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. Author manuscript; available in PMC: 2012 Sep 24.
Published in final edited form as: Biol Chem. 2010 Apr;391(4):333–343. doi: 10.1515/BC.2010.044

Prostate-specific antigen: an overlooked candidate for the targeted treatment and selective imaging of prostate cancer

Aaron M LeBeau 1,a,*, Maya Kostova 2, Charles S Craik 3, Samuel R Denmeade 1,2
PMCID: PMC3454521  NIHMSID: NIHMS336643  PMID: 20180648

Abstract

The role of prostate-specific antigen (PSA) or kallkirein-related peptidase 3 (KLK3) as a biomarker for prostate cancer is well known; however, the precise physiologic role of its serine protease activity in prostate cancer remains a mystery. PSA is produced at high levels by both androgen-dependent and -independent prostate cancers. Studies have documented high levels of active PSA in the milieu surrounding osseous and soft tissue metastases. This evidence, coupled with growing experimental evidence, suggests that PSA plays an important role in the pathobiology of prostate cancer. These observations support the development of PSA-selective inhibitors as useful tools for the targeted treatment and imaging of prostate cancer. Here we review the research that has been conducted to date on developing selective inhibitors for PSA. The different approaches used to determine PSA substrate specificity and for creating inhibitors is discussed. In addition, the unique active site characteristics of PSA and how these motifs aided our research in developing PSA targeted agents is highlighted.

Keywords: human kallikrein-related peptidase 3, inhibition, prostate cancer, prostate-specific antigen, serine protease, small molecule

Introduction

Prostate cancer is uniformly lethal once it has escaped the confines of the prostate gland, resulting in the death of over 25 000 American men each year (Jemal et al., 2007). While we have learned a great deal about the biochemistry and genetics of prostate cancer over the past decade, there are two critical needs associated with the treatment of metastatic prostate cancer that have yet to be met. First, all men undergoing androgen ablation therapy eventually relapse and no longer respond to androgen ablation, no matter how completely given (Crawford et al., 1989; Laufer et al., 2000). At this point in the disease process there is an urgent need for more effective non-hormonal therapies for patients with metastatic disease. Second, the clinical development of novel therapies is limited by the inability to adequately image the therapeutic response of prostate cancer metastases in patients. In men failing androgen ablative therapy, prostate cancer preferentially metastasizes to the bone resulting in the formation of predominantly osteoblastic lesions (O’Keefe and Guise, 2003; Roodman, 2004). Therapeutic response in the bone is difficult to evaluate with current imaging modalities. Thus, there is a compelling need for better imaging modalities for prostate cancer that can be used to detect disease recurrence at an earlier time point and to better evaluate the therapeutic efficacy of new treatments. A number of prostate cancer targets have been identified (e.g. androgen receptor, pTEN, etc.) and novel therapies directed at these targets are currently under development (Sarker et al., 2009; Vis and Schröder, 2009). One target for prostate cancer that has been overlooked is prostate-specific antigen (PSA) or human kallikrein-related peptidase 3 (KLK3). Prostate cancer cells, like normal prostate secretory-luminal epithelial cells, produce very high levels of the PSA (Lilja, 1985; Watt et al., 1986). PSA is a chymotrypsin-like kallikrein that is used extensively as a biomarker to screen for prostate cancer, to detect recurrence following local therapies and to follow response to systemic therapies for metastatic disease (Lilja, 1985; Watt et al., 1986; Denmeade and Isaacs, 2004). PSA is aptly named, in that it is produced at high levels exclusively by prostate cells and is not produced in significant amounts by any other normal tissue in men (Lilja et al., 2000).

Since the initial discovery of PSA, thousands of papers have been written about its role as a biomarker for prostate cancer. The functional role for PSA in the normal prostate and in prostate cancer is not known and has not been fully addressed in the literature. Thus, PSA is an enigmatic protease; it is well known, yet poorly understood. Recently, a number of studies have implicated a role for PSA in the pathobiology of prostate cancer. Accumulating evidence suggests that PSA is more than a tumor biomarker and may function as a salient player in prostate cancer growth, invasion and metastasis. To understand the role of PSA in prostate cancer, the field would be greatly aided by the development of a small-molecule inhibitor for the enzymatic activity of PSA. The selective inhibition of PSA could have important biological effects that would allow for the interrogation of PSA’s mechanism of action in promoting prostate cancer. In addition to defining the role of PSA in prostate cancer, small-molecule inhibitors of PSA’s enzymatic activity could hold promise as therapeutic agents against prostate cancer local growth, progression and/or metastasis. Such PSA inhibitors could also find a potential use as imaging agents due to the presence of high levels of enzymatically active PSA in the peritumoral fluid surrounding prostate cancers. In this review, we hope to shed some light on this enigma through an overview of current data addressing PSA’s role in prostate cancer pathobiology. In addition, we will review research efforts to develop inhibitors for PSA that could represent new tools to study, treat and/or image prostate cancer.

PSA in prostate cancer

A number of possible mechanisms whereby PSA may contribute to biological responses have been identified (Yousef and Diamandis, 2003). In in vitro studies, active PSA has been shown to directly cleave or release from binding proteins cytokines that are involved in growth stimulation and inflammation. PSA can cleave insulin-like growth factor binding proteins-3 (IGFBP-3) resulting in local release of IGF-1 (Williams et al., 2007a). The roles of IGF-1 and IGFBP-3 in prostate cancer are controversial. A number of studies have demonstrated an association between IGF-1 plasma levels and prostate cancer, while an equal amount have found that there are no increases in cleaved IGFBP-3 or IGF-1 in the plasma of men with prostate cancer when compared to healthy individuals (Cohen et al., 1992; Chan et al., 1998; Stattin et al., 2001; Koistinen et al., 2002). Despite this contradictory evidence in vivo, IGF-1 is known to have important effects in vitro. IGF-1 can have mitogenic and antiapoptotic effects on normal and transformed prostate epithelial cells as well as effect stromal-epithelial cells interactions (Iwamura et al., 1993; Culig et al., 1995). Additionally, in primary cultures of prostatic epithelial cells, PSA was found to specifically activate the small latent form of TGFβ2 suggesting that PSA may directly stimulate a variety of cellular processes through the release of active TGFβ2 stored within the matrix of the prostatic stroma (Dallas et al., 2005).

Unlike most solid tumor types, a hallmark of prostate cancer is the almost universal development of osteoblastic bone metastases in men failing androgen ablative therapy (O’Keefe and Guise, 2003; Roodman, 2004). The mechanism whereby PSA may stimulate bone adhesion or osteoblast growth and/or differentiation has not been clearly defined. As described above, active PSA can release cytokines involved in bone turnover such as IGF-1 and TGFβ2. For example, PSA cleavage of IGFBP-3 can release IGF-1 which can promote osteoblastic activity in bone. PSA activation of the small latent form of TGFβ2 in the bone microenvironment may also potentially contribute to the formation of osteoblastic lesions (Kanety et al., 1993). Previously, it had been demonstrated that PSA could hydrolyze PTHrP, an important mediator of osteolytic bone metastases, and inactivate PTHrP-stimulated cAMP accumulation in mouse osteoblasts (Iwamura et al., 1996; Dallas et al., 2005). PTHrP 1–141 can be cleaved by PSA to release a 22 and 23 amino acid fragments of PTHrP (Cramer et al., 1996). PTHrP stimulates osteoblastic production of the osteoclastic factor RANKL, but PTHrP has also been shown to also inhibit production of the osteoblastic factor endothelin-1 (Lawrence, 2001). This suggests PSA could alter the bone metastatic phenotype from osteoclastic to osteoblastic through PTHrP degradation.

PSA is known to interact or metabolize a number of factors involved in the production of osteoblastic lesions. For example, PSA can degrade extracellular matrix components fibronectin and laminin (Lilja et al., 2000). PSA-specific mAbs have been shown to block proteolysis of extracellular matrix components and decrease invasion of PSA-producing LNCaP cells in Matrigel invasion assays (Webber et al., 1995). Using phage display, Romanov et al. identified peptides with homology to the C-terminus of PSA that attenuated binding of a prostate cancer cell line to bone marrow endothelial cells (BMEC) (Romanov et al., 2004). In this study, PSA antibodies and exogenous enzymatically active PSA attenuated binding of prostate cancer cells to BMECs (Romanov et al., 2004). These results suggest a role for active PSA in preferential adhesion of prostate cancer cells to bone. Additionally, Gygi et al. (2002) demonstrated that exogenous PSA could stimulate modest proliferation of human osteosarcoma cells. Using this same osteosarcoma cell line, Nadiminty et al. showed that PSA-expressing SaOS-2 cells markedly upregulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin (Nadiminty et al., 2006).

To further add mystery and complexity to the role PSA plays in prostate cancer development, numerous studies have documented the anti-angiogenic properties of PSA. PSA has been shown to inhibit angiogenesis both in vitro and in vivo (Fortier et al., 1999, 2003). In early findings, Fortier et al. noted that purified PSA inhibited human umbilical vein endothelial cell (HUVEC) proliferation, migration, and invasion. Additionally, PSA inhibited endothelial cell response to the angiogenic stimulators FGF-2 and VEGF. Later the same group demonstrated in vivo using a murine model that PSA inhibited FGF mediated angiogenesis in a matrigel plug assay (Fortier et al., 2003). In an attempt to exploit the anti-angiogenic properties of enzymatically active, Wu et al. used phage display to discover PSA-activating peptides (Wu et al., 2000). Selected peptides from phage libraries were expressed as glutathione-S-transferase (GST) fusion peptides and tested for activity. The best peptide stimulated PSA activity five fold over the control and was highly selective for PSA when compared to proteases possessing similar specificity. Such peptides also inhibited HUVEC growth and are currently undergoing structural refinement in order to gain favorable pharmacokinetics and dynamics in vivo. (Mattsson et al., 2009; Koistinen et al., 2008a)

Peptide substrates for PSA

While the studies referenced above have identified a series of putative protein substrates for PSA, a number of studies have evaluated PSA substrate requirements using small peptide substrates. Initially, a variety of chymotrypsin substrates containing tyrosine in the P1 position were used to assay the enzymatic activity of PSA. These substrates, however, had relatively high Km values (1–2 mM), low kcat values for PSA and poor specificity (i.e., 10 000-fold more rapidly cleaved by chymotrypsin) (Watt et al., 1986; Lilja et al., 1989; Kurkela et al., 1995). The discovery that PSA could cleave the gel-forming proteins semenogelin I and II in human semen led Lilja et al. to map PSA-specific cleavage sites within these proteins. As expected, PSA cleaved after tyrosine and leucine residues (Lilja et al., 1985, 1989; Malm et al., 2000). Somewhat surprising was the finding that out of the nearly thirty peptides detailed in the cleavage map, nearly 40% of the peptides contained a glutamine residue in the P1, making PSA the only known serine protease with this substrate specificity in vivo.

With the goal of developing PSA-activated peptide prodrugs, Denmeade and colleagues used the semenogelin cleavage map to define selective PSA substrates (Denmeade et al., 1997). In this study, peptides of seven amino acids representing the sequence promixal to the PSA cleavage site (P7-P1) were synthesized with 7-amino-4-methylcoumarin (AMC) attached to the C-terminus of the P1 amino acid in the P-1 position (Denmeade et al., 1997). This method yielded PSA substrates with improved Km and kcat values and a high degree of specificity. Those substrates containing tyrosine in the P1 position were hydrolyzed efficiently by PSA, but the proteolysis rates were 10–20 higher with chymotrypsin and elastase. Substrates with glutamine in the P1 position were less efficiently hydrolyzed, but demonstrated better specificity for PSA. The P1 glutamine containing peptide EHSSKLQ-AMC was chosen from among the other sequences for additional characterization based on its PSA acitivity, its specificity for PSA and its stability in different sera. This sequence was used to generate substrates of varying lengths that were analyzed for PSA activity and specificity. It was found that PSA could only cleave substrates that were 4 amino acids or greater in length. From these studies the six amino acid substrate HSSKLQ-AMC (Km = 470 μM, kcat/Km = 23 M−1 s−1) was evaluated further based on a combination of efficiency and selectivity of hydrolysis by PSA. This PSA substrate sequence has been used to measure PSA activity in a variety of tissues (Denmeade et al., 1997). In addition it has been used to generate novel PSA activated prodrugs and protoxins for the treatment of prostate cancer and BPH (Denmeade et al., 1998, 2003a,b).

Other groups have sought to further define the substrate specificity of PSA using combinatorial methods and by refining sequences from naturally occurring substrates. Coombs et al. (1998) investigated the sequence characteristics of the substrate prime site binding pockets. Optimized semenogelin peptides and phage display were used to indentify PSA cleavable substrates containing P4 to P2′ residues. Five rounds of phage display yielded sequences were rich in hydrophobic residues populating the P1 and P2 positions with tyrosine and leucine being the most common P1 residues. When the residues contributed by the phage linker region were removed to prevent any bias towards serine, glycine or alanine, the resulting consensus sequence was: P5 (R, L > X), P4 (S > A), P3 (S > A, R, T), P2 (Y > X), P1 (Y > L), P1′ (S, T, A > Q), P2′ (S > A, R), P3′ (A, S). The hydrolysis rates of peptides selected from phage display were determined for PSA and chymotrypsin. In each case the kcat/Km value was found to be greater for chymotrypsin, often significantly (2.6 M−1s−1 for PSA versus 1.1↓105 M−1s−1). The substrates discovered were excellent PSA substrates, but even better substrates for chymotrypsin. This was most likely due to the fact that peptides identified by this method contained primarily Y or L in the P1 site. Taking advantage of the available crystal structure data showing that serine proteases as a group interact most strongly with positions P3-P3′, Yang et al. developed synthetic hexapeptide substrates for PSA using single position mini-libraries (Yang et al., 1999). P1′ and P2 mini-libraries were constructed based on a selected semenogelin cleavage peptide with tyrosine as the constant P1 residue. In this limited study, serine was found to be the preferred P1′ residue and phenylalanine the preferred P2 residue. The enzyme kinetics of the optimized substrate Mca-QFYSSNK(ε-Dnp) were measured for PSA and found to have a Km of 77μM, but a low kcat/Km of 39 M−1 s−1. Finally, Debela et al. recently determined the non-prime side specificity of several kallikreins, including PSA, using a positional scanning synthetic combinatorial library of tetrapeptide substrates (Debela et al., 2006). Using this method the order of preferred PSA P1 residues was found surprisingly to be Met > Nle = Ala > Tyr = Arg.

Structural features of the S1 pocket define PSA substrate specificity

The development of potent and selective PSA substrates and inhibitors requires a detailed understanding of the structure of the catalytic site of PSA. Particular attention must be paid to the S1 pocket of the protease, which in large part determines the specificity. Sequence alignment of PSA with all of the human kallikreins and bovine α-chymotrypsin (Figure 1A) reveals that the region spanning the S1 pocket contains a number of highly conserved residues that are critical for the structural integrity of the pocket and indirectly responsible for the maintenance of enzymatic activity. Specifically, residues such as Cys191, Cys220, Pro225 and Ser214 form crucial elements of the architecture surrounding the S1 pocket (Singh et al., 2009). Conversely, the variable regions of residues 183–190 and 221–224 within a given serine protease dictate the unique specificity of substrates and inhibitors for the individual protease. For all serine proteases, substrate and inhibitor recognition is mainly governed by the binding of the P1 amino acid residue to the S1 pocket of the enzyme (Figure 1A). The amino acid residue at the bottom of the S1 pocket is therefore the key determinant for the P1 preference. For the majority of the kallikreins, this residue is aspartic acid. Thus, the majority of kallikreins exhibit trypsin-like specificity, cleaving after basic amino acids such as arginine and lysine. In contrast, for PSA the residue at the bottom of the pocket is serine (i.e. Ser189). Chymotrypsin also has serine in this position and thus shares some substrate specificity with PSA, cleaving after tyrosine and phenylalanine. Two other kallikreins also have a unique (i.e. non-aspartic acid) amino acid in the S1 pocket with KLK7 having asparagine and KLK9 having glycine at this position.

Figure 1.

Figure 1

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Sequence alignment of human kallikreins with bovine α-chymotrypsin (A) and structure of the S1 specificity pocket of PSA (B).

(A) Amino acid sequence alignment of the 15 human kallikreins and bovine α-chymotrypsin. Only the residues spanning the S1 pocket of serine proteases are presented. The residues highlighted in yellow are critical for the P1 specificities of respective protease. The catalytic Ser195 residue is highlighted in red. (B) Ribbon representation of the S1 specificity pocket of prostate-specific antigen. The protease residues lining the pocket are shown in cyan while the catalytic histidine and serine residues are shown in magenta. The S1 tyrosine residue of peptide substrate KGISSQY is shown in yellow.

The Ser189 residue in the PSA S1 pocket is flanked by the polar side chains of Ser226 and Thr190 (Figure 1B). The alignment of the polar hydroxyl moieties of these residues at the bottom of the pocket results in a cavity that is polar at the bottom, but hydrophobic on the sides due to the aliphatic parts of the side chains forming the walls of the cavity. As shown in Figure 1B, the aromatic ring of a P1 tyrosine residue is optimally sandwiched between the hydrophobic cavity walls while making a hydrogen bond with the backbone carbonyl of the Ser217 residue via its hydroxyl moiety. Thus, a residue like tyrosine with a medium-sized hydrophobic side chain and a polar phenolic group is ideal for binding at the S1 pocket of PSA. Other medium sized hydrophobic side chains such as leucine and norleucine can also interact favorably with this pocket. The other residues, Thr190, Thr213 and Ser226, in the vicinity of the 189-position in the PSA protein are also responsible for the subtle variations in the polarity and hydrophobic character of the S1 binding site. These residues produce a pocket that is truly unique in its binding characteristics.

These insights into the S1 pocket of PSA came initially from models built using the specificity pocket of other similar tissue kallikreins (Adlington et al., 2001; Singh et al., 2008) These models were later modified once a partial crystal structure for PSA was solved (Menez et al., 2008). The first PSA inhibitors reported in the literature used such a homology model derived from porcine kallikein to design and characterize a series of monocyclic β-lactam compounds (Adlington et al., 2001). Prior to this study, it had been shown that β-lactams were efficient and specific inhibitors of several serine proteases, especially human leukocyte elastase (Knight et al., 1992; Green et al., 1995). The first generation of monocyclic β-lactam inhibitors for PSA began with a lead compound 1 (IC50 = 8.98 μM) developed by Adlington et al. (Adlington et al., 2001) (Table 1). This group then used the homology model to aid in the development of more potent PSA inhibitors 1–7 (Adlington et al., 1997). Using a low energy conformation of the azitidinone compound 1, initial modeling studies suggested that the (3S,4S) enantiomer should be used as the template for the further SAR studies. With this template the binding importance of the three side chains appended to the 2-azetidininone was investigated in the active site pocket of PSA homology model. The best inhibitor from these studies was the enantiomerically pure inhibitor 7 possessing an IC50 value of 0.226 nM for PSA (Adlington et al., 2001). The specificity of this compound and others in the class versus chymotrypsin and other proteases was, unfortunately, not reported this study.

Table 1.

Structures and IC50 values of the (β-lactam-based PSA inibitors developed by Adlington et al. (1997, 2001).

graphic file with name nihms336643u1.jpg
Compound R1 R2 R3 IC50 (μM)
1 H CO2Bn CO2H 8.98 ± 0.1
2 H P-CO2CH2-C6H4-CO2H CO2H 5.84 ± 0.9
3 H H CO2H 24.3 ± 3.9
4 H CO2H CH2NH2-TFA 1.34 ± 0.1
5 H CO2Bn H 1.43 ± 0.2
6a OH CO2Bn H 0.348 ± 0.1
7b OH CO2Bn H 0.226 ± 0.01
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a

Racemic mixture of compounds.

b

Homochiral 3S4S enantiomer.

To develop potent and selective β-lactam based inhibitors for PSA, our laboratory investigated the mechanism behind PSA inhibition by β-lactams and tested newly synthesized β-lactams for candidate inhibitors (Singh et al., 2008). It has been suggested that β-lactams inhibit PSA in a time dependant fashion with a 1:1 stoichiometry. To probe for the time dependent nature of β-lactam inhibition, time course assays in which the enzymatic reaction was allowed to run for 1h, after which a representative β-lactam 1 (IC50 = 8.98 μM) was added at a 1 mM final concentration and the assay was allowed to proceed for another hour. The inhibitory effect was not immediate and not observed until 24 minutes after the addition of the inhibitor, at which time the inhibition was complete. Based on these results, it was concluded that the β-lactams react with PSA in a time-dependent manner, competing with substrate, until a majority of PSA is converted into an irreversible inactive state. Inhibitor wash-out studies and MALDI mass spectrometry analysis indicated that a stable covalent complex formed between the β-lactam and the active site serine residue in PSA (Singh et al., 2008).

Other attempts have been made to find small-molecule inhibitors for PSA using compounds other than β-lactams. Azapeptides, that can inhibit both cysteine and serine proteases, have been developed and used to effectively inhibit PSA (Huang et al., 2001). The best inhibitor from this study had a Ki of 0.5 μM and a kinact/Ki of 32000 M−1s−1. To elucidate the role PSA in angiogenesis, Koistinen and co-workers screened a library of nearly 50 000 compounds to discover inhibitors of PSA (Koistinen et al., 2008b). Out of the compounds screened only two were identified that inhibited PSA in a dose dependent manner. The active compounds were either benzoxazinone or triazole derivatives. Focused mini-libraries of compounds similar in structure to the initial compounds were constructed resulting in the discovery of several compounds with low micromolar and nanomlar IC50 values for PSA. It was found that the benzoxazinone compounds were non-competitive inhibitors and the triazoles were competitive inhibitors of PSA. The most effective inhibitors were tested in a HUVEC angiogenesis model. Two of the compounds were found to abrogate the anti-angiogenic function of PSA as noted by the increased tube area and formation of tubular connections in the HUVEC model (Koistinen et al., 2008b).

Peptide-based PSA inhibitors

Peptide-based inhibitors have the advantage of ease of synthesis over the other small molecule classes described above. Peptides also provide a convenient platform for the addition of therapeutic and imaging moieties. On this basis, we used an iterative approach to generate a series of inhibitors. For screening purposes, peptide aldehydes were initially generated based on ease of synthesis (LeBeau et al., 2008, 2009a,b). Subsequently, boronic acid based inhibitors were generated from promising peptide aldehydes.

Since the recognition of glutamine in the P1 position is relatively unique to PSA, the first set of studies was designed to evaluate the specificity of glutamine containing peptide aldehyde inhibitors using the peptide substrate SSKLQ as a starting template. The evaluation of a P1 glutamine peptide aldehyde inhibitor is problematic, since glutamine aldehydes exist primarily in the cyclic hemiaminal form. While NMR analysis confirmed that the full length glutamine peptide aldehyde 8 was in equilibrium between a cyclic hemiaminal and an aldehyde, it could still act as a modest inhibitor of PSA with a Ki of 45.21 μM (Table 2). Although cyclization does occur and is thermodynamically favored, the side chain is still potent and able to form favorable interactions with the bottom of the S1 pocket. Therefore, this result suggested that a non-cyclizable glutamine aldehyde derivative would be an even more potent and viable PSA inhibitor. The non-cyclizable γ-N,N-dimethyl glutamine derivative 9 was a more potent inhibitor with a nearly 20 fold lower Ki of 2.53 μM (LeBeau et al., 2009a). PSA inhibitor 9 still possessed the desired predicted motif, a side chain with two hydrophobic methlyenes, an amide carbonyl and a nitrogen with a lone pair of electrons that were in the vicinity of other polar residues and could act as hydrogen bond acceptors. A shorter side chain 10 resulted in a less potent inhibitor with a Ki of 13.09 μM for PSA (Table 2).

Table 2.

The chemical structures, Ki values and GOLD scores of the PSA inhibitors used in this study.

graphic file with name nihms336643u2.jpg
Compound R Ki(μM) GOLD Score (PSA)
8 CH2CH2CONH2 45.21 ± 3.89 33.45
9 CH2CH2CON(CH3)2 2.53 ± 0.13 30.66
10 CH2CON(CH3)2 13.09 ± 0.88 19.82
11 CH2NHCHO 0.91 ± 0.06 29.54
12 CH2NHCOCH3 3.91 ± 0.35 29.56
13 CH2NHCOCH2CH3 9.84 ± 0.33 34.13
14 CH2NHCOCH(CH3)2 13.28 ± 0.93 33.25
15 CH2NHCO(C6H5) >25 27.55
16 CH2CH2SCH3 3.84 ± 0.21 34.77
17 CH2CH2SOCH3 7.25 ± 0.44 38.27
18 CH2CH2CN 8.14 ± 0.29 27.47
19 CH(CH3)2 >100 16.16
20 CH2CH(CH3)2 6.51 ± 0.25 26.37
21 CH2CH2CH2CH3 11.24 ± 0.26 30.93
22 (C6H5) >100 22.92
23 CH2(C6H5) 0.57 ± 0.04 38.84
24 CH2(C6H5)OH 0.37 ± 0.02 39.66
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Isostere replacement glutamine derivative peptide aldehydes (11–15) were tested and a pronounced increase in the ability to inhibit PSA was seen with some of the compounds (LeBeau et al., 2009a). The best compound of this series, 11 the N-formyl derivative, had a Ki of 0.909 μM for PSA. The rest of the compounds in the series all possessed Ki values of greater than 1 μM. Compound 12, with the larger acetyl group on the γ amine, had a higher Ki of 3.91 μM. The Ki values for the compounds (13–15) steadily increased as the acyl chain increased in size and branching, leading to unfavorable steric clashes with the S1 pocket wall. Based on positional scanning substrate studies documenting the ability of PSA to cleave after methionine residues (Debela et al., 2006), peptide aldehydes containing a P1 methionine 16 and methionine sulfoxide 17 were generated and also found to inhibit PSA, with 16 having a lower Ki of 3.84 μM and 17 possessing a nearly 2-fold higher Ki of 7.25 μM (LeBeau et al., 2009a) (Table 2).

Peptide aldehydes possessing hydrophobic P1 aldehydes containing natural and unnatural amino acids were next tested for their ability to inhibit PSA (LeBeau et al., 2009a; see Table 2). Compound 19, the full length valine peptide aldehyde, was not an inhibitor of PSA with a Ki>100 μM. By increasing the side chain of the valine by one carbon to the leucine peptide aldehyde 20, the Ki decreased to 6.51 μM, emphasizing that while β-branching is not well-tolerated, γ-branching is allowed. The norleucine peptide aldehyde 21, containing the longest hydrocarbon side chain out of the series, worked as an inhibitor, however with a higher Ki of 11.24 μM. The P1 phenylglycine peptide aldehyde derivative 22 did not inhibit PSA with a Ki>100. Moving from 22 to the phenylalanine peptide aldehyde 23, a marked decrease in the Ki is observed yielding a sub-micromolar Ki of 0.57 μM. The substitution of a phenolic side chain as in the tyrosine peptide aldehyde 24 produced an even more potent PSA inhibitor with a Ki of 0.37 μM. An explanation for this observation is that the tyrosine side chain makes sufficient hydrophobic interactions with the walls of the S1 pocket and its hydroxyl group is able to interact in a favorable manner with the polar residues at the bottom of the pocket.

To demonstrate the unique substrate specificity of PSA versus chymotrypsin, the best P1 peptide aldehyde inhibitors were tested against chymotrypsin (LeBeau et al., 2009a). The full length glutamine aldehyde 8, while not the best inhibitor but one that demonstrated the unique proteolytic activity of PSA, was tested against chymotrypsin and showed no ability to inhibit chymotrypsin with a Ki for chymotrypsin >1000 μM (Table 3). Likewise the non-cyclizable γ-N,N-dimethyl glutamine derivative 9 did not inhibit chymotrypsin to any degree nor did the glutamine isostere 11. Compounds 23 and 24 were almost equally as potent for PSA and chymotrypsin since both compounds were peptide aldehydes of canonical hydrophobic P1 residues. Having established a positive correlation between GOLD score and the inhibitory potency of the peptide aldehyde inhibitors, we set out to use the same methodology for elucidating the difference in potency of six peptide aldehyde inhibitors that were simultaneously tested against both PSA and chymotrypsin. Table 3 presents the respective Ki values and the GOLD docking scores of these compounds when their P1 side chain was docked in the S1 pocket of either protease. Remarkably, the difference in the potency of 8, 9, 11 and 16 against PSA versus chymotrypsin was consistent with the respective differences in GOLD scores, especially for compound 8, 9, and 11. Similarly, 23 and 24 were equally potent against both PSA and chymotrypsin also possessed the highest GOLD scores.

Table 3.

The specificity of six peptides aldehydes for PSA versus chymotrypsin and the corresponding GOLD scores for each protease.

Compound PSA Chymotrypsin Chy/PSA ratiob PSA Chymotrypsin
Ki (μM)a Ki (μM)a Gold score Gold score
8 45.21 ± 3.89 >1000 >22 33.45 16.53
9 2.53 ± 0.13 >100 >40 30.66 14.27
11 0.91 ± 0.06 >100 > 110 29.54 19.25
16 3.84 ± 0.21 >100 >26 34.77 23.07
23 0.57 ± 0.04 0.62 ± 0.05 1.08 38.84 28.38
24 0.37 ± 0.02 0.29 ± 0.03 0.78 39.66 28.86
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a

Value reported is for n=3 (±S.E.).

b

PSA selectivity in terms of (Chymotrypsin Ki/(PSA Ki).

The P1 residues of the most specific peptide aldehyde inhibitors 8, 9, 11, 16 could not easily be converted into boronic acids, thus, our next study consisted of evaluating peptide aldehdyes with fixed P1 side chains whose structures could be translated into boronic acids. We decided to fix the P1 position as leucine and norleucine due to the ease of synthesizing resin-bound leucine/norleucine peptide aldehydes and the short synthesis required to make boronc acid derivatives. The effect on substituting different amino acids in the P2 position and the peptide length was investigated (Table 4). The data from the P2 focused library provided evidence that PSA prefers hydrophobic residues in the P2 position (LeBeau et al., 2009b). Both negatively and positively charged residues were not tolerated. It was observed that residues containing β branched side chains, i.e. isoleucine, valine and threonine, were not effective inhibitors. On the inhibitor length, it was discovered the inhibitor had to be at least four residues in length for effective inhibiton with the P5 residue representing a variable position. Based on these findings the inhibitor Z-SSKLL-H (Ki = 6.5 μM) was selected as the best inhibitor chosen for further study.

Table 4.

Peptide aldehdye and boronic acid inhibitors of PSA.

Compound Ki(μM)
Leucine aldehydes
25 Z-SSKLL-H 6.51 ± 0.25
26 Z-SSKFL-H 11.9 ± 0.9
27 Z-SSKYL-H 13.09 ± 0.91
28 Z-SSKML-H 13.7 ± 1.2
29 Z-SSKQL-H 21.79 ± 0.51
30 Z-SSKIL-H 37.43 ± 2.43
31 Z-SSKKL-H 57.85 ± 1.14
32 Z-SSKVL-H 70.71 ± 1.77
33 Z-SSKTL-H >100
34 Z-SSKAL-H >100
35 Z-SSKF(Br)L-H >500
36 Z-SSKLD-H >1000
37 Z-SKLL-H 42.43 ± 2.13
38 Z-SSKL-H >1000
39 Z-KLL-H >100
40 Z-YL-H >500
41 Z-KL-H >1000
42 Z-QL-H >1000
43 Z-SSKLL-H >1000
Norleucine aldehdyes
44 Z-SSKLNle-H 11.24 ± 0.26
45 Z-KLNle-H 414.1 ± 22.49
46 Z-KNle-H >1000
47 Z-QNle-H >1000
48 Z-SSKNle-H >1000
49 Z-SSKLNle-H >1000
Peptidyl boronic acids
50 Z-Q(boro)L 265.15 ± 18.39
51 Z-K(boro)L 353.5 ± 23.68
52 Z-SSK(boro)L 18.49 ± 1.48
53 Z-SSK(boro)Nle 34.21 ± 0.83
54 Z-KL(boro)L 5.98 ± 0.24
55 Z-KL(boro)NLe 23.57 ± 0.47
56 Z-SSKL(boro)Nle 0.398 ± 0.020
57 Z-SSKL(boro)L 0.065 ± 0.005
D-isomer amino acids
58 Z-SSKL(boro)L 0.149 ± 0.009
59 Z-SSKL(boro)L 21.48 ± 2.28
60 Z-SSKL(boro)L 28.67 ± 1.32
61 Z-SSKL(boro)L 75.76 ± 5.21
62 Z-SSKL(boro)L 32.21 ± 1.73
63 Z-SSKL(boro)L 227.27 ± 11.61
64 Z-SSKL(boro)L 439.94 ± 19.04
Lysine derivatives
65 Z-SSL(boro)L 3.86 ± 0.29
66 Z-SSK(Ac)L(boro)L 0.294 ± 0.026
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Residues containing D-isomer amino acids are underlined.

Boronic acid-based PSA inhibitors

The ultimate goal of these studies was to identify a candidate PSA inhibitor that could be used to target therapeutic agents or modified to create imaging agents. While peptide aldehydes are useful screening tools, they would not be useful in complex biological matrixes. They are reactive with both cysteine and serine proteases and the aldehyde functionality can react with numerous nucleophiles in a biological milieu. To overcome this, our best aldehydes inhibitors would in turn be synthesized into boronic acid derivatives. Peptide boronic acids react only with nucleophilic serine residues and are potent serine proteases inhibitors due to the Lewis acid nature of the electron deficient boron (Bone et al., 1987). They are also stable at physiologic pH and in complex biological environments.

A strategy using peptidyl boronic acids was pursued using the sequence preferences discovered in the primary peptide aldehyde screen (LeBeau et al., 2008). A dramatic increased in inhibitory ability was seen when the sequences went from peptide aldeyhdes to boronic acids. For example the Ki of Z-SSKL(boro)L (57) was 65 nM which was a 100-fold improvement in Ki compared to the corresponding aldehyde (Table 4). D-isomer amino acids were incorporated into the sequence of our lead inhibitor Z-SSKL(boro)L (58–64) to determine the relative importance of each amino acid residue. It was found that incorporating one or multiple D-amino acids into the P2–P4 positions was highly deleterious to inhibitor function while D-amino acid substitution the P5 position was more tolerated as it only increased the Ki ~ 2-fold (LeBeau et al., 2008).

Next the inhibitor Z-SSKL(boro)L was tested for specificity against a panel of proteases consisting of chymotrypsin, trypsin, DPP-4, elastase, and the non-serine proteases cathepsin B and D (LeBeau et al., 2008). Although chymotrypsin is capable of cleaving after leucine residues, the Ki for inhibiting chymotrypsin was 60-fold higher than for PSA. Modest inhibition of elastase was observed, while the compounds did not inhibit trypsin or DPP-4 at all. No inhibition of either cathepsin B or D was observed over a range of concentrations, consistent with the fact that boronic acids alone are not effective inhibitors of cysteine or aspartyl protease.

The specificity of our inhibitor for PSA over chymotrypsin can be explained by observing the role of the P3 lysine residue in binding to PSA. In our model of the PSA catalytic site interaction with Z-SSKL(boro)L, the P3 residue serves as a lid to the specificity pocket and docks at a location just above the P1 binding site in the specificity pocket (Singh et al., 2008; LeBeau et al., 2009b; see Figure 2). In this model, we observed that the P3 lysine residue of the inhibitor interacts with a carboxyl group of the Glu208 side chain in the protease active site. The distance between the two groups (3.5Å) is close enough for the formation of a salt bridge, thus leading to favorable binding interactions between the protease and the inhibitor. Noted earlier, omission of the lysine residue (65) or replacing it with its mirror image, abrogated this important interaction and yielded poor inhibitors. The presence of an acidic residue at position 208 is not common in chymotrypsin family proteases.

Figure 2.

Figure 2

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Interactions of the Z-S5S4K3L2(boro)L1 inhibitor with the key residues in the catalytic pocket.

Catalytic triad residues are shown in yellow, the protease residues in magenta and the backbone of 204–206 residues in green. Dotted lines represent hydrogen bonds, and the dotted circle pinpoints the exact location of the boronic acid moiety.

Modeling studies further documented that the P4 pocket, located in the lower groove area, is mainly formed by His164, Pro165, Gln166 and Trp205 residues (Singh et al., 2008, 2009; Figure 2). The P4 binding pocket of PSA is bounded by the kallikrein loop from above but loop residues are not involved in any important energetic interactions. The Gln166 side chain is ideally placed to form a hydrogen bond with the hydroxyl of P4 serine (Singh et al., 2008, 2009). The presence of Gln166 at this position is unique for PSA and not common in other serine proteases from the kallikrein family. The importance of this interaction with S4 was demonstrated by marked increase in Ki for inhibitors lacking P4 Finally, the P5 side chain was found to dock in the lower groove area but it is exposed to the solvent and there are no specific interactions with the protease residue. This suggests that P5 and additional N-terminal amino acid residues should not provide a significant contribution towards the overall binding affinity of inhibitor molecule.

Subsequent to these studies and after completing an extensive aldehyde screen (LeBeau et al., 2009b), the refined sequence of the best PSA inhibitor was Z-SSQn(boro)L with glutamine in the P3 position and norleucine now in the P2 position. This new inhibitor, with an increased serum half life over 57, is the most potent described to date for PSA with a Ki = 25 nM, although it is somewhat less specific with a Ki of 211 nM for chymotrypsin. This inhibitor was subsequently coupled at the amino terminus to a single amino acid chelating group capable of chelating 99mTc without any impact on Ki for PSA inhibition (LeBeau et al., 2009b). Further studies are underway to determine the ability of such chelates to image PSA producing human prostate cancer xenografts in vivo.

Conclusions

PSA represents an excellent target for prostate cancer therapy and imaging. As prostate cancer progresses, the glandular architecture of the prostate becomes compromised allowing PSA in the tissue to leak into the circulation where it is found in high concentrations, thus becoming the basis for the PSA test. Low PSA concentrations in prostatic tissue are often associated with an increase in malignancy grade and tumor stage (Stege et al., 2000; Grande et al., 2000). PSA is produced at high levels by both androgen dependent and castrate resistant prostate cancer cells. Autopsy studies have demonstrated the homogeneity of PSA production in both bony and soft tissue metastases with the majority of cells in metastatic sites producing PSA (Roudier et al., 2003). In addition, PSA is aptly named as it is highly specific and not produced to any measurable degree by any other tissues in the adult male except by normal and malignant prostate epithelial cells (Lilja et al., 2000). As reviewed here and elsewhere, accumulating evidence suggests that this high level of PSA production may play a role in the growth and progression of prostate cancer (Williams et al., 2007a). These combined results provide the background rationale to support the further development of PSA activated therapies. In this regard, clinical studies have been performed using a PSA-activated peptide doxorubicin prodrug. This prodrug was not developed further due to poor specificity of the peptide and non-specific activation within the blood (Wong et al., 2001). However, a PSA-activated prodrug consisting of the more specific HSSKLQ peptide coupled to an analog of the highly potent natural product thapsigargin is also under development (Denmeade et al., 2003). The bacterial toxin proaerolysin has also been modified to a form that requires PSA for activation and this protoxin (Willams et al., 2007b), termed PRX302 has completed phase I studies as intraprostatic therapy for men with locally recurrent prostate cancer and phase I and II as therapy for BPH. Finally, the potent PSA inhibitors described here are also under study as potential targeted therapeutic agents and are also being modified to generate selective imaging agents for prostate cancer (LeBeau et al., 2009b).

Acknowledgments

The authors would like to acknowledge Daniel Hostetter, Amanda Vlasic, and Paco Rodriguez for their careful proof reading of the manuscript and for numerous stimulating discussions related to the topic. A.M. LeBeau is grateful to acknowledge the financial support of the US Department of Defense Postdoctoral Prostate Cancer Training Award PC094386.

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