Pseudogene Associated Recurrent Gene Fusion in Prostate Cancer.

We present the functional characterization of a pseudogene associated recurrent gene fusion in prostate cancer. The fusion gene KLK4-KLKP1 is formed by the fusion of the protein coding gene KLK4 with the noncoding pseudogene KLKP1. Screening of a cohort of 659 patients (380 Caucasian American; 250 African American, and 29 patients from other races) revealed that the KLK4-KLKP1 is expressed in about 32% of prostate cancer patients. Correlative analysis with other ETS gene fusions and SPINK1 revealed a concomitant expression pattern of KLK4-KLKP1 with ERG and a mutually exclusive expression pattern with SPINK1, ETV1, ETV4, and ETV5. Development of an antibody specific to KLK4-KLKP1 fusion protein confirmed the expression of the full-length KLK4-KLKP1 protein in prostate tissues. The in vitro and in vivo functional assays to study the oncogenic properties of KLK4-KLKP1 confirmed its role in cell proliferation, cell invasion, intravasation, and tumor formation. Presence of strong ERG and AR binding sites located at the fusion junction in KLK4-KLKP1 suggests that the fusion gene is regulated by ERG and AR. Correlative analysis of clinical data showed an association of KLK4-KLKP1 with lower preoperative PSA values and in young men (<50 years) with prostate cancer. Screening of patient urine samples showed that KLK4-KLKP1 can be detected noninvasively in urine. Taken together, we present KLK4-KLKP1 as a class of pseudogene associated fusion transcript in cancer with potential applications as a biomarker for routine screening of prostate cancer.

Internalization of secreted antigen-targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis.

Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen-targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue-specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.

Indirect Back-Titration ELISA: A New Format for Estimation of Human Tissue Kallikreins.

Enzyme-linked immunosorbent assay (ELISA) is either based on sandwich, competitive, or inhibition type of format. However, these formats need 2 or 3 monoclonal antibodies (moAB) to estimate 1 antigen. To get a cost-effective, high throughput, ELISA for estimation of human tissue kallikreins we have now developed an indirect, back-titration style, Time Resolved ImmunoFluorometric (TRIF) ELISA that uses only 1 antigen-specific moAB and a general polyclonal antibody. Polystyrene microtiter plate wells coated with a capture antibody, a mouse moAB prepared against a specific human tissue kallikrein are allowed to interact either with the corresponding pure antigen, as the calibrator, or with the corresponding antigen present in a biological fluid or tissue extract. The detection antibody, anti-mouse IgG conjugated with alkaline phosphatase, is added to find the antigen-free immobilized capture moAB. Conjugated enzyme is allowed to hydrolyze diflunisal phosphate to produce a highly fluorescent complex. The fluorescence measured in TRIF mode corresponds to the antigen-free immobilized capture moAB and is used to quantify antigen-bound capture moAB. The detection antibody binds with the antigen-free capture moAB and strength of the signal correlates inversely with the amount of antigen bound to the capture moAB. With a minimum detection level of 20 ng/L the assay has no cross-reactivity with several test molecules. The method is sensitive, specific, applicable to a variety of biological samples, and cost-effective as it uses only 1 moAB and a polyclonal antibody. Using this assay, a single epitope can be estimated without purification.

A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity.

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.

Finding off-targets, biological pathways, and target diseases for chymase inhibitors via structure-based systems biology approach.

Off-target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off-targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off-target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off-targets into biological pathways and to establish links between pathways and particular adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off-targets. These off-targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors.

A new method to assay protease based on amyloid misfolding: application to prostate cancer diagnosis using a panel of proteases biomarkers.

This paper reports a sensitive method with electrochemical technique to detect various proteases, which can be used for the diagnosis of prostate cancer. For the proposed assay method, the working electrode is modified with the peptide probes for the target proteases. These probes contain the substrate sequence of target proteases, as well as the seed peptide sequence that can accelerate the misfolding of amyloid-beta. If there are proteases in the test solution, after protease cleavage of the substrate peptides, the distal seed peptide will be removed from the electrode surface. So, in the absence of proteases, the seed peptides can initiate and accelerate amyloid-beta misfolding on the electrode surface. Consequently, the formed aggregates strongly block the electron transfer of the in-solution electroactive species with the electrode, resulting in suppressed signal readout. Nevertheless, in the presence of proteases, enzyme cleavage may lead to greatly mitigated protein misfolding and evident signal enhancement. Since the contrast in signal readout between the two cases can be amplified by using the protein misfolding step, high sensitivity suitable for direct detection of proteases in serum can be achieved. These results may suggest the feasibility of our new method for the detection of a panel of proteases in offering detailed diagnosis of prostate cancer and a better treatment of the cancer.

Magnetic-particle-based, ultrasensitive chemiluminescence enzyme immunoassay for free prostate-specific antigen.

We report a magnetic-particle (MMP)-based chemiluminescence enzyme immunoassay (CLEIA) for free prostate-specific antigen (f-PSA) in human serum. In this method, the f-PSA is sandwiched between the anti-PSA antibody coated MMPs and alkaline phosphatase (ALP)-labeled anti-f-PSA antibody. The signal produced by the emitted photons from the chemiluminescent substrate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2'-adamantane)) is directly proportional to the amount of f-PSA in a sample. The present MMP-based assay can detect f-PSA in the range of 0.1-30 ng mL(-1) with the detection limit of 0.1 ng mL(-1). The linear detection range could match the concentration range within the "diagnostic gray zone" of serum f-PSA levels (4-10 ng mL(-1)). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4 ng mL(-1)). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum.

Prostate-specific kallikrein-related peptidases and their relation to prostate cancer biology and detection. Established relevance and emerging roles.

Kallikreins are a family of serine proteases with a range of tissue-specific and essential proteolytic functions. Among the best studied are the prostate tissue-specific KLK2 and KLK3 genes and their secreted protease products, human kallikrein 2, hk2, and prostate-specific antigen (PSA). Members of the so-called classic kallikreins, these highly active trypsin-like serine proteases play established roles in human reproduction. Both hK2 and PSA expression is regulated by the androgen receptor which has a fundamental role in prostate tissue development and progression of disease. This feature, combined with the ability to sensitively detect different forms of these proteins in blood and biopsies, result in a crucially important biomarker for the presence and recurrence of cancer. Emerging evidence has begun to suggest a role for these kallikreins in critical vascular events. This review discusses the established and developing biological roles of hK2 and PSA, as well as the historical and advanced use of their detection to accurately and non-invasively detect and guide treatment of prostatic disease.

Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms.

Kallikreins are secreted serine proteases with important roles in human physiology. Human plasma kallikrein, encoded by the KLKB1 gene on locus 4q34-35, functions in the blood coagulation pathway, and in regulating blood pressure. The human tissue kallikrein and kallikrein-related peptidases (KLKs) have diverse expression patterns and physiological roles, including cancer-related processes such as cell growth regulation, angiogenesis, invasion, and metastasis. Prostate-specific antigen (PSA), the product of the KLK3 gene, is the most widely used biomarker in clinical practice today. A total of 15 KLKs are encoded by the largest contiguous cluster of protease genes in the human genome (19q13.3-13.4), which makes them ideal for evolutionary analysis of gene duplication events. Previous studies on the evolution of KLKs have traced mammalian homologs as well as a probable early origin of the family in aves, amphibia and reptilia. The aim of this study was to address the evolutionary and functional relationships between tissue KLKs and plasma kallikrein, and to examine the evolution of alternative splicing isoforms. Sequences of plasma and tissue kallikreins and their alternative transcripts were collected from the NCBI and Ensembl databases, and comprehensive phylogenetic analysis was performed by Bayesian as well as maximum likelihood methods. Plasma and tissue kallikreins exhibit high sequence similarity in the trypsin domain (>50%). Phylogenetic analysis indicates an early divergence of KLKB1, which groups closely with plasminogen, chymotrypsin, and complement factor D (CFD), in a monophyletic group distinct from trypsin and the tissue KLKs. Reconstruction of the earliest events leading to the diversification of the tissue KLKs is not well resolved, indicating rapid expansion in mammals. Alternative transcripts of each KLK gene show species-specific divergence, while examination of sequence conservation indicates that many annotated human KLK isoforms are missing the catalytic triad that is crucial for protease activity.

Structure of plasma and tissue kallikreins.

The kallikrein kinin system (KKS) consists of serine proteases involved in the production of peptides called kinins, principally bradykinin and Lys-bradykinin (kallidin). The KKS contributes to a variety of physiological processes including inflammation, blood pressure control and coagulation. Here we review the protein structural data available for these serine proteases and examine the molecular mechanisms of zymogen activation and substrate recognition focusing on plasma kallikrein (PK) and tissue kallikrein (KLK1) cleavage of kininogens. PK circulates as a zymogen bound to high-molecular-weight kininogen (HK). PK is activated by coagulation factor XIIa and then cleaves HK to generate bradykinin and factor XII to generate further XIIa.A structure has been described for the activated PK protease domain in complex with the inhibitor benzamidine. Kallikrein-related peptidases (KLKs) have a distinct domain structure and exist as a family of 15 genes which are differentially expressed in many tissues and the central nervous system.They cleave a wide variety of substrates including low-molecular-weight kininogen (LK) and matrix proteins. Crystal structures are available for KLK1, 3, 4, 5, 6 and 7 activated protease domains typically in complex with S1 pocket inhibitors. A substrate mimetic complex is described for KLK3 which provides insight into substrate recognition. A zymogen crystal structure determined for KLK6 reveals a closed S1 pocket and a novel mechanism of zymogen activation. Overall these structures have proved highly informative in understanding the molecular mechanisms of the KKS and provide templates to design inhibitors for treatment of a variety of diseases.

Release of proteolytic activity following reduction in therapeutic human serum albumin containing products: detection with a new neoepitope endopeptidase immunoassay.

Botulinum type A toxin (BoNT/A) is defined by its specific endopeptidase cleavage of SNAP25 between Gln(197) and Arg(198) under reducing conditions. The neurotoxin is widely used for therapeutic or cosmetic purposes, but should not contain other toxin serotypes or unwanted protease activities. Using a neoepitope endopeptidase immunoassay, additional cleavage between Arg(198) and Ala(199) was detected with a range of therapeutic BoNT/A products confirming an earlier report of an unidentified proteolytic component. By developing the assay and making it insensitive to BoNT/C1, any activity due to the type C1 toxin was excluded. Therapeutic preparations consist of ng quantities of toxin protein which are typically stabilised by 0.125-30 mg of HSA. An excellent correlation (R(2)=0.993) between HSA content per vial and measured activity was obtained within the therapeutic BoNT/A products tested. No activity was detected in any of the non-albumin formulated preparations, thereby identifying HSA as the source of the unknown protease for the first time. To investigate the cause of this activity, either as an intrinsic molecular activity of albumin or due to an albumin-associated purification contaminant, further studies on a variety of commercial plasma-derived HSA products or recombinant HSA materials free from potential plasma contaminants were carried out. The measured proteolytic levels were highly consistent amongst preparations, and could all be partially inhibited by the presence of zinc and blocked by PKSI-527 and aprotinin. By contrast, the data did not support the role of plasmin, kallikrein, trypsin, α(2)-antiplasmin-plasmin complexes or HSA purification contaminants, PKA (prekallikrein activator) or kallikrein-like activity. Taken together, these findings indicate a new intrinsic proteolytic activity of the albumin molecule revealed under reducing conditions as the source of the unexpected Arg-Ala cleaving activity.

Evolutionary history of tissue kallikreins.

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs.

Prostate specific antigen: one out of five disulfide bridges determines inactivation by reduction.

PSA (kallikrein hK3) proteolytic activity proved highly sensitive to reducing agents like dithiothreitol (DTT) and dihydrolipoic acid while beta-mercaptoethanol and glutathione were less effective. Ascorbate exhibited no significant inhibitory potential. Loss of activity by reduction could be readily reversed by re-oxidation. Inactivation was associated with the reduction of two out of five conserved disulfides. Mass spectrometry of differentially modified cysteines, and Edman degradation analyses identified Cys 22-Cys 157 and Cys 191-Cys 220 as DDT-sensitive. The highly homologous porcine pancreatic kallikrein (pK1) showed a completely different response: Even at 20 mM DDT, no inactivation was seen; and in this case, only one of the five disulfides (Cys 22-Cys 157) was opened. This indicated that it is the accessabilty of the Cys 191-Cys 220 disulfide near the catalytic serine 195 that decides on the ability of reductants to inactivate the proteolytic activity of PSA. A structural basis for this interpretation is provided when the two homologous proteins were compared with respect to the threedimensional architecture around the crucial disulfide Cys 191-Cys 220 where in the case of PK1, but not in PSA, the phenylalanine-residue (Phe 149) is in an interfering position.

Defining the extended substrate specificity of kallikrein 1-related peptidases.

Human kallikrein 1-related peptidases (KLKs) form a subfamily of 15 extracellular (chymo)tryptic-like serine proteases. KLKs 4, 5, 13 and 14 display altered expression/activity in diverse pathological conditions, including cancer. However, their distinct (patho)physiological roles remain largely uncharacterized. As a step toward distinguishing their proteolytic functions, we attempt to define their primary and extended substrate specificities and identify candidate biological targets. Heterologously expressed KLKs 4, 5, 13 and 14 were screened against fluorogenic 7-amino-4-carbamoylmethylcoumarin positional scanning-synthetic combinatorial libraries with amino acid diversity at the P1-P4 positions. Our results indicate that these KLKs share a P1 preference for Arg. However, each KLK exhibited distinct P2-P4 specificities, attributable to structural variations in their surface loops. The preferred P4-P1 substrate recognition motifs based on optimal subsite occupancy were as follows: VI-QSAV-QL-R for KLK4; YFWGPV-RK-NSFAM-R for KLK5; VY-R-LFM-R for KLK13; and YW-KRSAM-HNSPA-R for KLK14. Protein database queries using these motifs yielded many extracellular targets, some of which represent plausible KLK substrates. For instance, cathelicidin, urokinase-type plasminogen activator, laminin and transmembrane protease serine 3 were retrieved as novel putative substrates for KLK4, 5, 13 and 14, respectively. Our findings may facilitate studies on the role of KLKs in (patho)physiology and can be used in the development of selective KLK inhibitors.

Molecular structures and dynamics of the stepwise activation mechanism of a matrix metalloproteinase zymogen: challenging the cysteine switch dogma.

Activation of matrix metalloproteinase zymogen (pro-MMP) is a vital homeostatic process, yet its molecular basis remains unresolved. Using stopped-flow X-ray spectroscopy of the active site zinc ion, we determined the temporal sequence of pro-MMP-9 activation catalyzed by tissue kallikrein protease in milliseconds to several minutes. The identity of three intermediates seen by X-ray spectroscopy was corroborated by molecular dynamics simulations and quantum mechanics/molecular mechanics calculations. The cysteine-zinc interaction that maintains enzyme latency is disrupted via active-site proton transfers that mediate transient metal-protein coordination events and eventual binding of water. Unexpectedly, these events ensue as a direct result of complexation of pro-MMP-9 and kallikrein and occur before proteolysis and eventual dissociation of the pro-peptide from the catalytic site. Here we demonstrate the synergism among long-range protein conformational transitions, local structural rearrangements, and fine atomic events in the process of zymogen activation.

Activity and stability of human kallikrein-2-specific linear and cyclic peptide inhibitors.

Human glandular kallikrein (KLK2) is a highly prostate-specific serine protease, which is mainly excreted into the seminal fluid, but part of which is also secreted into circulation from prostatic tumors. Since the expression level of KLK2 is elevated in aggressive tumors and it has been suggested to mediate the metastasis of prostate cancer, inhibition of the proteolytic activity of KLK2 is of potential therapeutic value. We have previously identified several KLK2-specific linear peptides by phage display technology. Two of its synthetic analogs, A R R P A P A P G (KLK2a) and G A A R F K V W W A A G (KLK2b), show specific inhibition of KLK2 but their sensitivity to proteolysis in vivo may restrict their potential use as therapeutic agents. In order to improve the stability of the linear peptides for in vivo use, we have prepared cyclic analogs and compared their biological activity and their structural stability. A series of cyclic variants with cysteine bridges were synthesized. Cyclization inactivated one peptide (KLK2a) and its derivatives, while the other peptide (KLK2b) and its derivatives remained active. Furthermore, backbone cyclization of KLK2b improved significantly the resistance against proteolysis by trypsin and human plasma. Nuclear magnetic resonance studies showed that cyclization of the KLK2b peptides does not make the structures more rigid. In conclusion, we have shown that backbone cyclization of KLK2 inhibitory peptides can be used to increase stability without losing biological activity. This should render the peptides more useful for in vivo applications, such as tumor imaging and prostate cancer targeting.

Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences.

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.

High-speed biomarker identification utilizing porous silicon nanovial arrays and MALDI-TOF mass spectrometry.

Speed and accuracy are crucial prerequisites in the application of proteomic methods to clinical medicine. We describe a microfluidic-based nanovial array for rapid proteolytic processing linked to MALDI-TOF MS. This microscale format consumes only minute amounts of sample, and it is compatible with rapid bioanalytical protocols and high-sensitivity readouts. Arrays of vials (300 microm in diameter and 25 microm deep), isotropically etched in silicon wafers were electrochemically porosified. Automated picoliter microdispensing was employed for precise fluid handling in the microarray format. Vials were prefilled with trypsin solution, which was allowed to dry. Porosified and nonporosified nanovials were compared for trypsin digestion and subsequent MS identification of three model proteins: lysozyme, alcohol dehydrogenase, and serum albumin at levels of 100 and 20 fmol. In an effort to assess the rapid digestion platform in a context of putative clinical applications, two prostate cancer biomarkers, prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2), were digested at levels of 100 fmol (PSA), 20 fmol (PSA) and 8 fmol (hK2). All biomarker digestions were completed in less than 30 s, with successful MS identification in the porous nanovial setting.

Novel peptide inhibitors of human kallikrein 2.

Human kallikrein 2 (hK2) is a serine protease produced by the secretory epithelial cells in the prostate. Because hK2 activates several factors participating in proteolytic cascades that may mediate metastasis of prostate cancer, modulation of the activity of hK2 is a potential way of preventing tumor growth and metastasis. Furthermore, specific ligands for hK2 are potentially useful for targeting and imaging of prostate cancer and for assay development. We have used enzymatically active recombinant hK2 captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. Using libraries expressing 10 or 11 amino acids long linear peptides, we identified six different peptides binding to hK2. Three of these were shown to be specific and efficient inhibitors of the enzymatic activity of hK2 toward a peptide substrate. Furthermore, the peptides inhibited the activation of the proform of prostate-specific antigen by hK2. Amino acid substitution analyses revealed that motifs of six amino acids were required for the inhibitory activity. These peptides are potentially useful for treatment and targeting of prostate cancer.