Rosalina Wisastra

Inhibition of the PCAF histone acetyl transferase and cell proliferation by isothiazolones

Small molecule HAT inhibitors are useful tools to unravel the role of histone acetyl transferases (HATs) in the cell and have relevance for oncology. We present a systematic investigation of the inhibition of the HAT p300/CBP Associated Factor (PCAF) by isothiazolones with different substitutions. 5-chloroisothiazolones proved to be the most potent inhibitors of PCAF. The growth inhibition of 4 different cell lines was studied and the growth of two cell lines (A2780 and HEK 293) was inhibited at micromolar concentrations by 5-chloroisothiazolones. Furthermore, the 5-chloroisothiazolone preservative Kathon CG that is used in cosmetics inhibited PCAF and the growth of cell lines A2780 and HEK 293, which indicates that this preservative should be applied with care.

Inflammation, Cancer and Oxidative Lipoxygenase Activity are Intimately Linked

Cancer and inflammation are intimately linked due to specific oxidative processes in the tumor microenvironment. Lipoxygenases are a versatile class of oxidative enzymes involved in arachidonic acid metabolism. An increasing number of arachidonic acid metabolites is being discovered and apart from their classically recognized pro-inflammatory effects, anti-inflammatory effects are also being described in recent years. Interestingly, these lipid mediators are involved in activation of pro-inflammatory signal transduction pathways such as the nuclear factor κB (NF-κB) pathway, which illustrates the intimate link between lipid signaling and transcription factor activation. The identification of the role of arachidonic acid metabolites in several inflammatory diseases led to a significant drug discovery effort around arachidonic acid metabolizing enzymes. However, to date success in this area has been limited. This might be attributed to the lack of selectivity of the developed inhibitors and to a lack of detailed understanding of the functional roles of arachidonic acid metabolites in inflammatory responses and cancer. This calls for a more detailed investigation of the activity of arachidonic acid metabolizing enzymes and development of more selective inhibitors.

Antibody-Free Detection of Protein Tyrosine Nitration in Tissue Sections

Tyrosine nitration is a covalent post-translational protein modification that occurs in vivo as a consequence of oxidative stress. It has been shown that tyrosine nitration is caused by peroxynitrite, which is formed by reaction between superoxide anions (O ) and nitric oxide (NO). Other reactive nitrogen species, such as NO2 + , NO2Cl, and NO2, can also be involved in tyrosine nitration. Recent studies have shown that 3-nitrotyrosine is a biomarker for several inflammatory diseases, such as inflammatory bowel disease (IBD), rheumatoid arthritis, and asthma. Tyrosine nitration has often been regarded as nonselective oxidative damage to proteins; however, recently more specific roles have been described. For example, tyrosine nitration triggers dissociation of IkB from nuclear factorkB (NF-kB); this results in the activation of a pathway that plays an important role in cancer and inflammation. The importance of tyrosine nitration in the pathology of multiple diseases and its potential role in signal transduction highlights the urgent need for reliable analytical methods to study protein nitration. Several methods have been developed for the detection of nitrotyrosine; these employ antibodies, mass spectrometry, or fluorogenic tagging. Currently, tyrosine-nitrated proteins in tissue sections are detected by immunohistochemistry with antibodies directed at nitrotyrosine. Protein-bound nitrotyrosine in tissue homogenate and blood plasma is usually detected by immunoblot methods; these also rely on nitrotyrosine-directed antibodies. However, nitrotyrosine antibodies have disadvantages, such as a lack of selectivity for nitrotyrosine, high background signals in certain tissues, high costs, and limited stability upon prolonged storage. In this study, we introduce a novel method that does not rely on antibodies to detect nitrotyrosine in biological samples. This method involves two reaction steps to convert the 2-nitrophenol functionality of protein-bound nitrotyrosine chemoselectively into a fluorophore (Scheme 1). In the first reaction step, the 2-nitrophenol functionality is reduced to a 2-aminophenol functionality by sodium dithionite. In the second step, the 2-aminophenol functionality is converted into a fluorophore by reaction with salicylaldehyde and Al . This novel histochemical staining selectively detects 2-nitrophenol, as well as 2-nitrosophenol and 2-aminophenol functionalities. In this study, we describe the application of this methodology to detect nitrotyrosine in tissue sections by fluorescence microscopy. This method is also applicable for staining protein-bound 2-nitrophenol functionalities on Western blot membranes in complex biological samples such as blood plasma. Initially, we studied the fluorescence of the Schiff base of 2aminophenol and salicylaldehyde complexed with aluminium. The fluorescence spectra of this complex in solution showed an excitation wavelength (lex) of 412 nm and an emission wavelength (lem) of 520 nm in H2O and in MeOH (Figure S1 and S2 in the Supporting Information). The fluorescence proved to be linear between 0.31 and 10 mm in both solvents (Figure S3), and the fluorescence signal in H2O was eight times stronger than that in MeOH. Thus, H2O is the preferred solvent for the detection of nitrotyrosine in biological samples. The first reaction step for the histochemcial staining (Scheme 1) is reduction of 2-nitrophenol by a freshly prepared solution of sodium dithionite (Na2S2O4) in demineralised water. This reagent provides fast and complete conversion of 2-nitrophenol in an aqueous environment, thus demonstrating that sodium dithionite is an appropriate reducing agent for this reaction. Several control experiments were performed for the second staining reaction (Scheme 1), the formation of the Schiff base between the 2-aminophenol functionality and salicylaldehyde in complex with Al . Analysis of the fluorescence spectra of the reagents in the solution used for this step (containing salicylaldehyde and aluminium) gave very weak fluoresScheme 1. Reduction of a protein bound 2-nitrophenol functionality to a 2aminophenol functionality, and subsequent conversion into a fluorophore. a) aqueous Na2S2O4, b) aqueous AlCl3 and salicylaldehyde.

Anacardic acid derived salicylates are inhibitors or activators of lipoxygenases.

Lipoxygenases catalyze the oxidation of unsaturated fatty acids, such as linoleic acid, which play a crucial role in inflammatory responses. Selective inhibitors may provide a new therapeutic approach for inflammatory diseases. In this study, we describe the identification of a novel soybean lipoxygenase-1 (SLO-1) inhibitor and a potato 5-lipoxygenase (5-LOX) activator from a screening of a focused compound collection around the natural product anacardic acid. The natural product anacardic acid inhibits SLO-1 with an IC(50) of 52 μM, whereas the inhibitory potency of the novel mixed type inhibitor 23 is fivefold enhanced. In addition, another derivative (21) caused non-essential activation of potato 5-LOX. This suggests the presence of an allosteric binding site that regulates the lipoxygenase activity.

Enrichment and detection of tyrosine-nitrated proteins

Nitrotyrosine is a post‐translationally modified amino acid with distinctly different properties than tyrosine or any other of the genetically encoded amino acids. Detecting proteins containing nitrotyrosine is the first step towards a better understanding of the role of nitrotyrosine in health and disease. Moreover, quantifying the extent of nitrotyrosine and determining its location in a protein forms the basis for a better understanding of the effect of tyrosine nitration on biological function. Described in this unit is a method to detect tyrosine‐nitrated proteins in tissue sections and on western blots after creating a fluorescent complex between aminotyrosine, salicylaldehyde, and Al3+. In addition, an approach is detailed for labeling aminotyrosine with biotin to enrich peptides from complex samples. Both methods require reduction of nitrotyrosine to aminotyrosine, which can be achieved with sodium dithionite or hemin plus dithiothreitol. Curr. Protoc. Protein Sci. 69:14.13.1‐14.13.19.

Discovery of a novel activator of 5-lipoxygenase from an anacardic acid derived compound collection

Lipoxygenases (LOXs) and cyclooxygenases (COXs) metabolize poly-unsaturated fatty acids into inflammatory signaling molecules. Modulation of the activity of these enzymes may provide new approaches for therapy of inflammatory diseases. In this study, we screened novel anacardic acid derivatives as modulators of human 5-LOX and COX-2 activity. Interestingly, a novel salicylate derivative 23a was identified as a surprisingly potent activator of human 5-LOX. This compound showed both non-competitive activation towards the human 5-LOX activator adenosine triphosphate (ATP) and non-essential mixed type activation against the substrate linoleic acid, while having no effect on the conversion of the substrate arachidonic acid. The kinetic analysis demonstrated a non-essential activation of the linoleic acid conversion with a KA of 8.65 μM, αKA of 0.38μM and a β value of 1.76. It is also of interest that a comparable derivative 23d showed a mixed type inhibition for linoleic acid conversion. These observations indicate the presence of an allosteric binding site in human 5-LOX distinct from the ATP binding site. The activatory and inhibitory behavior of 23a and 23d on the conversion of linoleic compared to arachidonic acid are rationalized by docking studies, which suggest that the activator 23a stabilizes linoleic acid binding, whereas the larger inhibitor 23d blocks the enzyme active site.

Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCAF

Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak nitrogen-sulfur bond to form disulfides. They show selectivity for inhibition of the thiol-dependent cysteine protease cathepsin B and the histone acetyltransferase p300/CBP associated factor (PCAF) based on their substitution pattern. Furthermore, enzyme kinetics and mass spectroscopy indicate covalent binding of a 5-chloroisothiazolone to cathepsin B, which demonstrates their potential utility as probes for activity-based protein profiling.

A dual inhibitor of matrix metalloproteinases and a disintegrin and metalloproteinases, [18F]FB-ML5, as a molecular probe for non-invasive MMP/ADAM-targeted imaging

BACKGROUND Numerous clinical studies have shown a correlation between increased matrix metalloproteinase (MMP)/a disintegrin and metalloproteinase (ADAM) activity and poor outcome of cancer. Various MMP inhibitors (MMPIs) have been developed for therapeutic purposes in oncology. In addition, molecular imaging of MMP/ADAM levels in vivo would allow the diagnosis of tumors. We selected the dual inhibitor of MMPs and ADAMs, ML5, which is a hydroxamate-based inhibitor with affinities for many MMPs and ADAMs. ML5 was radiolabelled with (18)F and the newly obtained radiolabelled inhibitor was evaluated in vitro and in vivo. MATERIALS AND METHODS ML5 was radiolabelled by direct acylation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB) for PET (positron emission tomography). The resulting radiotracer [(18)F]FB-ML5 was evaluated in vitro in human bronchial epithelium 16HBE cells and breast cancer MCF-7 cells. The non-radioactive probe FB-ML5 and native ML5 were tested in a fluorogenic inhibition assay against MMP-2, -9, -12 and ADAM-17. The in vivo kinetics of [(18)F]FB-ML5 were examined in a HT1080 tumor-bearing mouse model. Specificity of probe binding was examined by co-injection of 0 or 2.5mg/kg ML5. RESULTS ML5 and FB-ML5 showed high affinity for MMP-2, -9, -12 and ADAM-17; indeed IC50 values were respectively 7.4 ± 2.0, 19.5 ± 2.8, 2.0 ± 0.2 and 5.7 ± 2.2 nM and 12.5 ± 3.1, 31.5 ± 13.7, 138.0 ± 10.9 and 24.7 ± 2.8 nM. Radiochemical yield of HPLC-purified [(18)F]FB-ML5 was 13-16% (corrected for decay). Cellular binding of [(18)F]FB-ML5 was reduced by 36.6% and 27.5% in MCF-7 and 16 HBE cells, respectively, after co-incubation with 10 μM of ML5. In microPET scans, HT1080 tumors exhibited a low and homogeneous uptake of the tracer. Tumors of mice injected with [(18)F]FB-ML5 showed a SUVmean of 0.145 ± 0.064 (n=6) which decreased to 0.041 ± 0.027 (n=6) after target blocking (p<0.05). Ex vivo biodistribution showed a rapid excretion through the kidneys and the liver. Metabolite assays indicated that the parent tracer represented 23.2 ± 7.3% (n=2) of total radioactivity in plasma, at 90 min post injection (p.i.). CONCLUSION The nanomolar affinity MMP/ADAM inhibitor ML5 was successfully labelled with (18)F. [(18)F]FB-ML5 demonstrated rather low binding in ADAM-17 overexpressing cell lines. [(18)F]FB-ML5 uptake showed significant reduction in the HT1080 tumor in vivo after co-injection of ML5. [(18)F]FB-ML5 may be suitable for the visualization/quantification of diseases overexpressing simultaneously MMPs and ADAMs.