Boronate-Based Oxidant-Responsive Derivatives of Acetaminophen as Proinhibitors of Myeloperoxidase.

Myeloperoxidase (MPO) is an important component of the human innate immune system and the main source of a strong oxidizing and chlorinating species, hypochlorous acid (HOCl). Inadvertent, misplaced, or excessive generation of HOCl by MPO is associated with multiple human inflammatory diseases. Therefore, there is a considerable interest in the development of MPO inhibitors. Here, we report the synthesis and characterization of a boronobenzyl derivative of acetaminophen (AMBB), which can function as a proinhibitor of MPO and release acetaminophen, the inhibitor of chlorination cycle of MPO, in the presence of inflammatory oxidants, i.e., hydrogen peroxide, hypochlorous acid, or peroxynitrite. We demonstrate that the AMBB proinhibitor undergoes conversion to acetaminophen by all three oxidants, with the involvement of the primary phenolic product intermediate, with relatively long half-life at pH 7.4. The determined rate constants of the reaction of the AMBB proinhibitor with hydrogen peroxide, hypochlorous acid, or peroxynitrite are equal to 1.67, 1.6 × 104, and 1.0 × 106 M-1 s-1, respectively. AMBB showed lower MPO inhibitory activity (IC50 > 0.3 mM) than acetaminophen (IC50 = 0.14 mM) toward MPO-dependent HOCl generation. Finally, based on the determined reaction kinetics and the observed inhibitory effects of two plasma components, uric acid and albumin, on the extent of AMBB oxidation by ONOO- and HOCl, we conclude that ONOO- is the most likely potential activator of AMBB in human plasma.

Fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid: a comparative study.

MPO-derived oxidants including HOCl contribute to tissue damage and the initiation and propagation of inflammatory diseases. The search for small molecule inhibitors of myeloperoxidase, as molecular tools and potential drugs, requires the application of high throughput screening assays based on monitoring the activity of myeloperoxidase. In this study, we have compared three classes of fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid, including boronate-, aminophenyl- and thiol-based fluorogenic probes and we show that all three classes of probes are suitable for this purpose. However, probes based on the coumarin fluorophore turned out to be not reliable indicators of the inhibitors' potency. We have also determined the rate constants of the reaction between HOCl and the probes and they are equal to 1.8 × 104 M-1s-1 for coumarin boronic acid (CBA), 1.1 × 104 M-1s-1 for fluorescein based boronic acid (FLBA), 3.1 × 104 M-1s-1 for 7-(p-aminophenyl)-coumarin (APC), 1.6 × 104 M-1s-1 for 3'-(p-aminophenyl)-fluorescein (APF), and 1 × 107 M-1s-1 for 4-thiomorpholino-7-nitrobenz-2-oxa-1,3-diazole (NBD-TM). The high reaction rate constant of NBD-TM with HOCl makes this probe the most reliable tool to monitor HOCl formation in the presence of compounds showing HOCl-scavenging activity.

Kinetic Study on the Reactivity of Azanone (HNO) toward Cyclic C-Nucleophiles.

Azanone (HNO) is an elusive electrophilic reactive nitrogen species of growing pharmacological and biological significance. Here, we present a comparative kinetic study of HNO reactivity toward selected cyclic C-nucleophiles under aqueous conditions at pH 7.4. We applied the competition kinetics method, which is based on the use of a fluorescein-derived boronate probe FlBA and two parallel HNO reactions: with the studied scavenger or with O2 (k = 1.8 × 104 M-1s-1). We determined the second-order rate constants of HNO reactions with 13 structurally diverse C-nucleophiles (k = 33-20,000 M-1s-1). The results show that the reactivity of HNO toward C-nucleophiles depends strongly on the structure of the scavenger. The data are supported with quantum mechanical calculations. A comprehensive discussion of the HNO reaction with C-nucleophiles is provided.

New uracil analog U-332 is an inhibitor of NF-κB in 5-fluorouracil-resistant human leukemia HL-60 cell line.

BACKGROUND: 5-Fluorouracil (5-FU) is an antimetabolite that interferes with DNA synthesis and has been widely used as a chemotherapeutic drug in various types of cancers. However, the development of drug resistance greatly limits its application. Overexpression of ATP-binding cassette (ABC) transporters in many types of cancer is responsible for the reduction of the cellular uptake of various anticancer drugs causing multidrug resistance (MDR), the major obstacle in cancer chemotherapy. Recently, we have obtained a novel synthetic 5-FU analog, U-332 [(R)-3-(4-bromophenyl)-1-ethyl-5-methylidene-6-phenyldihydrouracil], combining a uracil skeleton with an exo-cyclic methylidene group. U-332 was highly cytotoxic for HL-60 cells and showed similar cytotoxicity in the 5-FU resistant subclone (HL-60/5FU), in which this analog almost completely abolished expression of the ATP-binding cassette (ABC) transporter, multidrug resistance associate protein 1 (ABCC1). The expression of ABC transporters is usually correlated with NF-κB activation. The aim of this study was to determine the level of NF-κB subunits in the resistant HL-60/5-FU cells and to evaluate the potential of U-332 to inhibit activation of NF-κB family members in this cell line. METHODS: Anti-proliferative activity of compound U-332 was assessed by the MTT assay. In order to disclose the mechanism of U-332 cytotoxicity, quantitative real-time PCR analysis of the NF-κB family genes, c-Rel, RelA, RelB, NF-κB1, and NF-κB2, was investigated. The ability of U-332 to reduce the activity of NF-κB members was studied by ELISA test. RESULTS: In this report it was demonstrated, using RT-PCR and ELISA assay, that members of the NF-κB family c-Rel, RelA, RelB, NF-κB1, and NF-κB2 were all overexpressed in the 5-FU-resistant HL-60/5FU cells and that U-332 potently reduced the activity of c-Rel, RelA and NF-κB1 subunits in this cell line. CONCLUSIONS: This finding indicates that c-Rel, RelA and NF-κB1 subunits are responsible for the resistance of HL-60/5FU cells to 5-FU and that U-332 is able to reverse this resistance. U-332 can be viewed as an important lead compound in the search for novel drug candidates that would not cause multidrug resistance in cancer cells.

New Uracil Analogs with Exocyclic Methylidene Group as Potential Anticancer Agents.

BACKGROUND: Hybrid molecules combining uracil skeleton with methylidene exo-cyclic group were designed in the search for novel anticancer drug candidates. OBJECTIVE: Two series of racemic 5-methylidenedihydrouracils, either 1,3-disubstituted or 1,3,6-trisubstituted were synthesized and tested for their possible cytotoxic activity against two cancer cell lines (HL-60 and MCF-7) and two healthy cell lines (HUVEC and MCF-10A). The most cytotoxic analogs were re-synthesized as pure enantiomers. The analog designated as U-332 [(R)-3-(4-bromophenyl)-1-ethyl-5-methylidene-6-phenyldihydrouracil], which had a very low IC50 value in HL-60 cell line (0.77µM) and was the most selective towards cancer cells was chosen for further experiments on HL-60 cell line, in order to determine the possible mechanism involved in its antineoplastic action. METHODS: Cytotoxic activities of compound was assessed by the MTT assay. In order to explore the mechanism of U-332 activity, we performed quantitative real-time PCR analysis of p53 and p21 genes. Apoptosis, cell proliferation and DNA damage in HL-60 cells were determined using the flow cytometry. The ability of U-332 to determine GADD45ɑ protein level in HL-60 cells incubated with U-332 was analyzed by ELISA test. RESULTS: U-332 was shown to generate excessive DNA damage (70% of the cell population), leading to p53 activation, resulting in p21 down-regulation and a significant increase of GADD45α protein, responsible for the cell cycle arrest in G2/M phase. CONCLUSION: U-332 can be used as a potential lead compound in the further development of novel uracil analogs as anticancer agents.

New uracil analogs as downregulators of ABC transporters in 5-fluorouracil-resistant human leukemia HL-60 cell line.

Overexpression of ATP-binding cassette (ABC) transporters causing multidrug resistance (MDR) in cancer cells is one of the major obstacles in cancer chemotherapy. The 5-FU resistant subclone (HL-60/5FU) of the human HL-60 promyelocytic leukemia cell line was selected by the conventional method of continuous exposure of the cells to the drug up to 0.08 mmol/L concentration. HL-60/5FU cells exhibited six-fold enhanced resistance to 5-FU than HL-60 cells. RT-PCR and ELISA assay showed significant overexpression of MDR-related ABC transporters, ABCB1, ABCG2 but especially ABCC1 in the HL-60/5FU as compared with the parental cell line. Three novel synthetic 5-methylidenedihydrouracil analogs, U-236, U-332 and U-359, selected as highly cytotoxic for HL-60 cells in MTT test, showed similar cytotoxicity in the resistant cell line. When co-incubated with 5-FU, these analogs were found to down-regulate the expression of all three transporters. However, the most pronounced effect was caused by U-332 which almost completely abolished ABCC1 expression in the resistant HL-60/5FU cells. Additionally, U-332 inhibited the activity of ATPase, an enzyme which catalyzes hydrolysis of ATP, providing energy to efflux drugs from the cells through the cellular membranes. Taken together, the obtained data suggest that acquired 5-FU resistance in HL-60/5FU cells results from overexpression of ABCC1 and that targeting ABCC1 expression could be a potential approach to re-sensitize resistant leukemia cells to 5-FU. The synthetic uracil analog U-332, which can potently down-regulate ABC transporter expression and therefore disturb drug efflux, can be considered an efficient ABCC1 regulator in cancer cells.