SAR of non-hydrolysable analogs of pyridoxal 5'-phosphate against low molecular weight protein tyrosine phosphatase isoforms.

Kinases and phosphatases are key enzymes in cell signal transduction pathways. Imbalances in these enzymes have been linked to numerous disease states ranging from cancer to diabetes to autoimmune disorders. The two isoforms (IFA and IFB) of Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) appear to play a role in these diseases. Pyridoxal 5'-phosphate (PLP) has been shown to act as a potent but, impractical micromolar inhibitor for both isoforms. In this study, a series of non-hydrolysable phosphonate analogs of PLP were designed, synthesized and tested against the two isoforms of LMW-PTP. Assay results demonstrated that the best inhibitor for both isoforms was compound 5 with a Kis of 1.84 µM (IFA) and 15.6 µM (IFB). The most selective inhibitor was compound 16, with a selectivity of roughly 370-fold for IFA over IFB.

Identification of new inhibitors for low molecular weight protein tyrosine phosphatase isoform B.

The National Cancer Institute Diversity Set II (1356 compounds) and Diversity Set III (1597 compounds) were screened via in silico methods as potential inhibitors of low molecular weight protein tyrosine phosphatase (LWM-PTP) isoform B (EC 3.1.3.48). Those candidates that demonstrated comparable or better docking scores than that of pyridoxal 5'-phosphate (PLP), one of the most potent known inhibitors of LMW-PTP with a competitive inhibitor dissociation constant (Kis) of 7.6µM (pH 5.0), were analyzed via in vitro kinetic assays against LMW-PTP isoform B. While none of the compounds tested in vitro was significantly better that PLP, five compounds showed comparable inhibition. These five compounds are very diverse in structure and represent new therapeutic leads for inhibition of this isozyme.

Preferential glutathione conjugation of a reverse diol epoxide compared with a bay region diol epoxide of benzo[a]pyrene in human hepatocytes.

Many studies have examined the relationship between polymorphisms in glutathione S-transferase genes and cancer in people exposed to polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP), but the results to date have been modest. Missing from these studies has been an exploration of the formation of the appropriate glutathione conjugates in humans. We incubated human hepatocytes from 10 donors with racemic anti-BaP-7,8-diol-9,10-epoxide (BPDE), believed to be a major ultimate carcinogen of BaP, or with the noncarcinogenic reverse diol epoxide, racemic anti-BaP-9,10-diol-7,8-epoxide (rev-BPDE). Incubations were carried out for 12 or 24 h. We used high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464 --> m/z 317 to analyze the incubation mixtures for the mercapturic acid products that would result from glutathione conjugation. The standard mercapturic acids were synthesized by reaction of BPDE or rev-BPDE with N-acetylcysteine. We obtained convincing evidence in human hepatocytes for mercapturic acid formation from rev-BPDE in all 10 samples, in amounts up to 17 pmol/ml. However, we could detect mercapturic acids from BPDE in only 1 of 10 samples (0.05 pmol/ml). Taken together with our similar previous results of analyses of phenanthrene metabolites in human hepatocytes and human urine, the results of this study indicate that conjugation of BPDE with glutathione is a minor pathway in humans, indicating that glutathione S-transferase genotyping is not an effective method for assessing risk of PAH-induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis.

Identification of adducts formed in the reaction of 5'-acetoxy-N'-nitrosonornicotine with deoxyguanosine and DNA.

N'-Nitrosonornicotine (NNN) is believed to play an important role as a cause of cancer in people who use tobacco products and is considered to be a human carcinogen. NNN requires metabolism to form DNA adducts, which are absolutely critical to its carcinogenic properties. Previous studies have identified cytochrome P450-catalyzed 2'- and 5'-hydroxylation of NNN as potential DNA adduct forming metabolic pathways. 5'-Hydroxylation is the more prevalent of these in monkeys and humans and is known to generate mutagenic intermediates, but the DNA adducts formed by this pathway have never been characterized. In this study, we used 5'-acetoxyNNN as a stable precursor to 5'-hydroxyNNN and investigated its esterase-catalyzed reactions with deoxyguanosine (dGuo) and DNA. Adducts resulting from carbocation and oxonium ion intermediates, produced by the spontaneous decomposition of 5'-hydroxyNNN, were identified. The carbocation pathway resulted in the formation of 2-[2-hydroxy-5-(3-pyridyl)pyrrolidin-1-yl]deoxyinosine (12) which was characterized by comparison to an independently synthesized standard. Treatment of 12 with NaBH(3)CN produced two diastereomers of 2-[2-(3-pyridyl)pyrrolidin-1-yl]deoxyinosine (14), and their absolute configurations at the 2-position were determined by comparison to synthetic standards. The oxonium ion pathway produced diastereomers of N(2)[5-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (16), identified by comparison to synthetic standards. The absolute configuration at the 5-position was determined by establishing the stereochemistry of the enantiomers of 5-(3-pyridyl)-2-hydroxytetrahydrofuran at the 5-position and allowing these to react individually with dGuo. Treatment of 16 with NaBH(3)CN produced N(2)[4-hydroxy-4-(3-pyridyl)but-1-yl]dGuo (18) which was also synthesized independently. Using liquid chromatography-electrospray ionization-tandem mass spectrometry with selected reaction monitoring, we identified adducts 12 and 16 as products of the reactions of 5'-acetoxyNNN with dGuo. Similarly, adducts 14 and 18 were identified as products of the reaction of 5'-acetoxyNNN with DNA followed by NaBH(3)CN treatment and enzymatic hydrolysis. These results provide the first structural characterization of DNA adducts that can be formed by 5'-hydroxylation of NNN.

Phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates inhibit malignant progression of lung adenomas induced by tobacco carcinogens in A/J mice.

We have shown previously that naturally occurring isothiocyanates derived from cruciferous vegetables and their N-acetylcysteine conjugates inhibit lung adenoma formation induced by tobacco carcinogens in A/J mice at the post-initiation stage. The tumor-inhibitory activity by these compounds is linked with activation of activator protein and induction of apoptosis in lung tissues, suggesting that these compounds may also inhibit the development of adenomas to adenocarcinomas in lung. In this study, the chemopreventive activity of phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates during progression of lung adenomas to malignant tumors was investigated in A/J mice. Mice were divided into 14 groups and treated with a mixture of 3 micromol benzo(a)pyrene [B(a)P] and 3 micromol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) given by gavage once weekly for 8 weeks. Twenty weeks after the beginning of carcinogen administration, a total of 20 mice in the treatment groups were sacrificed with an average yield of 7.3 +/- 4.5 lung adenomas per mouse. The remaining mice in each group were fed diets containing phenethyl isothiocyanate (3 and 1.5 mmol/kg diet), sulforaphane (3 and 1.5 mmol/kg diet), phenethyl isothiocyanate-N-acetylcysteine (8 and 4 mmol/kg diet), sulforaphane-N-acetylcysteine (8 and 4 mmol/kg diet) during weeks 21 to 42. Four mice in each of the high-dose treatment groups were sacrificed during weeks 28 and 36 and the bioassay was terminated during week 42; lung tissues were harvested for histopathologic examination of tumors and for cell proliferation (proliferating cell nuclear antigen) and apoptosis (caspase-3) assays using immunohistochemical staining. At termination, the incidence of adenocarcinoma in the 3 mmol/kg diet phenethyl isothiocyanate group and 8 mmol/kg diet phenethyl isothiocyanate-N-acetylcysteine group was reduced to 19% and 13%, respectively, compared with 42% in the carcinogen-treated control group. At the lower doses, phenethyl isothiocyanate and its N-acetylcysteine conjugate also inhibited the incidences of lung adenocarcinoma, however, the decreases were not statistically significant. The lung tumor incidences in groups treated with sulforaphane-N-acetylcysteine in the diet were also significantly reduced to 11% or 16%. Furthermore, the malignant lung tumor multiplicity was significantly reduced from 1.0 tumor/mouse in the carcinogen-treated control group to 0.3 in the sulforaphane low-dose group, 0.3 and 0.4 in the two sulforaphane-N-acetylcysteine groups, and 0.4 in the phenethyl isothiocyanate high-dose group. The malignant tumor multiplicities in other treatment groups were also reduced (0.5-0.8 tumors/mouse), but not significantly. Unlike lung adenocarcinomas, both incidences and multiplicities of lung adenomas were not much affected by treatment with isothiocyanates or their conjugates. Immunohistochemical examination of the lung tumors from all time points indicated that significant reduction in proliferating cell nuclear antigen and induction of apoptosis (terminal nucleotidyl transferase-mediated nick end labeling and caspase-3) were observed in the isothiocyanate and isothiocyanate-N-acetylcysteine-treated groups that showed inhibition of the development of lung adenocarcinomas. The results of the study provide a basis for future evaluation of the potential of phenethyl isothiocyanate and sulforaphane and their conjugates as chemopreventive agents in smokers and ex-smokers with early lung lesions.

Stereospecific deuterium substitution attenuates the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Stereochemical determinants of the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were investigated using the stereospecifically deuterated isotopomers (4R)-[4-(2)H(1)]NNK and (4S)-[4-(2)H(1)]NNK. Upon ip administration to groups of 20 female A/J mice, NNK and (4S)-[4-(2)H(1)]NNK exhibited similar lung tumorigenicity at three different doses, whereas (4R)-[4-(2)H(1)]NNK was 2-fold less tumorigenic at all three doses. In a parallel experiment, levels of O(6)-methylguanine and 7-methylguanine were 2-fold lower in lung DNA of mice treated with (4R)-[4-(2)H(1)]NNK than in mice treated with NNK or (4S)-[4-(2)H(1)]NNK. To corroborate these in vivo data, the in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda (Sf9)-expressed mouse cytochrome p450s 2A4 and 2A5. Kinetic isotope effects on the apparent V(max) ((D)V) for the product of NNK 4-hydroxylation, OPB, were 2.7 +/- 0.2 and 2.8 +/- 0.4 when (4R)- and (4S)-[4-(2)H(1)]NNK were incubated with mouse lung microsomes, respectively. The (D)V values for OPB formation were 3.2 +/- 0.2 and 2.2 +/- 0.2 when (4R)-[4-(2)H(1)]NNK was the substrate for p2A4 and 2A5, respectively, whereas they were 1.3 +/- 0.1 and 1.1 +/- 0.1 when (4S)-[4-(2)H(1)]NNK was the substrate for these respective enzymes. Analysis of an OPB derivative (10) for deuterium content by LC/MS confirmed the results from the kinetic assays and indicated that p450s 2A4 and 2A5 preferentially abstract the pro-R 4-hydrogen of NNK. The results obtained using Sf9-expressed p450s provide a rationale for the differences observed in the lung tumor and DNA adduct experiments, namely, that the attenuated tumorigenicity of (4R)-[4-(2)H(1)]NNK relative to (4S)-[4-(2)H(1)]NNK is due to prochiral selectivity during p450-catalyzed metabolic activation.

Identification of adducts formed by pyridyloxobutylation of deoxyguanosine and DNA by 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a chemically activated form of tobacco specific carcinogens.

The tobacco specific carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are metabolically activated to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (7), which is known to pyridyloxobutylate DNA. A substantial proportion of the adducts in this DNA releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 11) under various hydrolysis conditions, including neutral thermal hydrolysis. These HPB-releasing DNA adducts have been detected in target tissues of animals treated with NNK and NNN as well as in lung tissue from smokers. Although their presence in pyridyloxobutylated DNA was conclusively demonstrated 15 years ago, their structures have not been previously determined. We investigated this question in the present study by determining the structures of products formed in reactions with dGuo and DNA of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc, 3), a stable precursor to 7. Reaction mixtures from NNKCH(2)OAc and dGuo were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) with selected ion monitoring at m/z 415. A major peak was detected and identified as 7-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (37) by its ESI-MS fragmentation pattern and by neutral thermal hydrolysis, which converted it to 11 and 7-[4-oxo-4-(3-pyridyl)but-1-yl]Gua (26). The latter was identified by comparison to synthetic 26 using LC-ESI-MS with selected ion monitoring at m/z 299, M + 1 of 26. Further evidence was obtained by NaBH(4) reduction of 26 to 7-[4-hydroxy-4-(3-pyridyl)but-1-yl]Gua, which was also matched with a standard. Adduct 37 was similarly identified in enzyme hydrolysates of DNA reacted with NNKCH(2)OAc, accounting for 30-35% of the HPB-releasing adducts in this DNA. Several other adducts resulting from pyridyloxobutylation of the N(2)- and O(6)-positions of Gua were also identified as products in the dGuo or DNA reactions by comparison to standards; their concentrations were considerably less than that of 37. These adducts were N(2)-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (23), N(2)-[4-oxo-4-(3-pyridyl)but-2-yl]dGuo (25), N(2)-[2-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (31a) (or its open chain tautomer 31b), and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (10). Adducts 23, 25, and 10 did not release HPB upon neutral thermal hydrolysis. The results of this study provide the first structural identification of an HPB-releasing DNA adduct of the tobacco specific nitrosamines NNK and NNN.

Reactions of formaldehyde plus acetaldehyde with deoxyguanosine and DNA: formation of cyclic deoxyguanosine adducts and formaldehyde cross-links.

We investigated the reactions of formaldehyde plus acetaldehyde with dGuo and DNA in order to determine whether certain 1,N(2)-propano-dGuo adducts could be formed. These adducts-3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-(3H)-one (1) and 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (3a,b)-have been previously characterized as products of the reaction of acrolein with dGuo and DNA. Adduct 1 predominates in certain model lipid peroxidation systems [Pan, J., and Chung, F. L. (2002) Chem. Res. Toxicol. 15, 367-372]. We hypothesized that this could be due to stepwise reactions of formaldehyde and acetaldehyde with dGuo, rather than by reaction of acrolein with dGuo. The results demonstrated that adducts 1 and 3a,b were relatively minor products of the reaction of formaldehyde and acetaldehyde with dGuo and that there was no selectivity in their formation. These findings did not support our hypothesis. However, substantial amounts of previously unknown cyclic dGuo adducts were identified in this reaction. The new adducts were characterized by their MS, UV, and NMR spectra as diastereomers of 3-(2'-deoxyribosyl)-6-methyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (10a,b). Adducts 10a,b were apparently formed by addition of formaldehyde to N1 of N(2)-ethylidene-dGuo, followed by cyclization. An analogous set of four diastereomers of 3-(2'-deoxyribosyl)-6,8-dimethyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (12a-d) were formed in the reactions of acetaldehyde with dGuo. These products are the first examples of exocyclic dGuo adducts of the pyrimido[1,2-a]purine type in which an oxygen atom is incorporated into the exocyclic ring. Formaldehyde-derived adducts were the other major products of the reactions of formaldehyde plus acetaldehyde with dGuo. Prominent among these were N(2)-hydroxymethyl-dGuo (9) and the cross-link di-(N(2)-deoxyguaonosyl)methane (13). We did not detect adducts 1, 3a,b, or 10a,b in enzymatic hydrolysates of DNA that had been allowed to react with formaldehyde plus acetaldehyde. However, we did detect substantial amounts of the formaldehyde cross-links di-(N(6)-deoxyadenosyl)methane (17), with lesser quantities of (N(6)-deoxyadenosyl-N(2)-deoxyguanosyl)methane (18), di-(N(2)-deoxyguanosyl)methane (13), and N(6)-hydroxymethyl-dAdo (19). Schiff base adducts of formaldehyde and acetaldehyde were also detected in these reactions. These results demonstrate that the reactions of formaldehyde plus acetaldehyde with dGuo are dominated by newly identified cyclic adducts and formaldehyde-derived products whereas the reactions with DNA result in the formation of formaldehyde cross-link adducts. The carcinogens formaldehdye and acetaldehyde occur in considerable quantities in the human body and in the environment. Therefore, further research is required to determine whether the adducts described here are formed in animals or humans exposed to these agents.

Inhibition of lung tumorigenesis in A/J mice by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine and myo-inositol, individually and in combination.

Isothiocyanates, their N-acetylcysteine conjugates, and myo-inositol (MI) are inhibitors of lung tumorigenesis in A/J mice. However, chemoprevention by combinations of these compounds in different temporal sequences has not been examined. This is important for developing practical approaches to lung cancer chemoprevention in smokers and ex-smokers. We used a tumor model in which A/J mice are treated with 8 weekly doses of benzo[a]pyrene (B[a]P) plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and killed 19 weeks after the final treatment. In Experiment 1, isothiocyanates or their N-acetylcysteine conjugates were added to the diet (1 or 3 micro mol/g) from 1 week before until 1 week after carcinogen treatment. The compounds were 2-phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), N-acetyl-S-(N-benzyl-thiocarbamoyl)-L-cysteine (BITC-NAC), N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine (PEITC-NAC), and N-acetyl-S-(N-3-phenylpropylthiocarbamoyl)-L-cysteine (PPITC-NAC). Significant reductions in lung tumor multiplicity were observed in mice treated with PEITC, PEITC-NAC, PPITC and PPITC-NAC. PEITC-NAC was chosen for combination studies with MI (Experiment 2). Mice were treated with B[a]P plus NNK without or with PEITC-NAC (3 micro mol/g diet), MI (55.5 micro mol/g diet), or PEITC-NAC plus MI (3 micro mol plus 55.5 micro mol/g diet). Different temporal sequences of dietary additions were investigated: carcinogen treatment phase; post-carcinogen treatment phase; entire experiment; 50% of carcinogen treatment phase until termination; and 75% of carcinogen treatment phase until termination. All treatments reduced lung tumor multiplicity except PEITC-NAC post-carcinogen or from 75% of the carcinogen treatment phase. Reduction of lung tumor multiplicity by PEITC-NAC plus MI was greater than that in the mice treated with the agents alone in all temporal sequences. When all results were combined, PEITC-NAC plus MI was significantly more effective than the agents alone. There was a significant trend for reduction in lung tumor multiplicity with increased duration of treatment by the chemopreventive agents. These results provide a basis for further development of mixtures of PEITC-NAC and MI for chemoprevention of lung cancer.