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Mol Cancer Ther. 2004;3:1439-1450
© 2004 American Association for Cancer Research

Screening a combinatorial peptide library to develop a human glandular kallikrein 2–activated prodrug as targeted therapy for prostate cancer

Samuel Janssen1, Carsten M. Jakobsen2, D. Marc Rosen1, Rebecca M. Ricklis1, Ulrich Reineke3, Soeren B. Christensen2, Hans Lilja4 and Samuel R. Denmeade1

1 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland; 2 Danish University of Pharmaceutical Sciences, Copenhagen, Denmark; 3 Jerini AG, Berlin, Germany; and 4 Memorial Sloan Kettering Cancer Center, New York, New York

Requests for reprints: Samuel Denmeade, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-502-3941; Fax: 410-614-8397. E-mail: denmesa{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation–independent apoptosis through dysregulation of intracellular calcium levels. Methods: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity. Results: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 µmol/L, kcat of 1.09 s–1, and a kcat/Km ratio of 41,132 s–1 mol/L–1. The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2. Conclusion: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Non-organ-confined prostate cancer is uniformly fatal once it reaches the hormone-refractory state because current therapies are unable to completely eliminate the androgen-independent prostate cancer cells present within metastatic sites. In previous studies, our laboratory and others have shown that metastatic androgen-independent prostate cancer cells have a remarkable low rate of cell proliferation (1, 2). This low proliferative rate could explain the relative unresponsiveness of these cells to standard antiproliferative chemotherapy. In vitro, however, highly proliferative androgen-independent prostate cancer cell lines remain exquisitely sensitive to induction of apoptosis.

In contrast to agents that activate apoptosis in proliferating cells, our laboratory has shown that thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pumps (3–5), has the dose-response ability to elevate intracellular calcium to sufficient levels to induce apoptosis in all of the rodent and human androgen-independent prostate cancer cell lines without requiring the cells to be proliferating (3, 6, 7). The cytotoxicity of thapsigargin, however, is not prostate cancer cell type specific (8, 9). Therefore, thapsigargin would be difficult to administer systemically without significant side effects. In addition, thapsigargin is sparingly water soluble due to its high lipophilicity. Therefore, a method is required that both better solubilizes thapsigargin and selectively targets the cytotoxicity of thapsigargin to metastatic deposits of androgen-independent prostate cancer cells systemically (10). To accomplish this, a primary amine-containing thapsigargin analogue can be coupled to a peptide carrier to produce water-soluble inactive prodrug that is selectively activated within sites of prostate cancer (8, 10). The peptide carrier in this approach is designed to be a selective substrate for prostate tissue–specific proteases such as prostate-specific antigen (PSA) or human glandular kallikrein 2 (hK2; ref. 10). PSA and hK2 are only produced in high levels by normal and malignant prostate cancer cells (11–14). In addition, metastatic prostate cancer cells continue to secrete enzymatically active PSA and hK2 into the extracellular fluid at high levels (11, 12, 15). Once in the extracellular fluid, enzymatically active PSA and hK2 eventually enter the blood where they are inactivated by binding to major serum protease inhibitors [i.e., {alpha}1-antichymotrypsin and {alpha}2-macroglobulin for PSA (14, 16–19) and {alpha}1-antichymotrypsin, {alpha}2-antiplasmin, antithrombin II, protein C inhibitor, and {alpha}2-macrogloblin for hK2 (20, 21)].

Ideally, the thapsigargin analogue prodrug is inactive until the thapsigargin analogue is cleaved in the presence of enzymatically active PSA or hK2. In previous studies, we synthesized and characterized a series of primary amine-containing thapsigargin analogues and identified 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT) as a highly potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump and as equally cytotoxic as thapsigargin (22, 23). In additional studies, we identified a six–amino acid peptide substrate that is efficiently hydrolyzed by PSA (24). The potent thapsigargin analogue and other cytotoxic agents have been coupled to this peptide to produce prodrugs that are selectively cytotoxic to PSA-producing prostate cancer cells in vitro (8, 10, 24, 25). Significant antitumor effects have been observed when these PSA-activated prodrugs have been given to animals bearing PSA-secreting prostate cancer xenografts without producing significant host toxicity (8, 25).

Enzymatically active PSA is found in high levels in the seminal fluid (0.3–5 mg/mL; refs. 11, 26) and in the extracellular fluid of both normal and malignant prostate cancer cells (i.e., 50–500 µg/mL; ref. 15). In contrast, levels of hK2 in the seminal fluid are ~1% of those of PSA (26), whereas hK2 levels in the extracellular fluid of prostate cancers have not been reported. Using a chromogenic substrate (i.e., Pro-Phe-Arg-pNa), however, Mikolajczyk et al. (27) showed that the enzymatic activity of hK2 was 20,000-fold higher than that of PSA on a comparable substrate containing a Tyr cleavage site. In addition, although PSA and hK2 are both found almost exclusively in the prostate, hK2 is more highly expressed by prostate cancer cells than by normal prostate epithelium. Unlike PSA, hK2 expression seems to increase in more poorly differentiated cancers, with the strongest staining observed in prostate cancer lymph node metastases (14). Intensity of staining for hK2 has been found to increase with increasing Gleason grade (14). In contrast, PSA staining tends to decrease with increasing Gleason grade (14). Thus, although hK2 is produced at a lower level than PSA in prostate tissue, the increased production in more poorly differentiated cancers coupled with the several orders of magnitude higher enzymatic activity suggest that total hK2 enzymatic activity in the extracellular fluid may be similar or even greater than that of PSA. Therefore, hK2 represents an attractive alternative candidate for prostate-targeted prodrug activation therapy.

Although the hK2 protein is ~80% identical to PSA in primary structure (13), the two are markedly different in their enzymatic properties (11). Whereas PSA is the only member of the kallikrein family with chymotrypsin-like substrate specificity, hK2 displays the trypsin-like specificity common to the kallikrein family (13). The goal of the present study was to identify specific hK2 peptide substrates that could be used to produce prodrugs that are selectively activated by enzymatically active hK2 present in the extracellular fluid of prostate cancer sites. In the present study, we have generated random combinatorial peptide libraries to rapidly screen a large number of sequences to identify putative hK2 substrates. A lead peptide substrate that is efficiently hydrolyzed by hK2 was identified and used to produce a hK2-activated L12ADT prodrug.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
A mutant form of hK2 was used for these studies in which the amino acids –5 to –1 of the propeptide sequence of hK2 (i.e., Leu-Ile-Gln-Ser-Arg) were mutated to Asp-Asp-Asp-Asp-Lys to generate a pro-hK2 protein that can be activated to functional hK2 by factor Xa (28). Compared with wild-type hK2, expression of the propeptide hK2 mutant increases the expression levels up to 15- to 40-fold (28). The generation and characteristics of this mutant hK2 have been described previously (28). Fmoc amino acids were purchased either from Advanced Chemtech (Louisville, KY) or Novabiochem (La Jolla, CA). Reagents were used without further purification. All other reagents were from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified in the text.

Cell Lines
The LNCaP human prostate cancer cell line was obtained from American Type Culture Collection (Rockville, MD). The C4-2B and CWR22R human prostate cancer cell lines and TSU human bladder cancer cells were obtained from Dr. John Isaacs (Johns Hopkins University, Baltimore, MD). These cell lines were maintained by serial passage in RPMI 1640-10% fetal bovine serum in 5% CO2/95% air at 37°C.

Immobilized Peptide Synthesis and Hydrolysis Determination
Peptides ("Protease Spots") were provided by Jerini AG (Berlin, Germany) and were synthesized on continuous cellulose assays using the SPOT synthesis technique (29). Each peptide contained a 2-aminobenzoic acid (Abz) moiety at the NH2 terminus. Abz is a fluorescent molecule with optimal excitation at 325 nm and emission maxima at 420 nm. Peptides were synthesized on cellulose membranes then punched out as small discs into 96-well microtiter plates. Peptides (~8 nmol) are synthesized per spot.

To perform protease assay, spots were rinsed first for 5 minutes with methanol to solubilize peptides. Spots were then rinsed four times for 10 minutes under gentle agitation with a buffer consisting of 50 mmol/L Tris and 0.1 mol/L NaCl (pH 7.8; buffer A). Fresh buffer A was added to each well along with an aliquot of purified protease (i.e., mutant hK2 or trypsin) or 50% human serum in buffer A. The plate was sealed with plastic and reaction was allowed to occur at room temperature without agitation. At described intervals (i.e., 1, 2, 4, 7, and 24 hours), an aliquot (50 µL) of the reaction mixture was transferred to a new 96-well microtiter plate. Fluorescence was then measured at room temperature using a 96-well fluorometric plate reader (Fluoroscan II, ICN Biomedicals, Costa Mesa, CA) with excitation of 355 nm and emission of 408 nm. Fluorescence at each point was plotted and reaction rates were determined from slope of the best-fit line. Rates are expressed in relative fluorescence units per hour per milligram of protease.

Combinatorial Libraries
Combinatorial peptides libraries were synthesized as described previously (30). Peptides were anchored to polyethylene glycol A (PEGA) support resin (Polymerlabs, Amherst, MA, 400 µm, 0.2 mmol/g) without a cleavable linker.

Amino acid couplings were done according to established Fmoc/t-butyl protocols using 1-hydroxybenzotriazole/N,N'-diisopropylcarbodiimide activation (31) and performing standard double couplings. Generally, completion of acylation reactions was verified by both ninhydrin (32) and fluorescamine testing (33). The Fmoc protecting group was removed with 25% piperidine in dimethylformamide (DMF). N-{alpha}-Fmoc-N-ß-t-Boc-L-diaminopropionic acid (Novabiochem) was used for the introduction of diaminopropionic acid (Dap). Three randomized positions were introduced using a Labmate Parallel Organic Synthesizer (4 x 6 vessels, Advanced Chemtech) according to the "split-and-mix" procedure (34). All natural amino acids, except for Cys, were used with the following side chain protection: Trt (Asn, Gln, and His), tBu (Tyr), OtBu (Asp, Glu, Ser, and Thr), Boc (Lys and Trp), and Pmc (Arg). Amino acid stock solutions (0.5 mol/L with 0.5 mol/L 1-hydroxybenzotriazole) were mixed with N,N'-diisopropylcarbodiimide for 20 minutes (4 eq. of each). The activated amino acids were added to the resin and 5% diisopropylethylamine (0.15 mL) in DMF was added. After 2 to 3 hours, the resin aliquots were washed (3x 1-methyl-2-pyrrolidone, 3x methanol, and 3x DMF) and couplings were repeated with 2 eq. amino acid for 1 to 2 hours. A resin sample of each aliquot was subjected to a ninhydrin test and a fluorescamine test, which showed completion of the acylation reactions in all cases. Next, the resin aliquots were pooled [Fmoc-X1-X2-X3-Dap-Phe-K(Abz)-PEGA] and deprotected with piperidine and the remaining four constant residues, Ala, Lys, Gly, and nitrotyrosine (Y', Fluka, Milwaukee, WI) were added as Fmoc amino acids in batch with 1-hydroxybenzotriazole/N,N'-diisopropylcarbodiimide activation as described above. For the final deprotection of the side chains, the resin was suspended in reagent K [trifluoroacetic acid (TFA)/thioanisole/water/phenol/1,2-ethanedithiol 82.5:5:5:5:2.5 (v/v), 1x 10 minutes, 1x 3 hours]. The resin was washed with 95% acetic acid (3x), dichloromethane (3x), DMF (3x), 5% diisopropylethylamine in DMF (3x), and DMF (6x). The resin was stored until screening suspended in DMF at –20°C.

For screening, resin (~1 mL, ~65,000 beads) was first suspended in methanol in a Petri dish and examined under transilluminant UV light (302 nm) to detect any false-positive fluorescent beads prior to addition of protease. After removal of 40 to 50 beads, the resin was washed with water and finally suspended in buffer (10 mL) in a glass Petri dish. After a final screen for false-positives, hK2 was added from a frozen stock solution to make a 4 µg/mL final concentration. Fluorescent beads were selected and removed with a micropipette; washed with NaCl (1 mol/L), water, DMF, methanol, and water; and stored in methanol at –20°C.

Peptide Sequencing
Peptide sequencing was completed at the University of Arizona Laboratory for Protein Sequencing and Analyses (Tucson, AZ) using an Applied Biosystems (Foster City, CA) 477A Protein/Peptide Sequencer (Edman chemistry) interfaced with a 120A high-performance liquid chromatography (HPLC; C18 phenylthiohydantoin column, reverse-phase chromatography) analyzer to determine phenylthiohydantoin amino acids.

Automated Synthesis of Fluorescence-Quenched Peptides
For validation of the Edman results, peptides were resynthesized using a Rainin PS3 peptide synthesizer with O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate/4-methylmorpholine activation. Peptides were synthesized on PEGA resin for on-bead analysis or on Fmoc-Lys(4-methyltrityl)-Wang resin for solution assays. The Fmoc-Lys(4-methyltrityl)-Wang resin was first deprotected with 2% TFA in DCM (3x 2 minutes). The {varepsilon}-amine of Lys was then acylated with Boc-Abz. Deprotection/cleavage was done in TFA/triisopropylsilane/water [95:2.5:2.5 (v/v)] for 2 to 3 hours. Peptides were ether precipitated, dried, and purified by C18-HPLC using a linear gradient of acetonitrile (0.1% TFA), lyophilized, and dissolved in DMSO. Peptide identities were confirmed by analysis on a PerSeptive Voyager DE-STR matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) using dihydroxybenzoic acid as a matrix. Fluorescence measurements were done on a Fluoroscan II 96-well plate reader (ICN Biomedicals, excitation 355 nm, emission 460 nm). Kinetic variables were calculated as described earlier (24).

Peptide-Prodrug Synthesis
The peptide sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR)-L was synthesized on a Rainin PS3 automated peptide synthesizer on Fmoc-Leu-Wang resin (100-µmol scale). The same protecting groups were used as during the combinatorial synthesis, except for the Lys, which was orthogonally protected with the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) group (Novabiochem). After deprotection of the NH2-terminal Gly, the amine was acetylated with acetic anhydride and 4-methylmorpholine. Deprotection of the acid-labile protecting groups and purification were done as outlined above. Boc-12ADT was synthesized as described previously (23). TFA treatment followed by semiprep HPLC and lyophilization afforded the amine-containing 12ADT. The protected peptide (Ac-GK(ivDde)AFRRL) was coupled to 12ADT after 1-hydroxybenzotriazole/N,N'-diisopropylcarbodiimide activation. After completion of the reaction, the ivDde group was removed by adding hydrazine to the reaction mixture (2% final concentration, 30 minutes). Semipreparative HPLC yielded Ac-GKAFRR-L12ADT typically in 60% to 70% yield. Product was confirmed by MALDI-TOF analysis.

Plasma Stability Assays
Mouse plasma was obtained from cardiac puncture of anesthetized mice prior to euthanization by CO2 overdose according to protocols approved by the Johns Hopkins Animal Care and Use Committee. Human plasma was obtained from discarded, pooled, and unlabeled clinical samples. Plasma was diluted to 50% in Tris buffer [50 mmol/L Tris-HCl, 100 mmol/L NaCl (pH 7.8)]. To this, test peptides/prodrugs were added to 250 or 500 µmol/L final concentration. After the incubation period, 1 volume of 1% TFA in acetonitrile was added to precipitate the protein fraction and the tube was centrifuged at 14,000 rpm for 2 minutes. The supernatant was analyzed by C18-HPLC and the collected peaks were analyzed by MALDI-TOF as described above.

Cytotoxicity Assays
Clonal survival of TSU (5 x 104 cells) following 48-hour exposure to varying concentrations of hK2-activated prodrugs or vehicle control with or without exogenously added hK2 was done as described previously (9). Percentage inhibition of clonal survival was calculated from the ratio of number of colonies observed in treated group to number of colonies in control group. Cytotoxic response to 7-day exposure to varying concentrations of hK2-activated prodrug in hK2-producing and nonproducing cell lines was determined using the Promega Cell Titer 96 Nonradioactive Cell Proliferation Assays (Promega, Madison, WI) according to the manufacturer's instructions as described previously (8).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a hK2-Specific Peptide Substrate
The critical requirement for a hK2-activated prodrug is identification of a peptide substrate that is efficiently and selectively hydrolyzed by the enzyme. Therefore, the first task was to define the peptide substrate requirements of hK2. Previously, Lovgren et al. (20) identified several hK2 cleavage sites within the human seminal proteins semenogelin I (SgI) and semenogelin II (SgII). Based on this hK2 cleavage map for SgI and SgII, small peptides were synthesized containing the amino acid sequence on the NH2-terminal side of the scissile bond (i.e., P6-P1, where P1 is the amino acid at the cleavage site). These peptides were synthesized using a technique called the SPOT technique (for a recent review, see ref. 29) in which the COOH terminus of the peptide is tethered to a cellulose membrane and the amine of the NH2 terminus of the peptide is labeled with the fluorophore Abz. hK2 digestion releases the Abz-containing portion of the peptide and the degree of hydrolysis can thus be monitored by measuring the increase in fluorescence of the supernatant over time.

Using this SPOT technique, the peptides representing hK2 cleavage sites within SgI and SgII were synthesized and screened for hK2 hydrolysis. Within the group of semenogelin cleavage sites defined by Lovgren et al. (20), the amino acid Leu was present in the P'1 position in 5 of the 10 sites. Therefore, for this assay, the P'1 position (COOH-terminal of scissile bond) was fixed as Leu. Gly was introduced at the NH2 terminus of all peptides to provide a linker between the Abz and the P6 position of the semenogelin cleavage site peptide sequence. The relative hydrolysis rates of these SgI and SgII native hK2 cleavage site peptides were then obtained by incubating enzymatically active hK2 with the cellulose membrane peptides and measuring fluorescent activity released into the supernatant following hK2 hydrolysis of the peptide (Table 1). The semenogelin sequences showed a range of hydrolysis rates and not all semenogelin sequences showed significant activity. As noted previously, many of the hK2 cleavage sites within semenogelin protein contain tandem basic amino acid residues at position P1 and P2, with the P1 position being Arg in all but one sequence and the P2 being either Arg, His, or Lys in 5 of 11 sites (20). In our analysis, the most active native SgI/SgII sequence (GGKAHRL) contains besides the Arg at P1 two more basic residues (Lys and His), suggesting a preference for positive charge by hK2 (see Table 1).


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  		Table 1. Relative hydrolysis rates of hK2 substrates by hK2 as determined by release of NH2-terminal conjugated Abz

 
The occurrence of Arg in the P1 position is a common phenomenon in the natural hK2 substrates (11). Therefore, in an attempt to produce better hK2 substrates, the native sequences (Table 1, bold) were modified by substituting the amino acid in the P2 position for Arg, thereby producing a R-R cleavage site. In each case, substitution of R-R for the native P2-P1 sequence resulted in peptides that were for the most part better (i.e., 1.2- to 74-fold) hK2 substrates. Next, based on hK2 hydrolysis rates, the two best substrates (i.e., GHEQKRRL and GGGKARRL) were subjected to further sequence modifications that included substitution of other amino acids in the P'1 position, substitution with D-amino acids, and substitution of Ala in each position (i.e., Ala screen; data not shown). From these experiments, it could be concluded that at the P'1 position Leu is preferred over Asp and Ser (data not shown). Substitution of L-Arg over D-Arg in P1 and/or P2 markedly decreased activity (i.e., dR-R = 4.4-fold, R-dR = 43-fold, and dR-dR = 130-fold decreased activity), whereas substitution of D-Leu in the P'1 position only decreased activity ~2-fold (data not shown).

To determine if there was a strict requirement for Arg in the P1 position of the SgI/SgII–based peptide sequences, additional peptides were synthesized with His substituted for Arg in the P1 position (Table 1). In all cases, these P1 His-containing substrates were markedly poorer substrates for hK2 hydrolysis (Table 1). In additional studies, soluble fluorescence-quenched peptide substrates based on the SgI/SgII sequence GSKGHFRL were produced in which the P1 Arg was substituted with the positively charged amino acid Lys (i.e., GSKGHFKL and GSKGPFKL). The Arg-free sequence GSKGHFHL identified as native substrate from the SgI/SgII hK2 cleavage map was also synthesized for testing in solution. In these studies, none of these three Arg-free peptides were appreciably digested by hK2 even after prolonged incubation. These results further support results from earlier studies using small peptide substrates and phage display and show that hK2 has a strong preference for Arg in the P1 position of peptide substrates. Overall, from these studies, it appeared that hK2 prefers polar, positively charged substrates with a monobasic or dibasic RR motif at the cleavage site.

Finally, to determine whether these SgI/SgII–based peptide substrates were selective for hK2 hydrolysis, each sequence was incubated with equimolar amount of trypsin. A comparison of hydrolysis rates for each individual peptide for hK2 versus trypsin showed that all of these peptides were better substrates for trypsin (Table 1). hK2 hydrolysis rates or this series of peptides ranged from 1% to 78% of trypsin hydrolysis rates.

Based on these studies, the GKAFR peptide was selected for further analysis based on high relative hK2 versus trypsin hydrolysis rates. A fluorescent substrate was synthesized by coupling the fluorophore 7-amino-4-methyl coumarin (AMC) to the COOH terminus of the peptides to produce the substrate Mu-GKAFR-AMC (where Mu is morpholinocarbonyl). Rates of hydrolysis by hK2 and trypsin were determined and compared with the kallikrein substrate PFR-AMC. In addition, stability to nonspecific hydrolysis in human plasma was also assayed (Table 2). These results show that the GKAFR-AMC substrate is a better substrate for hK2 than PFR-AMC; however, neither substrate was selective for hK2 hydrolysis nor were these substrates stable to hydrolysis in human plasma (Table 2).


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  		Table 2. Relative hydrolysis rates of hK2 substrates by hK2 and trypsin as determined by release of NH2-terminal AMC

 
Screening a Combinatorial Peptide Library to Identify hK2 Substrates
Trypsin and trypsin-like proteases have a defined preference for the amino acid Arg or Lys at the site of hydrolysis. There are a large number of trypsin-like proteases present in human blood including human kallikrein 1, plasmin, thrombin, and other members of the clotting factor cascade. The proteolytic activity of these proteases in the blood is tightly regulated and these proteases are present in the bloodstream predominantly as inactive zymogens. hK2 is also a trypsin-like protease with a preference or perhaps a requirement for Arg at the P1 hydrolysis site (21). As our experience with the peptide sequences defined above indicated, peptide substrates containing Arg seemed relatively unstable to nonspecific hydrolysis in the blood. Therefore, in an attempt to identify more selective hK2 peptide substrates and to determine if hK2 indeed has a strict requirement for Arg at site of cleavage, a fluorescent-quenched combinatorial peptide library was synthesized containing random amino acid in positions P3-P1 to allow for screening of all possible amino acid combinations (i.e., 193 or 6,859 sequences).

Previously, St Hilaire et al. (30) showed that protease substrate requirements can be routinely mapped by on-bead (i.e., resin) digestion of short peptides. By following the "split-and-mix" approach (34), a peptide library is generated on polymeric solid-phase synthesis resin "beads," so that each bead contains at the end a unique but random peptide sequence. These peptides are bracketed by a fluorophore at the COOH terminus (Abz coupled to the {varepsilon}-amino group of Lys) and a pairing quencher moiety at the NH2 terminus (3-nitrotyrosine; Fig. 1). On hydrolysis of any backbone amide bond, the quencher-containing NH2-terminal part of the peptide is liberated and diffuses into the solution, resulting in bright fluorescence due to unquenching of the remaining COOH-terminal part, still linked to the bead (30). The polymeric support has to swell sufficiently in water to allow diffusion of the protease into the bead. Our previous data using the SPOT-based peptides suggested that the positively charged tripeptide GKA would be a close-to-optimal P6-P4 amino acid sequence and that the COOH-terminal positions were of more significance for defining selectivity of hK2 activity. Therefore, this library was biased in that between the NH2-terminal Lys-Abz fluorophore and the COOH-terminal nitrotyrosine quencher a constant tripeptide (GKA) was inserted in positions P6-P4 followed by random amino acids in positions P3-P1 (i.e., GKAXXX; Fig. 1). PEGA (a mix of polyacrylamide and polyethylene glycol) resin was chosen as the solid-phase support based on preliminary studies, demonstrating superiority of this resin over alternative resin supports (e.g., TentaGel) for this application (data not shown).



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  		Figure 1. Chemical structure of fluorescence-quenched combinatorial "one bead, one peptide" library (Y'GKAXXX-Dap-F-K'-PEGA, where Y' is nitrotyrosine and K' is Abz-substituted Lys).

 
Additionally, Thorpe and Walle (35) published data from a small combinatorial library to find linkers for the optimal display of peptide ligands to various protein targets. They reported that the insertion of a dipeptide consisting of a cationic residue together with a hydrophobic residue presents a general method for optimizing peptide display on solid-phase beads. To test this observation in our own system, we incorporated a linker described by Thorpe and Walle (35) in which the cationic residue was Dap and the hydrophobic residue was phenylalanine (F). We synthesized the test peptide Y'GKAFRL-Dap-F-K'-PEGA and observed that the time required to generate clearly detectable fluorescence on the beads was reduced to 4 to 5 hours compared with 10 to 12 hours for the same peptide sequence lacking the Dap-F linker. Based on these results, the Dap-F dipeptide linker was included in all subsequent libraries.

Therefore, the final library used for screening with hK2 contained the general sequence Y'GKAXXX-Dap-F-K' PEGA, where X is any of 19 amino acids (Cys was excluded from library) and contained 193 peptide sequences on ~50,000 beads (i.e., ~7 beads for each unique peptide sequence; Fig. 1). After carefully removing any false-positive fluorescent beads from the library (~40–50 beads), purified, enzymatically active hK2 was added at a final concentration of 4 µg/mL. After 1 hour, the first positive bead was removed. Over the subsequent 3 hours, 9 more beads were selected. In total, 14 beads were selected over a period of 24 hours. Positive beads were sequenced by Edman degradation.

Seven of 14 peptides contained one or more Arg residues. The peptides lacking any Arg did not show a specific amino acid preference. One sequence (FRR) was similar to double Arg motif sequences identified previously as optimal in the SPOT assay.

To confirm that the selected sequences represented true hK2 substrates and not false-positives, the majority of the fluorescence-quenched peptides were resynthesized, cleaved from the resin, and tested for hydrolysis by hK2 in solution. After resynthesis, none of the soluble non-Arg-containing peptides were hydrolyzed by hK2 (data not shown), confirming that the Arg-free sequences were not hK2 substrates but false-positives. In contrast, each of the resynthesized Arg-containing peptide substrates was readily hydrolyzed by hK2. The combinatorial screen identified seven Arg-containing peptides. Four of these were resynthesized (X1-X2-X3 = RAF, KPR, FRR, and MRQ, respectively). All four Arg-containing sequences that were resynthesized reproduced fluorescence when these peptides were digested on-bead with hK2. For a more quantitative analysis, the fluorescence-quenched peptides were cleaved off the resin and purified by HPLC. The rate of hydrolysis was quantified by measuring increase in fluorescence (Fig. 2). The best substrate has proven to be the sequence with Arg at P1 and P2 (i.e., Y'GKAFRR-Dap-F-K'). In <5 minutes, >50% of the peptide were digested (500 µmol/L peptide, 4 µg/mL hK2). For the other peptides, digestion of the same amount of peptide took 19 to 29 minutes. Maximum digestion was only 70% to 75%, a value that was reached with Y'GKAFRR-Dap-F-K' in <15 minutes. In subsequent studies, hydrolysis rates using the Y'GKAFRR-Dap-F-K' peptide were analyzed by Lineweaver-Burke reciprocal plots. The Michaelis-Menten constant (Km) was determined at 26.5 µmol/L, the kcat was 1.09 s–1, and the kcat/Km ratio was 41,132 s–1 mol/L–1. These results compare favorable with those reported previously for the Pro-Phe-Arg-AMC substrate used to assay hK2 activity (Km, 40 µmol/L; kcat, 0.92 s–1; kcat/Km, 22,916 s–1 mol/L–1) and were superior to the GKAFR-AMC substrate we generated based on results of SPOT analysis (Km, 146 µmol/L; kcat, 0.13 s–1; kcat/Km, 895 s–1 mol/L–1).



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  		Figure 2. Hydrolysis of various lead substrates (250 µmol/L) by hK2 (8 mg/mL) in PBS buffer. Note that the double Arg substrate (Y'GKAFRR-Dap-FK') by far exceeded all other substrates. A substrate without Arg (Y'GSKGHFKL-Dap-F-K') did not show any hydrolysis. To determine 100% digestion, trypsin was added (50 mg/mL) and samples were incubated to 37°C for 30 minutes and the fluorescence was determined.

 
Stability of hK2 Peptides in Plasma
Arg-containing peptides are potential substrates for the wide variety of other trypsin-like proteases that are present in the blood and may have residual activity in the blood. The plasma stability of a hK2 peptide substrate may therefore be limited and this would have significant consequences related to the development of a hK2-activated prodrug. Therefore, two fluorescence-quenched hK2 peptide substrates were incubated in 50% mouse or human plasma (diluted in PBS buffer) to determine stability using a plate reader. Similar to results with Arg-containing peptides from the SPOT analysis, none of these Arg-containing, fluorescence-quenched hK2 peptide substrates identified from the combinatorial screening were stable in human or mouse plasma (data not shown). Fluorescence-quenched Y'GKAFRR-Dap-GK' and Y'GKAFRR-LGK' (500 µmol/L each) were hydrolyzed when incubated in 50% mouse or human plasma (Fig. 3). Mouse plasma degraded the peptides faster than human plasma (Fig. 3). The Leu-containing peptide was less stable than the Dap-containing peptide in both plasma types. HPLC analysis of the peptides after 3 hours of incubation confirmed that the fluorescence generated during this plate reader assay corresponded with proteolysis, and in each case, several degradation products were generated; almost no parent peptide remained after 3 hours in mouse plasma (~5%), whereas in human plasma ~25% of intact peptide remained after 3 hours. Overnight incubation in human plasma resulted in complete degradation of both peptides. To determine whether acetylation of the NH2 terminus of the hK2 peptide would enhance stability, the acetylated fluorescence-quenched peptide, Ac-Y'GKAFRR-LGK', was synthesized and plasma stability was compared with that of the nonacetylated peptide (Y'GKAFRR-LGK'). Hydrolysis of these two peptides was completely identical as judged from the generation of fluorescence in the plate reader assay and by HPLC analysis. These results indicate that nonspecific hydrolysis of the hK2 peptide substrate is not due to degradation by plasma aminopeptidases.



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  		Figure 3. Hydrolysis of hK2 peptide substrates in plasma. Arg-containing lead hK2 substrates (500 µmol/L) were incubated in 50% mouse or human plasma. Generation of fluorescence indicated that the fluorescence-quenched peptides are unstable in plasma. Hydrolysis of the substrates was confirmed by HPLC. Comparison of mouse and human plasma for the same substrate suggests higher proteolytic activity in mouse plasma compared with human plasma.

 
Synthesis and Characterization of a hK2-Activated Thapsigargin Prodrug
The preceding results would suggest that the development of a hK2-activated peptide-based prodrug might not be feasible due to poor stability of Arg-containing peptides in plasma. However, the possibility remained that the introduction of a bulky hydrophobic moiety like an analogue of thapsigargin might alter the relative rate of hK2 hydrolysis and/or stability in plasma. Therefore, we proceeded to synthesize a putative hK2-activated prodrug by coupling the GKAFRR peptide sequence to a primary amine-containing thapsigargin analogue (Fig. 4). In previous studies, we had identified L12ADT as a potent amino acid–containing thapsigargin analogue that was as cytotoxic as thapsigargin with a LD50 value of ~30 nmol/L against human prostate cancer cells in vitro (23). Previously, the L12ADT analogue has been coupled to a PSA-specific peptide to produce a prodrug that is selectively cytotoxic to PSA-producing prostate cancer cells in vitro and in vivo (8).



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  		Figure 4. Chemical structure of hK2 prodrug, AcGKAFRR-L12ADT. HK2 cleavage sites are indicated. The ratio of R-L12ADT/L12ADT generated by hK2 digestion was 1:1.8.

 
Based on these results, the prodrug Ac-GKAFRR-L12ADT was synthesized (Fig. 4). This putative hK2 prodrug was incubated with enzymatically active hK2 (4 µg/mL) to determine extent of hydrolysis over time. HPLC analysis of aliquots of the incubation mixture indicated that the hK2 prodrug is rapidly cleaved by hK2 (Fig. 5). MALDI-TOF analysis of the digestion products indicated that cleavage occurred after each Arg residue generating both Arg-Leu-12ADT (RL12ADT) and L12ADT. In 25 minutes, 50% was hydrolyzed; after 1 hour, >80% of the starting prodrug were hydrolyzed (Fig. 5). The ratio of the products RL12ADT/L12ADT was 1:1.8 as determined by HPLC integration. In previous studies, the IC50 of L12ADT against human TSU bladder cancer cell line was ~30 nmol/L. R-L12ADT was ~5-fold less potent against this cell line in growth inhibition assays in vitro (data not shown).



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  		Figure 5. HK2-mediated hydrolysis of Ac-GKAFRR-L12ADT prodrug. Hydrolysis of the prodrug (125 µmol/L) was analyzed by HPLC and quantified by HLPC integration of appropriate peak areas. Percentage hydrolysis was calculated from the ratio of area of starting material substrate at indicated time points/peak area of substrate at time 0 min.

 
To determine the plasma stability for the hK2 prodrug, we incubated the prodrug in 50% fresh heparinized (1%) plasma for 24 hours at room temperature, precipitated the proteins, and analyzed the supernatant by HPLC. Unexpectedly, HPLC analysis after 24-hour incubation in plasma yielded only a single peak corresponding to the hK2 prodrug. MALDI-TOF mass spectrometry of the isolated single peak confirmed the plasma stability of the prodrug.

hK2 Levels in Prostatic Tissues and Human Prostate Cancer Cell Lines
The levels of hK2 production in benign and malignant prostate tissues have not been as well characterized as PSA levels. In a previous study, Lovgren et al. showed that the average hK2 levels in the seminal plasma was 6.4 µg/mL (26). In this study, the level of PSA in the seminal plasma was 820 µg/mL (26). We have isolated prostatic fluid taken directly from radical prostatectomy specimens (i.e., without contamination from seminal vesicle fluid) and measured PSA and hK2 levels. In this prostatic fluid, PSA levels were 696 ± 305 µg/mL (n = 4), whereas hK2 levels were 165-fold lower at 4.2 ± 0.5 µg/mL (n = 5). Haese et al. (36) evaluated preoperative PSA and hK2 levels in men with stage T2 or T3 prostate cancers undergoing radical prostatectomy. Men were selected with PSA values <10 ng/mL. In this study, the PSA level for men with T2 versus T3 was not significantly different (~6 ng/mL). In contrast, hK2 levels in the serum of these patients were 50-fold (stage T3) to 75-fold (stage T2) lower (i.e., 0.08–0.12 ng/mL, respectively; ref. 36).

In contrast, hK2 levels in conditioned medium of human prostate cancer cell lines are several orders of magnitude lower (i.e., 0.1–60 ng/mL after 4 days of conditioning). For example, in one experiment, LNCaP human prostate cancer cells (6 x 106) produce hK2 levels in the conditioned medium of ~16.7 ng/mL, whereas PSA levels were 166 ng/mL after 4 days of conditioning (data not shown). Thus, in vitro assessment of hK2-selective cytotoxicity of a hK2-activated prodrug is problematic because the available cell models produce much lower levels (i.e., ~100-fold) of hK2 than estimated levels in extracellular fluid of human prostate cancers.

Cytotoxicity of hK2-Activated Prodrug In vitro
To determine the in vitro efficacy and selectivity of the hK2-activated prodrug, cytotoxicity against a series of hK2-producing cell lines (i.e., CWR22R, LNCaP, and C4-2B) was compared with cytotoxicity against TSU a non-hK2-producing human bladder cancer cell line (Fig. 6). In a previous study, the levels of hK2 production by these human prostate cancer cell lines were determined (37). The lowest levels were found in the CWR22R line, which produced 1.1 ± 0.2 ng hK2/106 cells/d (37). LNCaP cells produced 2.2 ± 0.6 ng hK2/106 cells/d and C4-2B cells produced 14.9 ± 3.0 ng hK2/106 cell/d, the highest level of all the cell lines tested (37). In this experiment, the hK2 prodrug had a similar inhibitory effect on cell growth after 7-day exposure at concentrations ≥1.25 µmol/L in all cell lines tested (Fig. 6). A modest difference in effect was observed for all hK2-producing cell lines at lower concentrations of prodrug. C4-2B cells, the line that produces highest levels of hK2, seemed to be the most sensitive to the prodrug (Fig. 6). The estimated IC50 for TSU in this study was ~1.25 µmol/L, whereas the IC50 for the highest hK2-producing line (C4-2B) was ~0.3 µmol/L.



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  		Figure 6. Cytotoxicity of Ac-GKAFRR-L12ADT against hK2-producing CWR22R, LNCaP, and C4-2B human prostate cancer cell lines and hK2 nonproducing TSU human bladder cancer cell line. Cell lines were incubated in the presence of indicated concentrations of prodrug for 7 days at which point cell proliferation assay (Promega) was done to determine viable cell number. Columns, percentage growth inhibition compared with vehicle-treated controls from each cell line; bars, SE. Results of eight replicate wells from duplicate experiments.

 
The modest ~4-fold difference in activity between a hK2-producing and a nonproducing cell line in this experiment may be secondary to production of low levels of hK2 by these human prostate cancer cell lines. Therefore, to determine the relative efficacy and specificity of a hK2-activated prodrug requires conditions that more closely mimic levels of enzymatically active hK2 found in extracellular fluid of human prostate cancers. In previous studies, we showed that PSA levels in extracellular fluid of human prostate cancers was 69 ± 12 µg/mL (15). However, hK2 levels in the extracellular fluid of human prostate cancers have not been reported. To determine approximate levels of hK2 in relation to PSA, we measured hK2 and PSA levels in the human PC-82 prostate cancer xenograft model. PC-82 does not grow as a cell line in vitro and is maintained through serial passage in nude mice. In previous studies, we determined that PSA levels in PC-82 xenografts were only ~3-fold lower than those found in human prostate cancers (15). In contrast, in LNCaP xenografts, the levels of PSA were ~45-fold lower than those observed in human prostate cancers (15). Therefore, the PC-82 represents a more relevant model for estimating levels of hK2 in human prostate cancers. PC-82 xenografts were harvested and homogenized; then, total PSA and hK2 levels in tumor lysates were determined by ELISA assay (Hybritech, San Diego, CA). The concentration of PSA in these PC-82 lysates was 5.15 ± 0.35 µg/g of tissue, whereas the levels of hK2 were ~12-fold lower at 0.41 ± 0.06 µg/g of tissue.

On this basis, we estimated that a level of hK2 of ~1 µg/mL in the conditioned medium would roughly approximate levels of enzymatically active hK2 found in extracellular fluid of human prostate cancer xenografts. Therefore, in the next series of experiments, non-hK2-producing human TSU cells were treated in serum-containing medium in the absence or presence of purified, enzymatically active hK2 (1 µg/mL). Clonal survival assays were then done after 5-day exposure to fhK2-activated prodrug (Fig. 7). In these experiments, there was ~10-fold enhancement of efficacy (i.e., IC50 = 0.5 µmol/L in the presence of hK2 versus ~5 µmol/L in the absence of hK2) of the hK2-activated drug in the presence of enzymatically active hK2 (1 µg/mL) in the serum-containing tissue culture medium.



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  		Figure 7. Cytotoxicity of Ac-GKAFRR-L12ADT in the presence of hK2-containing medium. Human TSU bladder cancer cells were exposed for 5 days to indicated concentrations of prodrug in either standard serum-containing medium or same medium plus enzymatically active hK2 (1 µg/mL). Clonal survival was then compared with survival of vehicle-treated controls.

 
Hydrolysis of hK2 Peptide and Prodrug by Other Potential Tumor-Associated Proteases
Due to their short length, the hK2 peptide substrates could potentially be substrates for other trypsin-like proteases as well. Although a rather specific protease substrate can be defined with seven amino acids, there is a lack of higher-order structural information by which natural protein substrates normally impose high specificity. Cancer progression is often correlated with increased protease activity (38). These activities could be potentially beneficial because they could broaden the scope of applications for protease-activated prodrugs. To test the hypothesis that other tumor-associated proteases could activate the Ac-GKAFRR-L12ADT prodrug, we selected several known proteases (Table 3) implicated in cancer progression to determine if our lead substrate, GKAFRR-L, could be efficiently hydrolyzed by any of these proteases. For this analysis, we selected trypsin-like Ser proteases plasmin and urokinase, cathepsin B, and cathepsin D. We analyzed hydrolysis of both the fluorescence-quenched peptide substrate and the thapsigargin prodrug. No appreciable hydrolysis of either substrate was observed following incubation with cathepsin D or cathepsin B. Urokinase showed low activity on the fluorescence-quenched peptide substrate but not on the prodrug (Table 3). Plasmin had a >10-fold slower rate of hydrolysis of the peptide substrate than hK2. However, with the prodrug, plasmin had ~6-fold higher hydrolysis rate than hK2. Analysis of the cleavage products showed that, with plasmin, proteolysis occurs between the two arginines, generating the less potent cytotoxin Arg-Leu-12ADT. Plasmin therefore could be a valid target for selective activation of the GKAFRR-L12ADT prodrug in other types of cancer where plasmin activation may play an important role. Further studies are under way in our laboratory to characterize in vitro activation of the GKAFRR-L12ADT prodrug using non–prostate cancer cell lines that display increased plasmin activity.


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  		Table 3. Relative hydrolysis rates normalized to hydrolysis rate of cathepsin B

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The peptide substrate requirements of hK2 have been characterized to identify peptides that are readily cleaved by this trypsin-like Ser protease that could be used to produce prodrugs that are targeted to hK2-producing prostate cancers. Previous to our studies, several groups have evaluated the substrate requirements for hK2. Mikolajczyk et al. (21) used a few peptide substrates and concluded that hK2 had P1 Arg-restricted specificity. More recently, Cloutier et al. (39) recently used phage display technology to study the substrate specificity of hK2 using many peptide sequences and also reported that hK2 has a strict preference for Arg in the P1 position. Substitution of the P1 Arg with Lys or His normally abolished all hK2 hydrolysis. Despite many attempts, including a combinatorial peptide library screen, we did not identify an Arg-free hK2 substrate, confirming the findings of these previous investigators. We further showed that hK2 prefers dibasic Arg-Arg at the P1-P2 positions, an observation that was initially reported by Lovgren et al. Additionally, we observed that substitution of D-Arg in the P1 and/or P2 position significantly decreases hK2 hydrolysis.

Previously, several studies have shown that inactivation of peptide hormones such as vasopressin as well as major clotting factors occurs by cleavage after Arg by proteolytic activity present in plasma (40, 41). Because these hK2-selective peptides will be incorporated into prodrugs that will need to be given systemically via the blood, our main concern was that Arg-containing peptide substrates for a trypsin-like protease such as hK2 would have limited stability in plasma. This would severely limit the feasibility of a hK2-targeted prodrug. Our initial results were discouraging in that all of the Arg-containing hK2 fluorescence-quenched peptide substrates were rapidly hydrolyzed in non-hK2-containing human and mouse plasma. However, the L12ADT-containing hK2 prodrug was completely stable in human and mouse plasma possibly due to binding of this prodrug to serum proteins, making it inaccessible to active plasma proteases. Currently, studies are under way in our laboratory to elucidate the mechanisms for this paradoxical plasma stability of the prodrug versus the unconjugated peptide substrates.

Several laboratories are currently developing protease-targeted prodrugs using similar types of combinatorial or phage-based screens to identify peptide substrates. Our results show that the peptide carrier may behave differently when unconjugated to drug than the subsequent prodrug in terms of hydrolysis rates and stability in plasma. This difference most likely is due to certain characteristics (e.g., hydrophobicity, molecular weight, and degree of binding to serum proteins) of the drug being targeted. Therefore, the prescreening of lead peptides unconjugated to drug for plasma stability may not be useful for predicting plasma stability of the subsequent prodrugs.

In this study, there was only ~4-fold difference in cytotoxicity between the highest hK2-producing cell line (C4-2B) and a non-hK2-producing cell line (TSU). This limited differential cytotoxicity most likely is due to the low level of hK2 production by the human prostate cancer cell lines evaluated in this study (37). In this regard, when non-hK2-producing TSU cells were incubated in the presence of higher levels of hK2 that are more representative of levels that may be present in extracellular fluid of human prostate cancers, the differential cytotoxicity increased to 10-fold. Additionally, Kumar et al. (42) showed that, in serum-containing conditioned medium from LNCaP cells, most of the hK2 produced was present as enzymatically inactive pro-hK2, whereas, if these cells were grown in serum-free medium supplemented with androgen, all of the hK2 was present as mature hK2. Finally, enzymatically active hK2 rapidly forms complexes with serum protease inhibitors such as {alpha}2-macroglobulin, {alpha}1-antichymotrypsin, {alpha}2-antiplasmin, protein C inhibitor (20, 21). Thus, in vitro and in vivo assessment of hK2-selective cytotoxicity of the hK2-activated prodrug will be difficult because the available cell models produce much lower levels (i.e., ~100-fold) of enzymatically active hK2 than estimated levels in extracellular fluid of human prostate cancers, which in serum-containing medium may be enzymatically inactive either through lack of proper processing (i.e., pro-hK2) or through binding to serum protease inhibitors. Therefore, we are currently measuring levels of hK2 in the extracellular fluid of human prostate cancers to generate human prostate cancer cell lines that produce similar levels of enzymatically active hK2 that can then be used to better assess the cytotoxicity of the hK2-activated prodrug both in vitro and in vivo.


   Footnotes
 
Grant support: NIH Prostate SPORE P50 CA58236 (S.R. Denmeade), Aegon Scholarship in Oncology (S. Janssen), and Danish Cancer Society (C.M. Jakobsen).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/ 9/04; revised 8/12/04; accepted 8/27/04.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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