Marcin Sieńczyk

Synthesis and Structure−Activity Relationships of Phosphonic Arginine Mimetics as Inhibitors of the M1 and M17 Aminopeptidases from Plasmodium falciparum

The malaria parasite Plasmodium falciparum employs two metallo-aminopeptidases, PfA-M1 and PfA-M17, which are essential for parasite survival. Compounds that inhibit the activity of either enzyme represent leads for the development of new antimalarial drugs. Here we report the synthesis and structure-activity relationships of a small library of phosphonic acid arginine mimetics that probe the S1 pocket of both enzymes and map the necessary interactions that would be important for a dual inhibitor.

Human Neutrophil Elastase Phosphonic Inhibitors with Improved Potency of Action

Herein, we present the synthesis and the measurement of the inhibitory activity of novel peptidyl derivatives of α-aminoalkylphosphonate diaryl esters as human neutrophil elastase inhibitors. Their selectivity against other serine proteases, including porcine pancreatic elastase, chymotrypsin, and trypsin, was also demonstrated. We also describe the preparation of single peptide diastereomers. The most active and selective compound developed possessed a k(inact)/K(I) of 2353000 M(-1) s(-1), which is the most potent irreversible peptidyl inhibitor of human neutrophil elastase reported to date. The peptidyl inhibitors were demonstrated to be stable in PBS buffer and human plasma, as were their complexes with HNE.

New potent cathepsin G phosphonate inhibitors

Cathepsin G is an enzyme with dual chymotrypsin and trypsin-like specificity. As a leukocyte proteinase it is involved in the early stages of the immune response. In this work the synthesis and inhibitory activity of diaryl phosphonic-type irreversible cathepsin G inhibitors are described. Modification of the lead structure Z-Phg(P)(OPh)2 (k(obs)/I=91 M(-1)s(-1)) in phenyl ester moieties followed by incorporation of the basic functional group into the aromatic side chain yielded highly potent cathepsin G inhibitor Z-(4-guanidine)Phg(P)(OC6H4-4-S-Me)2 with the apparent second-order inhibition value at 15,600 M(-1)s(-1). Further elongation of the obtained compound by tripeptide resulted in the inhibitor Ac-Phe-Val-Thr-(4-guanidine)Phg(P)(OC6H4-4-S-Me)2 with the highest k(obs)/I value ever reported in literature (256,000 M(-1)s(-1)).

Surface plasmon resonance imaging biosensor for cathepsin G based on a potent inhibitor: development and applications.

A specific surface plasmon resonance imaging (SPRI) array biosensor for the determination of the enzymatically active cathepsin G (CatG) has been developed. For this purpose, a specific interaction between an inhibitor immobilized onto a chip surface and CatG in an analyzed solution was used. The MARS-115 CatG peptidyl inhibitor containing the 1-aminoalkylphosphonate diaryl ester moiety at the C terminus and N-succinamide with a free carboxylic function was synthesized and covalently immobilized onto the gold chip surface via the thiol group (cysteamine). Atomic force microscopy was used for the observation of surface changes during the subsequent steps of chip manufacture. Optimal detection conditions were chosen. High specificity of synthesized inhibitor to CatG was proved. The precision, as well as the accuracy, was found to be well suited to enzyme determination. The sensor application for the determination of CatG in white blood cells and saliva was shown for potential diagnosis of leukemia and oral cavity diseases during the early stages of those pathological states.

Application of specific cell permeable cathepsin G inhibitors resulted in reduced antigen processing in primary dendritic cells.

The serine protease cathepsin G (CatG) is expressed in primary antigen-presenting cells and regulates autoantigen processing in CatG pre-loaded fibroblasts. To further investigate the function of CatG in the major histocompatibility complex (MHC) class II loading compartments, a specific, cell permeable CatG-inhibitor is needed. In this study, several CatG-inhibitors were tested for their ability to penetrate the cell membrane of peripheral blood mononuclear cells (PBMC). We find that the commercially available reversible CatG-specific inhibitor I (CatG inhibitor) and the irreversible Suc-Val-Pro-Phe(P) (OPh)(2) (Suc-VPF) are both cell permeable and specifically inhibit intracellular CatG in the PBMC. Furthermore, selective inhibition of CatG resulted in reduced tetanus toxin C-fragment (TTC) and hemagglutinin (HA) processing and presentation to CD4(+) T cells. We conclude that these CatG inhibitors can be used for both antigen-processing studies and for modulation of T cell response in situ and in vivo.

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.

Phosphonic esters and their application of protease control.

α-Aminoalkylphosphonate diaryl esters are potent, irreversible, and highly selective site-directed inhibitors of serine proteases. The structure of the phosphonate group resembles the transition state observed during a peptide bond hydrolysis and therefore phosphonates are referred as transition state analogues. They react with the hydroxyl group of the active site serine residue leading to formation of a stable enzyme-inhibitor complex. Moreover, incorporation of a peptidyl chain at the N-terminus as well as an introduction of electron withdrawing or electron donating substituents within the ester ring structure allows for a generation of specific inhibitors that react only with target serine protease. The great advantage of the aminophosphonate diaryl esters over other classes of inhibitors is their stability in aqueous solutions, no toxicity and lack of reactivity with cysteine, threonine, aspartyl and metalloproteinases. The above resulted in their application as convenient tools to study proteases function and activity using in vivo and in vitro assays of different pathological disorders (diabetes, cancer metastasis, pulmonary diseases or hypertension); to determine the cellular localization of the proteinases (activity based probes), to be used in proteomic approach or as the reactive antigens to develop a catalytic function within the antibodies binding site. Herein we present the development of α-aminoalkylphosphonate diaryl esters as inhibitors of several serine proteases including dipeptidyl peptidase IV, cathepsin G, human neutrophil elastase, mast cell chymase and urokinase-type plasminogen activator. We have provided a historical perspective as well as a comprehensive report of the most recent studies in this field.

Simple phosphonic inhibitors of human neutrophil elastase

Herein, we describe the synthesis and resulting activity of a complex series of α-aminophosphonate diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase, trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple Cbz-protected α-aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where one of the obtained compounds Cbz-Val(P)(OC(6)H(4)-4-COOMe)(2) displayed an apparent second-order inhibition value at 33,015 M(-1) s(-1).

Application of a novel highly sensitive activity-based probe for detection of cathepsin G

Cathepsins are crucial in antigen processing in the major histocompatibility complex class II (MHC II) pathway. Within the proteolytic machinery, three classes of proteases (i.e., cysteine, aspartic, and serine proteases) are present in the endocytic compartments. The combined action of these proteases generates antigenic peptides from antigens, which are loaded to MHC II molecules for CD4+ T cell presentation. Detection of active serine proteases in primary human antigen-presenting cells (APCs) is restricted because of the small numbers of cells isolated from the peripheral blood. For this purpose, we developed a novel highly sensitive α-aminoalkylphosphonate diphenyl ester (DAP) activity-based probe to detect the serine protease cathepsin G (CatG) in primary APCs and after Epstein-Barr virus (EBV) exposure. Although CatG activity was not altered after short-term exposure of EBV in primary myeloid dendritic cells 1 (mDC1s), the aspartic protease cathepsin D (CatD) was reduced, suggesting that EBV is responsible for mitigating the presentation of a model antigen tetanus toxoid C-fragment (TTCF) by reduction of CatD. In addition, CatG activity was reduced to background levels in B cells during cell culture; however, these findings were independent of EBV transformation. In conclusion, our activity-based probe can be used for both Western blot and 96-well-based high-throughput CatG detection when cell numbers are limited.

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

Background: Proteinase 3 activity is poorly controlled by physiological inhibitors, and its biological function is not well understood. Results: We have designed irreversible phosphonate inhibitors based on structural differences between proteinase 3 and elastase. Conclusion: They selectively inhibit proteinase 3 in biological fluids and can act as activity-based probes. Significance: These inhibitors will help clarify proteinase 3 function. 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-peptidylP(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.