Synthesis of 5- and 6-Carboxy-X-rhodamines

An efficient route is reported to 5- and 6-carboxy-X-rhodamines (compounds <b>1</b> and <b>2</b>) that contain multiple <i>n</i>-propylene or γ,γ-dimethylpropylene groups bridging terminal nitrogen atoms and the central xanthene core. Gram quantities of these dyes are synthesized from inexpensive starting materials. The isolated products are activated by selective transformation of the carboxylic acid group into <i>N</i>-hydroxysuccinimidyl esters in situ and then conjugated with an amino group of a molecule of interest

Protein-Selective Capture to Analyze Electrophile Adduction of Hsp90 by 4-Hydroxynonenal

The analysis of protein modification by electrophiles is a challenging problem. Most reported protein–electrophile adducts have been characterized from in vitro reactions or through affinity capture of the adduct moiety, which enables global analyses but is poorly suited to targeted studies of specific proteins. We employed a targeted molecular probe approach to study modifications of the molecular chaperone heat shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affinity capture with a biotinyl–geldanamycin probe isolated both isoforms of the native protein (Hsp90α and Hsp90β) from human RKO colorectal cancer cells. Geldanamycin–biotin capture afforded higher purity Hsp90 than did immunoprecipitation and enabled detection of endogenously phosphorylated protein by liquid chromatography–tandem mass spectrometry (LC-MS/MS). We applied this approach to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. LC-MS/MS analyses of tryptic digests by identified His⁴⁵⁰ and His⁴⁹⁰ of Hsp90α as having a 158 Da modification, corresponding to NaBH₄-reduced HNE adducts. Five histidine residues were also adducted on Hsp90β: His¹⁷¹, His⁴⁴², His⁴⁵⁸, His⁶²⁵, and His⁶³². The rates of adduction at these sites were determined with Hsp90 protein in vitro and with Hsp90 in HNE-treated cells with a LC-MS/MS-based, label-free relative quantitation method. During in vitro and cell treatment with HNE, residues on Hsp90α and Hsp90β displayed adduction rates ranging from 3.0 × 10^–5 h^–1 to 1.08 ± 0.17 h^–1. Within the middle client-binding domain of Hsp90α, residue His⁴⁵⁰ demonstrated the most rapid adduction with <i>k</i>_obs of 1.08 ± 0.17 h^–1 in HNE-treated cells. The homologous residue on Hsp90β, His⁴⁴², was adducted more rapidly than the N-terminal residue, His¹⁷¹, despite very similar predicted p<i>K</i>_a values of both residues. The Hsp90 middle client-binding domain thus may play a signicant role in HNE-mediated disruption of Hsp90–client protein interactions. The results illustrate the utility of a protein-selective affinity capture approach for targeted analysis of electrophile adducts and their biological effects

Characterization of an AM404 Analogue, <i>N</i>-(3-Hydroxyphenyl)arachidonoylamide, as a Substrate and Inactivator of Prostaglandin Endoperoxide Synthase

<i>N</i>-(4-Hydroxyphenyl)arachidonoylamide (AM404) is an inhibitor of endocannabinoid inactivation that has been used in cellular and animal studies. AM404 is a derivative of arachidonic acid and has been reported to inhibit arachidonate oxygenation by prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and -2, respectively). While examining the structural requirements for inhibition of PGHS, we discovered that the <i>meta</i> isomer of AM404, <i>N</i>-(3-hydroxyphenyl)arachidonoylamide (3-HPAA), is a substrate for purified PGHS. PGHS-2 efficiently oxygenated 3-HPAA to prostaglandin and hydroxyeicosatetraenoate products. No oxidation of the phenolamide moiety was observed. 3-HPAA appeared to be converted by PGHS-1 in a similar manner; however, conversion was less efficient than that by PGHS-2. PGHS-2 was selectively, dose-dependently, and irreversibly inactivated in the presence of 3-HPAA. Complete inactivation of PGHS-2 was achieved with 10 μM 3-HPAA. Preliminary characterization revealed that 3-HPAA inactivation did not result from covalent modification of PGHS-2 or damage to the heme moiety. These studies provide additional insight into the structural requirements for substrate metabolism and inactivation of PGHS and report the first metabolism-dependent, selective inactivator of PGHS-2

Mass Spectrometric Methods for the Analysis of Nucleoside–Protein Cross-Links: Application to Oxopropenyl-deoxyadenosine

Electrophilic DNA adducts produced following oxidative stress can form DNA–protein cross-links (DPCs), dramatically altering genomic maintenance pathways. Complete characterization of DPCs has been hindered, in part, because of a lack of comprehensive techniques for their analysis. We have, therefore, established a proteomics approach to investigate sites of cross-link formation using <i>N</i>^<i>6</i>-(3-oxo-1-propenyl)-2′-deoxyadenosine (OPdA), an electrophilic DNA adduct produced from oxidative stress. OPdA was reacted with albumin and reduced with NaBH₄ to stabilize DPCs. Using LC-MS/MS proteomics techniques, high-resolution peptide sequence data were obtained; however, using a database searching strategy, adducted peptides were only identified in samples subjected to chemical depurination. This strategy revealed multiple oxopropenyl adenine-lysine adducts and oxopropenyl-lysine adducts with the most reactive lysines identified to be Lys256 and Lys548. Manual interrogation of the mass spectral data provided evidence of OPdA deoxynucleoside conjugates to lysines and cross-links that underwent facile collision-induced dissociation to release an unmodified peptide without subsequent fragmentation. These fragmentations precluded adduct detection and peptide sequencing using database searching methods. Thus, comprehensive analysis of DPCs requires chemical depurination of DNA–protein reaction mixtures followed by a combination of database-dependent and manual interrogation of LC-MS/MS data using higher-energy collision-induced dissociation. In the present case, this approach revealed that OPdA selectively modifies surface lysine residues and produces nucleoside–protein cross-links and oxopropenyl lysine

Electrophilic Modification of PKM2 by 4‑Hydroxynonenal and 4‑Oxononenal Results in Protein Cross-Linking and Kinase Inhibition

Rapidly proliferating cells require an increased rate of metabolism to allow for the production of nucleic acids, amino acids, and lipids. Pyruvate kinase catalyzes the final step in the glycolysis pathway, and different isoforms display vastly different catalytic efficiencies. The M2 isoform of pyruvate kinase (PKM2) is strongly expressed in cancer cells and contributes to aerobic glycolysis in what is commonly termed the Warburg effect. Here, we show that PKM2 is covalently modified by the lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE modify multiple sites on PKM2 <i>in vitro</i>, including Cys424 and His439, which play a role in protein–protein interactions and fructose 1,6-bis-phosphate binding, respectively. Modification of these sites results in a dose-dependent decrease in enzymatic activity. In addition, high concentrations of the electrophile, most notably in the case of ONE, result in substantial protein–protein cross-linking <i>in vitro</i> and in cells. Exposure of RKO cells to electrophiles results in modification of monomeric PKM2 in a dose-dependent manner. There is a concomitant decrease in PKM2 activity in cells upon ONE exposure, but not HNE exposure. Together, our data suggest that modification of PKM2 by certain electrophiles results in kinase inactivation

Differential Sensitivity and Mechanism of Inhibition of COX-2 Oxygenation of Arachidonic Acid and 2-Arachidonoylglycerol by Ibuprofen and Mefenamic Acid

Ibuprofen and mefenamic acid are weak, competitive inhibitors of cyclooxygenase-2 (COX-2) oxygenation of arachidonic acid (AA) but potent, noncompetitive inhibitors of 2-arachidonoylglycerol (2-AG) oxygenation. The slow, tight-binding inhibitor, indomethacin, is a potent inhibitor of 2-AG and AA oxygenation whereas the rapidly reversible inhibitor, 2′-<i>des</i>-methylindomethacin, is a potent inhibitor of 2-AG oxygenation but a poor inhibitor of AA oxygenation. These observations are consistent with a model in which inhibitors bind in one subunit of COX-2 and inhibit 2-AG binding in the other subunit of the homodimeric protein. In contrast, ibuprofen and mefenamate must bind in both subunits to inhibit AA binding

Structure−Activity Analysis of Diffusible Lipid Electrophiles Associated with Phospholipid Peroxidation: 4-Hydroxynonenal and 4-Oxononenal Analogues

Electrophile-mediated disruption of cell signal-ing is involved in the pathogenesis of several diseases including atherosclerosis and cancer. Diffusible and membrane bound lipid electrophiles are known to modify DNA and protein substrates and modulate cellular pathways including ER stress, antioxidant response, DNA damage, heat shock, and apoptosis. Herein we report on a structure−activity relationship for several electrophilic analogues of 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) with regard to toxicity and anti-inflammatory activity. The analogues studied were the oxidation products of HNE and ONE, HNEA/ONEA, the <i>in vivo</i> hydrolysis products of oxidized phosphatidylcholine, COOH-HNE/COOH-ONE, and their methyl esters, COOMe-HNE/ONE. The reactivity of each compound toward <i>N</i>-acetylcysteine was determined and compared to the toxicity toward a human colorectal carcinoma cell line (RKO) and a human monocytic leukemia cell line (THP-1). Further analysis was performed in differentiated THP-1 macrophages to assess changes in macrophage activation and pro-inflammatory signaling in response to each lipid electrophile. HNE/ONE analogues inhibited THP-1 macrophage production of the pro-inflammatory cytokines, IL-6, IL-1β, and TNFα, after lipopolysaccharide (LPS)/IFNγ activation. Inhibition of cytokine production was observed at submicromolar concentrations of several analogues with as little as 30 min of exposure. Phagocytosis of fluorescent beads was also inhibited by lipid electrophile treatment. Lipid electrophiles related to HNE/ONE are both toxic and anti-inflammatory, but the anti-inflammatory effects in human macrophages are observed at nontoxic concentrations. Neither toxicity nor anti-inflammatory activity are strongly correlated to the reactivity of the model nucleophile, <i>N</i>-acetylcysteine

[¹²³I]-Celecoxib Analogues as SPECT Tracers of Cyclooxygenase-2 in Inflammation

We report the synthesis and evaluation of a series of iodinated celecoxib analogues as cyclooxygenase-2 (COX-2)-targeted single photon emission computerized tomography (SPECT) imaging agents for the detection of inflammation. The structure−activity relationship identified 5-(4-iodophenyl)-1-{4-(methylsulfonyl)phenyl}-3-(trifluoromethyl)-1<i>H</i>-pyrazole (<b>8</b>) as a promising compound with IC₅₀ values of 0.05 μM against purified COX-2 and 0.03 μM against COX-2 in activated macrophages. The arylstannane of <b>8</b> undergoes facile radio-[¹²³I]-iodination upon treatment with Na¹²³I/NaI and chloramine T using an EtOAc/H₂O two-phase system. The [¹²³I]-<b>8</b> was produced in a radiochemical yield of 85% and a radiochemical purity of 99%. In vivo SPECT imaging demonstrated that the radiotracer was taken up by inflamed rat paws with an average 1.7-fold enrichment over contralateral noninflamed paws. This study suggests that conversion of celecoxib into its isomeric iodo-[¹²³I]-analogues is a useful approach for generating novel and efficacious agents for COX-2-targeted SPECT imaging of inflammation

Translesion DNA Synthesis by Human DNA Polymerase η on Templates Containing a Pyrimidopurinone Deoxyguanosine Adduct, 3-(2′-Deoxy-β-d-<i>erythro</i>-pentofuranosyl)pyrimido-[1,2-<i>a</i>]purin-10(3<i>H</i>)-one

M₁dG (3-(2′-deoxy-β-d-<i>erythro</i>-pentofuranosyl)pyrimido[1,2-<i>a</i>]purin-10(3<i>H</i>)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M₁dG to dT and M₁dG to dA) and frameshift mutations. M₁dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M₁dG in <i>Escherichia coli</i> is dependent on the SOS response, specifically the umuC and umuD gene products, suggesting that mutagenic lesion bypass occurs by the action of translesion DNA polymerases, like DNA polymerase V. Bypass of DNA lesions by translesion DNA polymerases is conserved in bacteria, yeast, and mammalian cells. The ability of recombinant human DNA polymerase η to synthesize DNA across from M₁dG was studied. M₁dG partially blocked DNA synthesis by polymerase η. Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M₁dG lesion (800-fold less efficient than opposite dG). Extension from M₁dG·dC was equally as efficient as from control primer-templates (dG·dC). dATP insertion opposite M₁dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M₁dG·dA was 20-fold less efficient than dG·dC. The sequences of full-length human DNA polymerase η bypass products of M₁dG were determined by LC-ESI/MS/MS. Bypass products contained incorporation of dA (52%) or dC (16%) opposite M₁dG or −1 frameshifts at the lesion site (31%). Human DNA polymerase η bypass may lead to M₁dG to dT and frameshift but likely not M₁dG to dA mutations during DNA replication

Nuclear Oxidation of a Major Peroxidation DNA Adduct, M₁dG, in the Genome

Chronic inflammation results in increased production of reactive oxygen species (ROS), which can oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3-(2-deoxy-β-d-erythro-pentofuranosyl)­pyrimido­[1,2-α]­purin-10­(3<i>H</i>)-one (M₁dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M₁dG is mutagenic in bacterial and mammalian cells and is repaired via the nucleotide excision repair system. Here, we report that M₁dG levels in intact DNA were increased from basal levels of 1 adduct per 10⁸ nucleotides to 2 adducts per 10⁶ nucleotides following adenine propenal treatment of RKO, HEK293, or HepG2 cells. We also found that M₁dG in genomic DNA was oxidized in a time-dependent fashion to a single product, 6-oxo-M₁dG (to ∼5 adducts per 10⁷ nucleotides), and that this oxidation correlated with a decline in M₁dG levels. Investigations in RAW264.7 macrophages indicate the presence of high basal levels of M₁dG (1 adduct per 10⁶ nucleotides) and the endogenous formation of 6-oxo-M₁dG. This is the first report of the production of 6-oxo-M₁dG in genomic DNA in intact cells, and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis, and repair