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.

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.

Phosphorus-containing isothiocyanate-derived mercapturic acids as a useful alternative for parental isothiocyanates in experimental oncology.

A series of phosphonates, phosphinates and phosphine oxides isothiocyanate-derived mercapturic acids were synthesized. A temperature dependence dynamic proton decoupled 31P NMR studies indicated that in most cases the compounds were obtained as a mixture of rotamers. Moreover, biologically relevant reversibility of mercapturic acids synthesis from the parental isothiocyanates was confirmed. All compounds were evaluated ashighly active antiproliferative agents in vitro in human colon cancer cell lines (LoVo and its doxorubicin-resistant subline LoVo/DX). The cell cycle progression and caspase-3 activity analyses revealed compounds moderate activity as apoptosis inducers and their poor influence on cell cycle progression in the LoVo cells. Our results confirm that isothiocyanate-derived mercapturic acids present a reasonable alternative for the parental compounds, and can replace them in the future studies on isothiocyanates potential as anticancer agents.

Synthesis and biological activity of diisothiocyanate-derived mercapturic acids.

This Letter deals with new non-natural diisothiocyanates, their mercapturic acid derivatives-conjugated with N-acetylcysteine as well as their antiproliferative activity towards human colon cancer cell lines and their inhibitory potency towards histone deacetylase activity. The activity of analysed isothiocyanates is not significantly different than their N-acetylcysteine conjugates. In comparison to simple mono-isothiocyanate analogues, aliphatic diisothiocyanates and their conjugates are much more active than the simple presence of two isothiocyanate functionalities could indicate.

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.

Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis.

The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.

Phosphonic analogues of glutamic acid as irreversible inhibitors of Staphylococcus aureus endoproteinase GluC: an efficient synthesis and inhibition of the human IgG degradation.

Endoproteinase GluC (V8 protease) is one of many virulence factors released by the Staphylococcus aureus species in vivo. The V8 protease is able to hydrolyze some serpins and all classes of mammalian immunoglobulins. The application of specific and potent inhibitors of V8 protease may lead to the development of new antibacterial agents. Herein, we present the synthesis and the inhibitory properties of novel peptidyl derivatives of a phosphonic glutamic acid analogue. One of the compounds Boc-Phe-Leu-Glu(P)(OC(6)H(4))(2) displayed an apparent second-order inhibition rate value of 8540 M(-1)s(-1). The Boc-Phe-Leu-Glu(P)(OC(6)H(4))(2) compound with the highest inhibitory potency showed the ability to prevent V8-mediated human IgG proteolysis in vitro.

Diisothiocyanate-Derived Mercapturic Acids Are a Promising Partner for Combination Therapies in Glioblastoma.

Glioblastoma represents the most aggressive tumor of the central nervous system. Due to invasion of glioblastoma stem cells into the healthy tissue, chemoresistance, and recurrence of the tumor, it is difficult to successfully treat glioblastoma patients, which is demonstrated by the low life expectancy of patients after standard therapy treatment. Recently, we found that diisothiocyanate-derived mercapturic acids, which are isothiocyanate derivatives from plants of the Cruciferae family, provoked a decrease in glioblastoma cell viability. These findings were extended by combining diisothiocyanate-derived mercapturic acids with dinaciclib (a small-molecule inhibitor of cyclin-dependent kinases with anti-proliferative capacity) or temozolomide (TMZ, standard chemotherapeutic agent) to test whether the components have a cytotoxic effect on glioblastoma cells when the dosage is low. Here, we demonstrate that the combination of diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ had an additive or even synergistic effect in the restriction of cell growth dependent on the combination of the components and the glioblastoma cell source. This strategy could be applied to inhibit glioblastoma cell growth as a therapeutic interference of glioblastoma.

Invariant chain processing is independent of cathepsin variation between primary human B cells/dendritic cells and B-lymphoblastoid cells.

As part of the endocytic antigen processing pathway, proteolytic cleavage of the invariant chain (Ii) is important for the generation of class II-associated invariant chain peptide (CLIP). CLIP remains associated with the major histocompatibility complex (MHC) class II molecule to prevent premature loading of antigenic peptides. Cysteine proteases, such as Cathepsin S (CatS), CatL, or CatV, play a pivotal role in the final stage of Ii degradation depending on the cell type studied. Less is known regarding the early stages of Ii processing. We therefore explored whether the serine protease CatG is involved in the initial step of Ii degradation in primary antigen presenting cells (APC), since the cathepsin distribution differs between primary APC and cell lines. While primary human B cells and dendritic cells (DC) do harbor CatG, this protease is absent in B-lymphoblastoid cells (BLC) or monocyte-derived DC generated in vitro. In addition, other proteases, such as CatC, CatL, and the asparagine endoprotease (AEP), are active in BLC and monocyte-derived DC. Here we demonstrate that CatG progressively degraded Ii in vitro resulting in several intermediates. However, pharmacological inhibition of CatG in primary B cells and DC did not alter Ii processing, indicating that CatG is dispensable in Ii degradation. Interestingly, stalling of cysteine proteases by inhibition in BLC vs. primary B cells and DC did not result in any differences in the generation of distinct Ii intermediates between the cells tested, suggesting that Ii processing is independent of the cathepsin variation within professional human APC.

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).

Synthesis and antiproliferative activity of novel α- and ß-dialkoxyphosphoryl isothiocyanates.

A series of 15 mostly new dialkoxyphosphoryl alkyl and aralkyl isothiocyanates were synthesized using two alternative strategies, and their in vitro antiproliferative activity against several cancer cell lines (including drug resistant) is here demonstrated. The IC(50) values measured for the new compounds are within the range of 6.3-21.5 µM, and they are quite similar to the activity of two best and most extensively investigated natural benzyl isothiocyanate (A) and phenethyl isothiocyanate (B). Preliminary studies utilizing the cell cycle and reduced glutathione level analysis performed on A549 lung cancer cell line using representative compounds revealed important differences in the mechanism of action possibly correlated with their chemical properties. Hydrophobic compounds react mainly with the cytosolic glutathione reduced leading to its depletion, causing an oxidative stress and cell cycle arrest in G0/G1 phase. On the other hand, hydrophilic compounds cause moderate cell cycle arrest and massive cell death associated with moderate reduced glutathione depletion. These suggest that significant changes in the chemical structure of isothiocyanates, which do not lead to the significant changes in antiproliferative activity, but simultaneously cause a differences in the mechanism of action are possible.

Phosphonic pseudopeptides as human neutrophil elastase inhibitors--a combinatorial approach.

Here we present a simple and rapid method for the construction of phosphonic peptide mimetic inhibitor libraries-products of Ugi and Passerini multicomponent condensations-leading to the selection of new biologically active phosphonic pseudopeptides. As the starting isonitriles, 1-isocyanoalkylphosphonate diaryl ester derivatives were applied. The structure of the synthesized inhibitors was designed to target human neutrophil elastase, a serine protease whose uncontrolled activity may lead to development of several pathophysiological states such as rheumatoid arthritis, cystic fibrosis or tumor growth and invasion. After screening the inhibitory activity of our constructed libraries, the most active compounds were synthesized as single molecules. One of the obtained inhibitors, Cbz-Met-O-Met-Val(P)(OC(6)H(4)-p-Cl)(2), displayed apparent second-order inhibition value at 40,105M(-1)s(-1) as the diastereomers mixture. Inhibition potency and selectivity of action toward other serine proteases as well as the results of initial in vitro experiments regarding inhibitors influence on cancer cell proliferation are presented.

Identification of very potent inhibitor of human aminopeptidase N (CD13).

In this Letter we describe broad comparision studies toward rat, pig, and human aminopeptidase N (CD13) orthologs using phosphinate inhibitors related in structure to hydroxamic acids. This SAR approach yielded a very potent inhibitor of human aminopeptidase N: alpha(1)-amino-3-phenylpropyl(alpha(2)-hydroxy-3-phenylpropyl)phosphinic acid with an IC(50)=60 nM.

New aromatic monoesters of alpha-aminoaralkylphosphonic acids as inhibitors of aminopeptidase N/CD13.

A series of new aromatic monoesters of alpha-aminoaralkylphosphonic acids were synthesized by selective hydrolysis of corresponding aromatic diesters of alpha-aminoaralkylphosphonic acids. New potential inhibitors of aminopeptidase N/CD13, an enzyme important in tumour angiogenesis, were developed. Some derivatives of the homophenylalanine and norleucine related monoaryl phosphonates displayed higher inhibition potency than corresponding alpha-aminoaralkylphosphonic acids toward aminopeptidase N/CD13. The effect of one of the new inhibitors on the growth of human PANC-1 and HT-1080 cell lines was examined, either alone or in combination with TNF-alpha.

Novel diphenyl esters of peptidyl alpha-aminoalkylphosphonates as inhibitors of chymotrypsin and subtilisin.

The activities of novel Cbz-N-protected alpha-aminophosphonic phenyl esters, analogs of leucine (1-15) and phenylalanine (17-29), which are substituted at the phenyl ester rings, as well as of their peptidic derivatives (31-43), were investigated for their inhibitory effects on chymotrypsin and subtilisin. The chemical nature and position of the examined substituents clearly demonstrated a strong structure-activity relationship. Among all synthesized compounds the most potent phosphonic-type inhibitors of subtilisin and chymotrypsin were identified, with k(2)/K(i) values 114,380 M(-1)s(-1) and 307,380 M(-1)s(-1), respectively.

Comparision of the cytotoxic effects of birch bark extract, betulin and betulinic acid towards human gastric carcinoma and pancreatic carcinoma drug-sensitive and drug-resistant cell lines.

Betulin and betulinic acid are naturally occurring pentacyclic triterpenes showing cytotoxicity towards a number of cancer cell lines. These compounds can be found in the bark of the many plants. In this report we have compared the cytotoxic activity of crude birch bark extract and purified betulin and betulinic acid towards human gastric carcinoma (EPG85-257) and human pancreatic carcinoma (EPP85-181) drug-sensitive and drug-resistant (daunorubicin and mitoxantrone) cell lines. Our results show significant differences in sensitivity between cell lines depending on the compound used, and suggest that both betulin and betulinic acid can be considered as a promising leads in the treatment of cancer.

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.

3,4-dimethoxybenzyl isothiocyanate enhances doxorubicin efficacy in LoVoDX doxorubicin-resistant colon cancer and attenuates its toxicity in vivo.

AIMS: The aim of the study was to evaluate the potential of naturally occurring isothiocyanates and doxorubicin in combined treatment of doxorubicin-resistant colon cancer. Doxorubicin is a cytostatic commonly used to treat many different types of cancer but its usage is often abrogated by severe side-effects and drug-induced resistance. MAIN METHODS: The antiproliferative potential of the combined treatment was analyzed in vitro by the SRB method (sulforhodamine B) and further evaluated for the mechanisms that determine the treatment outcome using a series of assays which included oxidative stress, apoptosis and compounds accumulation assessment. Ultimately, a combined treatment potential was assessed in vivo utilizing doxorubicin-resistant colon cancer model. KEY FINDING: The results indicate that naturally occurring isothiocyanates, represented by 3,4-dimethoxybenzyl isothiocyanate (dMBITC) increase doxorubicin the efficacy in doxorubicin-resistant human colon adenocarcinoma model by attenuated drug efflux, an increased reactive oxygen species production and an increased rate of apoptosis. In in vitro studies, over a 3-fold decrease in doxorubicin IC50 value was observed on the LoVoDX cell line when used in combination with suboptimal concentrations of dMBITC. The combined therapy exhibited a significantly higher efficacy than doxorubicin-alone treatment (c.a. 50% tumor growth inhibition in comparison to c.a. 25% for doxorubicin-alone treatment) in vivo. At the same time, the combined treatment attenuates doxorubicin toxicity as evidenced by improved animals body mass, main organs weight and biochemical markers of toxicity. SIGNIFICANCE: The adopted approach provides evidence that isothiocyanates can be successfully applied in the treatment of doxorubicin-resistant colon cancer, which warrants further studies.

First synthesis of alpha-aminoalkyl-(N-substituted)thiocarbamoyl-phosphinates: inhibitors of aminopeptidase N (APN/CD13) with the new zinc-binding group.

OO-Di-trimethylsilyl esters of alpha-N-benzyloxycarbonylaminoalkylphosphinates (III) undergo triethylamine catalyzed addition to isothiocyanates to give after hydrolysis, a series of new alpha-aminoalkyl-(N-substituted)thiocarbamoyl-phosphinates. Thiocarbamoyl-phosphinate moiety can be included in the structures of the metalloproteinase inhibitors as the zinc-binding group and the new compounds reported here are good inhibitors of important aminopeptidase N(CD13) with IC(50) in range of 10.56-0.25 microM.