), rate
constant that is dependent only on inhibitor concentration
P¼ ðvz=kobsÞ ½1 eðkobstÞ ð1Þ
For each inhibitor, a kobswas calculated for four different
concentrations of inhibitors via a nonlinear regression of
the data using Eqn 1 By plotting the kobs vs inhibitor
concentration [I], a second-order rate constant, k¢, equal to
the slope of the curve (k¢ ¼ Dkobs/D[I]), was determined
Due to the competition between inhibitor and the substrate,
Eqn 2 below is used to correct the second-order rate constant
k¢ by taking into account the substrate concentration [S] and
the Kmof the enzyme for its substrate, giving the ka
ka¼ ð1 þ ½S=KmÞ k0 ð2Þ The Kmof hK2 for Z-FR-AMC, chymotrypsin for
Suc-AAPF-AMC, PK for Z-FR-AMC and HNE for
MeOSuc-AAPV-AMC were 67 lM, 145 lM, 170 lM and 130 lM,
respectively
Western blot analysis of complex formation
and inhibitor degradation
Kallikrein hK2 was incubated 3 h at 37C with different
recombinant ACTs at a [I]o/[E]oratio of 100 : 1 in 50 mM
Tris, 200 mM NaCl, 0.05% (v/v) Triton X-100 Protein
samples were heated at 95C for 5 min, separated by SDS/
PAGE [12% (v/v) acrylamide, 19 : 1; T/C ratio) and then
electroblotted onto Hybond-ECL (Amersham Pharmacia) nitrocellulose The free-hK2 and hK2-ACT complexes were detected using a mouse anti-hK2 monoclonal Ig and an alkaline phosphatase-conjugated goat anti-mouse secon-dary Ig Western blot was visualized using the ECL detection kit (Amersham Pharmacia Biotech) hK2 was also incubated with ACT8.3 or ACT6.7 30 min at 25C (kinetic conditions) at a [I]o/[E]oratio of 10 : 1 in 50 mM Tris, 200 mM NaCl, 0.05% Triton X-100 Proteins were detected by Western blot, using an anti-His6monoclonal Ig followed by detection with the secondary antibody and protocol described above
Results
Production of soluble recombinant wild-type and variant ACTs
Wild-type serpin a1-antichymotrypsin was used to develop specific inhibitors of the kallikrein hK2 Residues P3-P3¢ located in the RSL structure of rACTWT were replaced
by substrate pentapeptides previously selected by phage-display technology [16] Six variants of rACT have been designed and constructed (Table 1) The scissile bond in substrate peptides was aligned according to Leu358-Ser359 into RSL of the serpin rACTWT and its variants were expressed in E coli TG1 as fusion proteins containing a His tag in the N-terminal position Each of them was produced
at low temperature allowing protein accumulation, mainly
as the active soluble form Purified under native conditions, the level of production varied between 1.0 and 2.5 mgÆL)1 The purity of serpins was estimated by SDS/PAGE analysis and was more than 98% (Fig 1)
rACT variants are specific mainly to kallikrein hK2
A panel of enzymes including human neutrophil elastase, chymotrypsin-like (Chtr, PSA) and trypsin-like (hK2, hK1,
PK, uPA) proteases have been screened to determine inhibitory specificity of rACT variants (Table 2) Incubating with an excess of inhibitors ([I]o/[E]oof 100 : 1) for 30 min, hK2 is completely inhibited by rACT6.2, rACT8.3, rACT6.7 and rACT6.1, whereas rACT8.20 and rACT5.18 inhibited 95% and 73% of enzyme activity, respectively Under these
Fig 1 SDS/PAGE analysis of purified recombinant ACT under redu-cing conditions Variant 6.1 (lane 1) and wild-type ACT (lane2).
Trang 4conditions, wild-type rACT showed no inhibitory activity
toward hK2 Among these variants, two are specific to hK2
(rACT8.3and rACT5.18), inhibiting no other tested enzyme
Two other variants, rACT6.7 and rACT6.2, also inhibited
PK at 36% and 100%, respectively As with wild-type ACT,
variant rACT8.20inhibited the two chymptrypsin-like
pro-teases Chtr and PSA but additionally also PK and HNE
None of the recombinant serpins showed inhibitory activity
against the kallikrein hK1 and uPA
Stoichiometries of inhibition for variant ACTs for
hK2 are improved in comparison to wild-type ACT
The determination of the stoichiometry of inhibition was
accomplished under physiological conditions of pH and
ionic strength for all enzymes to ensure the most valuable
comparison Recombinant wild-type ACT gave an SI value
of 2 (Table 3) with chymotrypsin, which is identical to the
value obtained with commercial ACT under similar
condi-tions (data not shown) All newly constructed variants of
ACT showed lower SI values with hK2 than wild-type ACT
(Fig 2) From these variants, rACT6.7, rACT 6.1 and
rACT6.2 had the lowest stoichiometry of inhibition values
for hK2 (9, 19 and 25, respectively) Whereas rACT6.2 and
rACT6.1 also had the lowest SI values (18 and 16) for PK,
the SI for rACT6.7 was much higher (277) The two
recombinant ACTs specific for hK2, rACT8.3 and rACT5.18 had higher SI ratios of 34 and 139, respectively The SI value of rACT8.20 inhibitor was superior to 100 for all tested proteases including hK2
Variant ACTs form stable complexes with hK2 without degradation of inhibitors
Western blot analysis of the reaction products of rACTs with hK2 was performed to determine the fate of inhibitors after the interaction with the enzyme Figure 3A shows that when hK2 is incubated with ACT variants, free hK2 (E) disappeared completely to form a covalent complex (E-I) This covalent complex demonstrated high stability; no breakdown over a 16 h incubation period (data not shown) Wild-type ACT inhibited hK2 more slowly, which was mainly uncomplexed after 3 h of incubation Elevated SI values measured with hK2 were not due to noncomplex forming degradation of ACT variant inhibitors rACT6.7 with the lowest SI for hK2 of all ACT variants and the highly hK2 specific variant rACT8.3were complexed with hK2 and analyzed by Western blotting (Fig 3B) All inhibitor proteins were either complexed with hK2 or present in the uncleaved form, indicating that the possible substrate pathway for the serpin–enzyme interaction is marginal [21]
Table 2 Inhibitoryprofile of rACT WT and its variants The scissile bond by hK2 in substrate peptides is designated by fl Amino acid sequence cleaved in RSL (reactive serpin loop) of recombinant ACTs corresponding to selected substrate peptide by hK2 Protease and serpins were incubated for 30 min at 25 C (90 min at 37 for PSA) at an [I] o /[E] o ratio of 100 : 1 Percentage inhibiton corresponds to 100 · [1 – (velocity in presence of inhibitor/velocity of unhibited control)].
Protease
Inhibition percentage
ACT 8.20
(LRflSRA)
ACT 6.2 (RRflSID)
ACT 8.3 (RGRflSE)
ACT 6.7 (KLRflTT)
ACT 6.1 (MTRflSN)
ACT 5.18 (ERflVSP)
ACT WT (LLflSA)
Table 3 Comparison of stoichiometryof inhibition values and second-order rate constants (k a ) for the reaction of rACT WT and its variants with hK2 and others proteases SI values reported were determined using linear regression analysis to extrapolate the I/E ratio (see Fig 1) Second-order rate constants for serpin–protease reactions were measured under pseudo first- or second-order conditions as described in Materials and methods Parentheses, amino acid sequence of P3-P3¢ residues in RSL (reactive serpin loop) of recombinant ACT corresponding to selected substrate peptide
by hK2; –, No detectable inhibitory activity, k a is measured in M )1 Æs)1.
Protease
ACT 8.20
(LRflSRA)
ACT 6.2 (RRflSID)
ACT 8.3 (RGRflSE)
ACT 6.7 (KLRflTT)
ACT 6.1 (MTRflSN)
ACT 5.18 (ERflVSP)
ACT WT (LLflSA)
Trang 5Variant ACTs showed highest association constants
with hK2
The rate of the inhibitory reaction with variant ACTs was
determined for each protease showing reactivity with these
inhibitors After determination of kobs(Fig 4), association
constants (ka) were calculated using the Kmof the proteases
for their corresponding substrates (Table 3) The ka value
of wild-type ACT with chymotrypsin was identical to the
previously published ka [22] The recombinant rACT6.7
showed the highest ka (8991M )1Æs)1) with hK2 whereas
that obtained with PK was 45-fold less In contrast,
recombinant rACT6.2gave an equivalent kawith hK2 and
PK, demonstrating a lack of discrimination between the
two proteases ka Values of hK2 specific recombinant
inhibitors rACT8.3 and rACT5.18 were lower (2439 and
595M )1Æs)1, respectively,) whereas nonspecific ACT8.20
exhibited a kaof 1779M )1Æs)1, for hK2, superior compared
to Chtr, PK and HNE One of the recombinant serpins,
rACT6.1, possessed a higher velocity with PK than with
hK2
Discussion
The major challenge in the development of hK2 inhibitors is
the design of highly selective, potent and bioavailable
compounds that could be used for in vivo investigations We
have previously used substrate phage-display to identify
peptide sequences that are efficiently and selectively cleaved
by hK2 [16] The current study proposes the use of peptide
substrates selected by phage-display technology to change
the specificity of serpin ACT which is known to inhibit a
large panel of human enzymes such as chymotrypsin, mast
cell chymase [23], cathepsin G [24], prostatic kallikreins hK2
[14] and PSA [25]
Production of ACT in a bacterial recombinant system has already been published by several groups and allows the production of active inhibitors in soluble form [26] In the present work, reduction of temperature during induction to
16C allowed the production of fully intact ACTs purified
in one step by affinity chromatography The efficiency of the bacterial recombinant system to produce active ACT was proved by the stoichiometry of inhibition of recombinant wild-type ACT with chymotrypsin and its constant of association which were similar to those obtained with natural ACT [13] All variants gave a production yield of around 2 mgÆL)1of culture We conclude that the bacterial system is capable of a suitable-level of production of functionally and structurally intact ACT variants
Serpins trap their target proteases in the form of an acyl– enzyme complex However, the trap is kinetically controlled, and the serpin–protease complexes can, in some cases, ultimately break down, releasing a cleaved inactive serpin and an active protease [10] ACT can also have substrate behaviour for some proteases For example, Cathepsin
D [27] and Pseudomonas human elastase [28] hydrolyse the RSL loop of ACT without formation of a covalent complex Thus, swapping of the amino acid sequences of the reactive site loop does not guarantee maintenance of
Fig 2 Stoichiometryof inhibition (SI) of hk2 byrACT WT and its
variants hK2 (5 n M ) was incubated with different concentrations
(6.25–500 n M ) of rACT 8.20 (·), rACT 6.2 (h), rACT 8.3 (n), rACT 6.7
(e), rACT 6.1 ( ), rACT 5.18 (s), rACT WT (+), at 25 C for 30 min in
reaction buffer Residual activities (velocity) for hK2, were assayed by
adding the fluorescent substrate (10 l M ) Z-FR-AMC Fractional
velocity corresponds to the ratio of the velocity of inhibited enzyme (v i )
to the velocity of the uninhibited control (v o ) The SI was determined
using linear regression analysis to extrapolate the I/E ratio (i.e the
x intercept).
Fig 3 Formation of complex between hK2 and recombinant inhibitors (A) hK2 was incubated 3 h at 37 C with rACT 8.20 (lane 1), rACT 6.2 (lane 2), rACT 8.3 (lane 3), rACT 6.7 (lane 4), rACT 6.1 (lane 5), rACT 5.18 (lane 6) and wild-type rACT (lane 7), at an I/E ratio of 100 : 1 The complex formation was analyzed by Western blot under reducing conditions using a mouse anti-hK2 Ig (B) ACT 8.3 (lane 1) or ACT 6.7 (lane 3) were incubated with hK2 (lane 2 and 4, respectively) under kinetic conditions (30 min at 25 C) at an I/E ratio of 10 : 1 The complex formation was analyzed by Western blot under reducing conditions using a mouse monoclonal anti-His tag Arrows indicate hK2 (E), inhibitor (I), and hK2–ACT complex (E-I).
Trang 6inhibitory activity of a serpin, which could be turned into
substrate All variants developed from hK2 selected
sub-strates [16] form a stable covalent complex and are not
converted into substrate The maintenance of the cleavage
axis in modified serpins is probably one of the essential rules
to respect to keep the inhibitory activity Plotnick et al [13]
reported that relocation of the RSL changes the complex
stability, which can lead to a complete loss of inhibitory
activity or inversely to an increase of inhibitory potential
A SI value superior to one is generally interpreted as a
substrate with the behaviour of serpin In this scheme, after
formation of an initial Michaelis complex and cleavage in
the reactive site loop, most of the complex is broken down
into active enzyme and the cleaved inhibitor, which is
inactivated We analyzed ACT-hK2 reactions for
noncom-plex forming cleavage of the inhibitor, incubating the
samples at a 10 : 1 excess of inhibitor to protease These
conditions, where SI values are close to or below those
calculated for the tested ACT variants (Table 3), normally
favour proteolysis of serpins or serpin–protease complexes Surprisingly, we observe a discrepancy to this hypothesis as degradation of variant ACTs by hK2 was not observed despite high SI values A possible explanation for the lack of ACT degradation is the condition under which the SI determination was performed Covalent ACT–hK2 com-plexes form very slowly in vitro [14] This is in agreement with our observation that after 30 min of incubation at
25C, no inhibition of hK2 with wild-type ACT can be detected (Table 2) and that even after prolonged incubation
at 37C hK2 is only partially complexed with wild-type ACT (Fig 3)
In this study, we have also assessed the specificity of new inhibitors toward other proteases The evaluation was performed under the same conditions for all proteases (pseudo-physiological conditions) in order to ensure a better translation for further in vivo applications The permuta-tions of RSL cleavage site for hK2 phage-display selected substrates changed wild-type ACT into highly sensitive inhibitors for hK2 In addition, two of these inhibitors showed a unique reactivity with hK2 and not with other studied enzymes known to target similar biological sub-strates, such as plasma kallikrein, hK1, PSA, urokinase, and elastase To our knowledge, this is the first report detailing the development of a specific inhibitor for hK2 The fact that four variants of ACT also inhibited plasma kallikrein
to some degree is not surprising taking into account their homology of substrate specificity Plasma kallikrein and kallikrein hK2, are trypsin-like serine proteases and show kininogenase activity [29] However, variants of ACT are more sensitive to hK2 than to plasma kallikrein, except rACT6.1, which is the best inhibitor of PK This data could
be explained by previous experiments designed to evaluate the specificity of plasma kallikrein, which demonstrated that specific elements are important for interaction with its active site and notably hydrophobic amino acids in P¢2 [30]), whereas, hK2 is more associated with small and noncharged amino acids in this position [16,31] Interestingly, besides hK2 rACT8.20also inhibits chymotrypsin, and more weakly, plasma kallikrein and human elastase This large spectrum
of specificity is probably due to the presence of arginine, leucine and alanine residues that are known to be suitable for trypsin-like enzymes, chymotrypsin-like enzymes and elastase, respectively
We have developed different variants of ACT some of which selectively inhibit human kallikrein hK2 The main advantage of protein inhibitors such as serpins over small chemical inhibitors is their high molecular mass and a long half-life In addition, as serpins are natural proteins present
in the blood circulation, they are expected to be less toxic than chemical compounds These novel inhibitors of hK2 will be useful for further experiments which would allow a better understanding of the role of hK2 in prostate cancer progression In vivo evaluation of these inhibitors will permit
an evaluation of their potential as prostate cancer treat-ments with xenografted animal models and indicate if human kallikrein hK2 is a promising therapeutic target
Acknowledgements
This work is supported by a grant from OPO Foundation (Zurich, Switzerland).
Fig 4 Inhibition of hK2 byrACT WT and its variants under pseudo-first
order conditions The interaction of hK2 and recombinant serpins was
measured under pseudo first-order conditions using the progress curve
method hK2 (2 n M ) and substrate Z-FR-AMC (10 l M ) were added to
varying amounts (20–800 n M ) of inhibitors (A) rACT 8.20 (e),
rACT 5.18 (+) (B) rACT 6.2 (s), rACT 8.3 (h), rACT 6.7 (n), rACT 6.1
(·) Representative progress curves were subjected to nonlinear
regression analysis using Eqn (1) and the rate (k obs ) was plotted against
the serpin concentrations A second-order rate constant (k¢) was
obtained from the slope of this line Using Eqn (2) and K m of the
enzyme for this substrate (K m ¼ 67 l M ), a corrected second-order rate
constant was calculated (Table 3).
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R.L., Saeid, M.S., Young, C.Y., Klee, G.G., Tindall, D.J &
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