Phosphonic Analogs of Alanine as Acylpeptide Hydrolase Inhibitors.

Acylpeptide hydrolase is a serine protease, which, together with prolyl oligopeptidase, dipeptidyl peptidase IV and oligopeptidase B, belongs to the prolyl oligopeptidase family. Its primary function is associated with the removal of N-acetylated amino acid residues from proteins and peptides. Although the N-acylation occurs in 50-90 % of eukaryotic proteins, the precise functions of this modification remains unclear. Recent findings have indicated that acylpeptide hydrolase participates in various events including oxidized proteins degradation, amyloid ß-peptide cleavage, and response to DNA damage. Considering the protein degradation cycle cross-talk between acylpeptide hydrolase and proteasome, inhibition of the first enzyme resulted in down-regulation of the ubiquitin-proteasome system and induction of cancer cell apoptosis. Acylpeptide hydrolase has been proposed as an interesting target for the development of new potential anticancer agents. Here, we present the synthesis of simple derivatives of (1-aminoethyl)phosphonic acid diaryl esters, phosphonic analogs of alanine diversified at the N-terminus and ester rings, as inhibitors of acylpeptide hydrolase and discuss the ability of the title compounds to induce apoptosis of U937 and MV-4-11 tumor cell lines.

Structure-based design, synthesis, and evaluation of the biological activity of novel phosphoroorganic small molecule IAP antagonists.

One of the strategies employed by novel anticancer therapies is to put the process of apoptosis back on track by blocking the interaction between inhibitor of apoptosis proteins (IAPs) and caspases. The activity of caspases is modulated by the caspases themselves in a caspase/procaspase proteolytic cascade and by their interaction with IAPs. Caspases can be released from the inhibitory influence of IAPs by proapoptotic proteins such as secondary mitochondrial activator of caspases (Smac) that share an IAP binding motif (IBM). The main purpose of the present study was the design and synthesis of phosphorus-based peptidyl antagonists of IAPs that mimic the endogenous Smac protein, which blocks the interaction between IAPs and caspases. Based on the structure of the IAP antagonist and recently reported thiadiazole derivatives, we designed and evaluated the biochemical properties of a series of phosphonic peptides bearing the N-Me-Ala-Val/Chg-Pro-OH motif (Chg: cyclohexylglycine). The ability of the obtained compounds to interact with the binding groove of the X-linked inhibitor of apoptosis protein baculovirus inhibitor of apoptosis protein repeat (XIAP BIR3) domain was examined by a fluorescence polarization assay, while their potential to induce autoubiquitination followed by proteasomal degradation of cellular IAP1 was examined using the MDA-MB-231 breast cancer cell line. The highest potency against BIR3 was observed among peptides containing C-terminal phosphonic phenylalanine analogs, which displayed nanomolar Ki values. Their antiproliferative potential as well as their proapoptotic action, manifested by an increase in caspase-3 activity, was examined using various cell lines.

Human proteinase 3 resistance to inhibition extends to alpha-2 macroglobulin.

Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1-PI and MNEI but not by SLPI. α2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a kass in the ≤ 105  m-1 ·s-1 range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-M bait region (residues 690-728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of α2-M (39pep-α2-M). Since the 39pep-α2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.

Temporal inhibition of mouse mammary gland cancer metastasis by CORM-A1 and DETA/NO combination therapy.

Carbon monoxide and nitric oxide are two of the most important vasoprotective mediators. Their downregulation observed during vascular dysfunction, which is associated with cancer progression, leads to uncontrolled platelet activation. Therefore, the aim of our studies was to improve vasoprotection and to decrease platelet activation during progression of mouse mammary gland cancer by concurrent use of CO and NO donors (CORM-A1 and DETA/NO, respectively). Methods: Mice injected intravenously with 4T1-luc2-tdTomato or orthotopically with 4T1 mouse mammary gland cancer cells were treated with CORM-A1 and DETA/NO. Ex vivo aggregation and activation of platelets were assessed in the blood of healthy donors and breast cancer patients. Moreover, we analyzed the compounds' direct effect on 4T1 mouse and MDA-MB-231 human breast cancer cells proliferation, adhesion and migration in vitro. Results: We have observed antimetastatic effect of combination therapy, which was only transient in orthotopic model. During early stages of tumor progression concurrent use of CORM-A1 and DETA/NO demonstrated vasoprotective ability (decreased endothelin-1, sICAM and sE-selectin plasma level) and downregulated platelets activation (decreased bound of fibrinogen and vWf to platelets) as well as inhibited EMT process. Combined treatment with CO and NO donors diminished adhesion and migration of breast cancer cells in vitro and inhibited aggregation as well as TGF-ß release from breast cancer patients' platelets ex vivo. However, antimetastatic effect was not observed at a later stage of tumor progression which was accompanied by increased platelets activation and endothelial dysfunction related to a decrease of VASP level. Conclusion: The therapy was shown to have antimetastatic action and resulted in normalization of endothelial metabolism, diminution of platelet activation and inhibition of EMT process. The effect was more prominent during early stages of tumor dissemination. Such treatment could be applied to inhibit metastasis during the first stages of this process.

Development and Evaluation of an Immunoglobulin Y-Based ELISA for Measuring Prostate Specific Antigen in Human Serum.

BACKGROUND: Measurement of serum prostate specific antigen (PSA) concentrations remains one of the leading methods for diagnosing prostate cancer. We developed and evaluated an immunoglobulin Y (IgY)-based ELISA to measure total PSA (tPSA) concentrations in human serum that could be used as an alternative to commercially available in vitro diagnostic assays that rely on mouse monoclonal IgG. METHODS: A sandwich ELISA based on an anti-PSA IgY antibody was developed. We evaluated the ability of the anti-PSA IgY antibody to detect free and complexed PSA at the same molar ratio. The assay was optimized, and its analytical performance was verified by calculating limit of background (LoB), limit of detection (LoD), and limit of quantification (LoQ). We performed correlation and regression analyses between tPSA concentrations measured by our ELISA and those from commercial assays: Cobas 6000 (Roche Diagnostics, Warszawa, Poland) and PSA total ELISA (IBL International, Hamburg, Germany). RESULTS: LoB, LoD, and LoQ, were 0.061, 0.083, and 0.100 ng/mL, respectively, and linearity range was 0.100-3.375 ng/mL. tPSA concentrations from our IgY-based ELISA strongly correlated with those from the commercial assays. CONCLUSIONS: Our IgY-based ELISA is an efficient equivalent to the above commercial assays. The use of IgY as the detecting agent could reduce the risk of false positive results, as well as decrease the overall cost of analysis.

Phosphonate inhibitors of West Nile virus NS2B/NS3 protease.

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)P(OPh)2 displaying Ki and k2/Ki values of 0.4 µM and 28 265 M-1s-1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease.

The Lord of the Bacteria: The Fellowship of the Leader and Other Serine Protease Inhibitors.

Since antibiotics use is currently limited due to undesired side effects and the increasing antibiotic resistance of various bacteria strains, there is a pressing need to develop new strategies and methods preventing epidemic outbreaks. The virulent potency of bacteria relies on a number of different extracellularly secreted factors among which proteases considered as promising, novel drug targets are of special interest. The first evidence that bacterial cysteine, serine and metalloproteinases contributed to the progression of infection was found in the early 70's. This extracellular proteolytic system allows bacteria to penetrate into tissues, escape detection by the host's immune mechanisms and grow despite limited access to nutrition. A molecule able to selectively inhibit the activity of bacterial proteases in the spread of infection may lead to designing novel therapeutics. Moreover, due to their mechanism of action, bacterial protease inhibitors can be used to fight antibiotic-resistant strains. Herein, we undertake a review of various bacterial proteases together with the design and development of their inhibitors (excluding ß-lactams) for the last ten years, and introduce the reader to a brief history of the subject.

Exploiting the S4-S5 Specificity of Human Neutrophil Proteinase 3 to Improve the Potency of Peptidyl Di(chlorophenyl)-phosphonate Ester Inhibitors: A Kinetic and Molecular Modeling Analysis.

The neutrophilic serine protease proteinase 3 (PR3) is involved in inflammation and immune response and thus appears as a therapeutic target for a variety of infectious and inflammatory diseases. Here we combined kinetic and molecular docking studies to increase the potency of peptidyl-diphenyl phosphonate PR3 inhibitors. Occupancy of the S1 subsite of PR3 by a nVal residue and of the S4-S5 subsites by a biotinylated Val residue as obtained in biotin-VYDnVP(O-C6H4-4-Cl)2 enhanced the second-order inhibition constant kobs/[I] toward PR3 by more than 10 times ( kobs/[I] = 73000 ± 5000 M-1 s-1) as compared to the best phosphonate PR3 inhibitor previously reported. This inhibitor shows no significant inhibitory activity toward human neutrophil elastase and resists proteolytic degradation in sputa from cystic fibrosis patients. It also inhibits macaque PR3 but not the PR3 from rodents and can thus be used for in vivo assays in a primate model of inflammation.

Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application.

Adenosine deaminase (ADA) is currently used as a diagnostic marker for tuberculous pleuritis. Although ADA has been suggested as a potential marker for several types of cancer, the importance of each of ADA isoforms as well as their levels and enzymatic activities in tumors need to be further investigated. Herein we developed avian immunoglobulin Y highly specific to human ADA via hens immunization with calf adenosine deaminase. The obtained antibodies were used for the development of a sensitive double-egg yolk immunoglobulin (IgY) sandwich ELISA assay with an ADA detection limit of 0.5 ng/ml and a linearity range of up to 10 ng/ml. Specific, affinity-purified IgYs were able to recognize human recombinant ADA and ADA present in human cancer cell lines. In addition, antigen-specific IgY antibodies were able to inhibit catalytic activity of calf ADA with an IC50 value of 47.48 nM. We showed that generated IgY antibodies may be useful for ADA detection, thus acting as a diagnostic agent in immunoenzymatic assays.

Selection of Effective HTRA3 Activators Using Combinatorial Chemistry.

Herein, we report selection, synthesis, and enzymatic evaluation of a peptidomimetic library able to increase proteolytic activity of HtrA3 (high temperature requirement A) protease. Iterative deconvolution in solution of synthesized modified pentapeptides yielded two potent HtrA3 activators acting in the micromolar range (HCOO-CH2O-C6H4-OCH2-CO-Tyr-Asn-Phe-His-Asn-OH and HCOO-CH2O-C6H4-OCH2-CO-Tyr-Asn-Phe-His-Glu-OH). Both compounds increased proteolysis of an artificial HtrA3 substrate over 40-fold in a selective manner. On the basis of molecular modeling, the selected compounds bind strongly to the PDZ domain.

Development of the first internally-quenched fluorescent substrates of human cathepsin C: The application in the enzyme detection in biological samples.

Cathepsin C is a widely expressed cysteine exopeptidase that is mostly recognized for the activation of the granule-associated proinflammatory serine proteases in neutrophils, cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted extracellularly; however, its occurrence in human bodily fluids/physiological samples has not been thoroughly studied. In the course of this study, the first fluorescence resonance energy transfer peptides for the measurement of the activity of human cathepsin C were designed and synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S' specificity study of cathepsin C, which has been examined for the first time. The extensive enzymatic studies of the obtained compounds resulted in the selection of the highly specific and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO2)-NH2, which was successfully employed for the detection of cathepsin C activity in complex biological samples such as cell lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate was performed in order to better understand the binding mode of the substrates in the active site of cathepsin C.

Cell surface cathepsin G activity differs between human natural killer cell subsets.

Natural killer (NK) cells are critical in diverse defense mechanisms, including elimination of viral infected cells and destruction of tumor cells. NK cells are characterized by the ability to initiate apoptosis in target cells when their cell surface major histocompatibility complex class I (MHC I) repertoire is missing. On the other hand, NK cells are not activated when MHC I or non-classical MHC molecules are found on the respective cells. It was demonstrated that cathepsin G (CatG) binds to the cell surface of NK cells; however, the distribution of this protease on the cell surface of NK cell subsets has not been identified. Here, we show that CatG cell surface level differs between NK cell subsets. CatG was determined on the protein- and activity level (activity-based probe MARS116) by using flow cytometry. Thus, MARS116 is a novel reporter of cell surface CatG activity and can be used to differentiate between distinct NK cell subsets.

Bladder cancer detection using a peptide substrate of the 20S proteasome.

The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods.

Cathepsin G-mediated proteolytic degradation of MHC class I molecules to facilitate immune detection of human glioblastoma cells.

To mount an adaptive immune response, MHC I molecules present antigenic peptides to CTLs. Transcriptional reduction of MHC I molecules is a strategy of immune evasion, which impairs the detection of infected or tumorous cells by CTLs. Natural killer (NK) cells, on the other hand, eliminate target cells specifically in the absence of MHC I. Consequently, infected or tumorous cells partly retain their MHC I at the cell surface to avoid NK recognition. However, it remains unclear which protease degrades MHC I molecules and how these cells maintain a limited set of MHC I at the cell surface. Here, we demonstrate that cathepsin G (CatG), a serine protease, found in the endocytic compartment of APCs and, to a lesser extent, CatD and CatS proteolytically degrade MHC I molecules. Inhibition of CatG boosted MHC I expression at the cell surface of primary human immune cells. In contrast, human glioblastoma cells do not harbor active CatG and might have lost the ability to proteolytically degrade MHC I during tumorigenesis to avoid NK-mediated killing. Overexpression of CatG in glioblastoma cells resulted in a rapid and efficient MHC I degradation. In conclusion, CatG is an essential protease for regulating MHC I molecules and thus modulation of CatG activity might present a new avenue for therapeutic intervention.

Method for generation of peptide-specific IgY antibodies directed to Staphylococcus aureus extracellular fibrinogen binding protein epitope.

The IgY antibodies offer an attractive alternative to mammalian IgGs in research, diagnosis and medicine. The isolation of immunoglobulin Y from the egg yolks is efficient and economical, causing minimal suffering to animals. Here we present the methodology for the production of IgY antibodies specific to Staphylococcus aureus fibrinogen binding protein (Efb) and its peptidyl epitope (spanning residues 127-140). The Efb is an extracellular, adhesion protein which binds both human fibrinogen and complement C3 protein thus contributing to the high infectious potential of this pathogen. The selected epitope of Efb protein is responsible for the interaction with C3. The immunochemical characterization of both anti-Efb and epitope-specific IgY antibodies revealed their similar avidity, titer, and reactivity profile, although some differences in the hen's immune response to administered antigens is discussed.

Design and synthesis of new substrates of HtrA2 protease.

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO2)-NH2, which displayed a specificity constant kcat/KM value of 14,535M(-1)s(-1).

Generation and application of polyclonal IgY antibodies specific for full-length and nicked prostate-specific antigen.

BACKGROUND: The prostate-specific antigen (PSA) is considered an important serum marker for prostate cancer detection, monitoring and staging. The purpose of this study was to generate IgY class antibodies that recognize native PSA and selected epitopes. METHODOLOGY: Hens immunized with either full-length human PSA or its peptidyl fragment-conjugates produced specific antibodies that were isolated from egg yolks. We developed a monoclonal/IgY sandwich ELISA with a PSA detection limit of 50 pg/ml and a linear range of 0.05-1.0 ng/ml. CONCLUSION: Because the signal observed for the PSA-specific IgY antibodies by ELISA and the reactivity profile of the epitope-derived IgYs were comparable to those of mouse monoclonal IgG antibodies, avian antibodies may be a cost-effective alternative to mammalian antibodies for prostate cancer diagnostics.

New selective peptidyl di(chlorophenyl) phosphonate esters for visualizing and blocking neutrophil proteinase 3 in human diseases.

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl(P)(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.

Determination of cathepsin G in endometrial tissue using a surface plasmon resonance imaging biosensor with tailored phosphonic inhibitor.

OBJECTIVE: Cathepsin G is a serine peptidase whose physiological role is mainly associated with an early immune response, anti-microbial activity as well as platelet activation or hydrolysis of coagulation factors. In addition, since the activity of cathepsin G has been associated with the development of various pathological disorders, the measurement of its activity in patient samples is of high interest. Unfortunately, the usefulness of common immunological methods is limited, since they cannot distinguish between catalytically active and inactive protease. STUDY DESIGN: Here we present the application of recently developed Surface Plasmon Resonance-based biosensor for the detection of active cathepsin G in human endometrium samples. The key element of the system is based on the irreversible binding of cathepsin G to its specific phosphonic-type inhibitor immobilized on the surface of the gold chip. The concentration of cathepsin G was measured in tissue samples from the group of patients with endometriosis as well as in the control group. RESULTS: The level of cathepsin G ascertained in endometrium tissue samples was over twice as high for the group of patients suffering from endometriosis as compared to the control group, with the median values of 0.5 pmol/mg and 0.2 pmol/mg, respectively. CONCLUSION: The SPR sensor armed with a specific irreversible phosphonic inhibitor represents a highly useful tool for the determination of catalytically active cathepsin G concentration in endometrial tissue.

Highly sensitive detection of cancer antigen 15-3 using novel avian IgY antibodies.

Early detection of cancer development is crucial for successful therapy and for monitoring patient outcome. Various immunodiagnostic methods are able to detect pathological changes in the human body ahead of symptomatic manifestation of the disease. Most immunological examinations are based on the detection of specific tumor markers in body fluids. Of the various cancer-specific proteins used for breast cancer diagnostics, one of the most commonly applied is the cancer antigen 15-3 (CA 15-3). An elevation in its serum level (>25-40 U/ml) usually correlates with tumor malignancy. The CA 15-3 antigen is also used for monitoring patients after surgical treatment and for measuring therapeutic efficacy. Herein, we present the generation of polyclonal IgY antibodies isolated from egg yolks of immunized hens and their application for CA 15-3 detection. The developed sandwich ELISA assay showed a detection limit of 0.028 U/ml, thus demonstrating its potential for clinical applications.