Correction: A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D.

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A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D.

The PPM1D gene is aberrantly amplified in a range of common cancers and encodes a protein phosphatase that is a potential therapeutic target. However, the issue of whether inhibition of PPM1D in human tumour cells that overexpress this protein compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of PPM1D can indeed reduce the viability of human tumour cells and that this effect is selective; tumour cell lines that overexpress PPM1D are sensitive to PPM1D inhibition whereas cell lines with normal levels are not. Loss of viability associated with PPM1D RNAi in human tumour cells occurs via the activation of the kinase P38. To identify chemical inhibitors of PPM1D, a high-throughput screening of a library of small molecules was performed. This strategy successfully identified a compound that selectively reduces viability of human tumour cell lines that overexpress PPM1D. As expected of a specific inhibitor, the toxicity to PPM1D overexpressing cell lines after inhibitor treatment is P38 dependent. These results further validate PPM1D as a therapeutic target and identify a proof-of-principle small molecule inhibitor.

Solid-phase synthesis of novel inhibitors of farnesyl transferase.

A novel diphosphate mimic, the 2,3,6-trifluoro-5-hydroxy-4-nitrophenoxy group (1), has been employed as the template in the solid-phase synthesis of novel farnesyl transferase inhibitors using the Mitsunobu reaction. The most potent inhibitor (farnesyloxy-5-hydroxy-2,3,6-trifluoro-4-nitrobenzene) displayed an IC50 of 6.3 microM versus farnesyl transferase.

Inhibition of protein prenylation by metabolites of limonene.

The monoterpenes limonene and perillyl alcohol are undergoing clinical evaluation in cancer patients. In this paper, we report the chemical synthesis, characterisation, and quantitation in patients' plasma of a novel human metabolite of limonene, which is identified as an isomer of perillic acid. The synthesis of R-perillic acid is also described, because previous reports on the activity of perillic acid against isoprenylation enzymes refer to the S-enantiomer, although it is the R-enantiomer which is the metabolite of R-limonene. The above monoterpenes, with several related compounds, were assayed for inhibitory activity towards the isoprenylation enzymes in rat brain cytosol. Although R- and S-limonene are only weak inhibitors of the isoprenylation enzymes, their major metabolites, perillic acid and perillyl alcohol, are more potent inhibitors, with IC50 values in the low mM range. The metabolites possess greater activity towards the geranylgeranyltransferase type I enzyme than farnesyltransferase, while the novel metabolite displays IC50 values similar to those of perillic acid suggesting that it may contribute to the in vivo activity of limonene.

Comparison of biochemical aromatase activity with aromatase immunohistochemistry in human breast carcinomas.

The role of intratumoural aromatase in human breast cancer growth remains controversial. At the same time as the use of aromatase inhibitors in the clinical setting continues to increase, so does the need for a tool to predict the likely response to this treatment. Intratumoural aromatase is a candidate predictive marker. The presently accepted 'gold standard' methods of assessment of aromatase activity are biochemical assays. However, these are time-consuming and require relatively large amounts of fresh or frozen tissue which are frequently not available. The development of a reliable immunohistochemical technique for the assessment of intratumoural aromatase which could be applied rapidly to more readily available paraffin-embedded material is therefore highly desirable. Unfortunately aromatase immunohistochemistry is also an area of controversy; some authors describe localisation to the stromal compartment but others to the malignant epithelial cells themselves. The aim of this study was therefore to compare immunohistochemical scores using two different antibodies with biochemical aromatase activity. Taking a group of 29 human breast carcinomas we demonstrated a strong correlation between immunoreactivity with a monoclonal antibody (p = 0.01) but not with a polyclonal (p = 0.16). The monoclonal produced reactivity in both epithelial and stromal cells but the polyclonal in only stromal cells. The implications of these results are discussed together with the need for further studies.

Antagonism of estrogen receptor and calmodulin association by antiestrogens is not dependent on an interaction with calmodulin.

Previously, two antiestrogens estradiol derivatives (3 and 4) bearing the basic side chain of tamoxifen were shown to impede the binding of the estrogen receptor (ER) to calmodulin (CaM)-Sepharose. In this study, the interaction of these and related compounds with calmodulin was examined using the cyclic AMP phosphodiesterase (cAMP-PDE) assay. Neither of the steroids gave any significant inhibition of the calmodulin dependent cAMP-PDE activity up to a final concentration of 20 microM. For comparison, tamoxifen and nafoxidine produced IC50 values of 6.7 microM +/- 1.0 and 7.4 microM +/- 1.1, respectively. In addition, a comparison was made of the activity of some triphenylethylene derivatives against CaM dependent cAMP-PDE and the ER-CaM Sepharose assays, but no relationship was observed. Overall, these results demonstrate that inhibition of the ER-CaM association by various steroidal and triphenylethylene antiestrogens does not relate to antagonism of calmodulin function or their binding affinity for the estrogen receptor.

3- and 4-pyridylalkyl adamantanecarboxylates: inhibitors of human cytochrome P450(17 alpha) (17 alpha-hydroxylase/C17,20-lyase). Potential nonsteroidal agents for the treatment of prostatic cancer.

Various 3- and 4-pyridylalkyl 1-adamantanecarboxylates have been synthesized and tested for inhibitory activity toward the 17 alpha-hydroxylase and C17,20-lyase activities of human testicular cytochrome P450(17 alpha). The 4-pyridylalkyl esters were much more inhibitory than their 3-pyridylalkyl counterparts. The most potent was (S)-1-(4-pyridyl)ethyl 1-adamantanecarboxylate (3b; IC50 for lyase, 1.8 nM), whereas the (R)-enantiomer 3a was much less inhibitory (IC50 74 nM). Nearly as potent as 3b was the dimethylated counterpart, the 2-(4-pyridylpropan-2-yl) ester 5 (IC50 2.7 nM), which was also more resistant to degradation by esterases. In contrast to their 4-pyridyl analogs, the enantiomers of the 1-(3-pyridyl)ethyl ester were similarly inhibitory (IC50 for lyase; (R)-isomer 8a 150 nM, (S)-isomer 8b 230 nM). Amides corresponding to the 4-pyridylmethyl ester 1 and the (S)-1-(4-pyridyl)ethyl ester 3b, respectively 11 and 15b, were much less inhibitory than their ester counterparts. On the basis of a combination of inhibitory potency and resistance to esterases, the ester 5 was the best candidate for further development as a potential nonsteroidal inhibitor of cytochrome P450(17 alpha) for the treatment of prostate cancer.

Identification and characterization of limonene metabolites in patients with advanced cancer by liquid chromatography/mass spectrometry.

Limonene is a farnesyl transferase inhibitor that has shown antitumor properties. The drug had been given orally to cancer patients. Plasma and urine samples collected from the patients were examined by reversed-phase HPLC-atmospheric pressure chemical ionization and electrospray ionization MS. The drug underwent rapid conversion to hydroxylated and carboxylated derivatives. Characterization and structural elucidation of the metabolites were achieved by LC/MS and NMR. Five major metabolites were detected in the plasma extracts, namely limonene-1,2-diol, limonene-8,9-diol, perillic acid, an isomer of perillic acid, and dihydroperillic acid. Urinary metabolites comprised the glucuronides of the two isomers of perillic acid, dihydroperillic acid, limonene-8,9-diol, and a monohydroxylated limonene.

Homologs of idoxifene: variation of estrogen receptor binding and calmodulin antagonism with chain length.

A series of homologs of idoxifene [1a, (E)-1-[4-(N-pyrrolidinoethoxy)phenyl]-1-(4-iodophenyl)-2-phenyl-1-butene ] and selected homologs of 4-iodotamoxifen [2a,(E)-1-[4-(N-dimethylamino)-ethoxy]phenyl]-1-(4-iodophenyl)-2-phenyl -1-butene] with the side chain (CH(2))(n) varying in length from n=3 (1b,2b) to n=10(1i,2i) have been synthesized and tested for antagonism of of the calmodulin-dependent activity of cAMP phosphodiesterase and for binding affinity to rat uterine estrogen receptor. Compared with 1a (IC(50) =1.5 microM), the homologs showed a progressive increase in calmodulin antagonism with a maximum inhibition at n=7-9 (1f-h)(IC(50)=0.2 microM), declining at n=10 (1i) to IC(50) =1.6 microM. In the pyrrolidino series, estrogen receptor binding affinity peaked at n=3 (1b, RBA= 23; estradiol = 100), declining by n=10 (1i) to RBA = 0.4, but the homolog n=8 (1g, RBA = 3.5) was still comparable to tamoxifen (RBA = 3.9). A similar pattern of activity was seen for the dimethylamino counterparts. These compounds represent a new class of antiestrogens with potent calmodulin antagonism.

Esters of 3-pyridylacetic acid that combine potent inhibition of 17 alpha-hydroxylase/C17,20-lyase (cytochrome P45017 alpha) with resistance to esterase hydrolysis.

Esters of 3- and 4-pyridylacetic acid have been prepared and tested for inhibitory activity toward the human testicular 17 alpha-hydroxylase/C17,20-lyase and human placental aromatase enzymes. The structural features required for optimal inhibition of the hydroxylase/lyase enzyme were a 3-pyridine ring, methyl substitution alpha to the carbonyl group, and a bulky alkoxycarbonyl substituent. The compounds with the greatest selectivity were isopinocampheyl 2-methyl-2-(3-pyridyl)propanoate, 9, 1-adamantyl 2-methyl-2-(3-pyridyl)propanoate, 12, and 2-methyl-2-adamantyl 2-methyl-2-(3-pyridyl)propanoate, 14, which, while inhibiting the aromatase activity with IC50 values of 30, 35, and 40 microM, respectively, exhibited IC50 values toward hydroxylase/lyase of between 13 and 90 nM. For comparison, ketoconazole gave an IC50 value of 15 microM against aromatase and values of 65 and 26 nM for inhibition of the hydroxylase and lyase activities, respectively. Some of the structural features required for enzyme inhibition also conferred resistance to esterase hydrolysis, in vitro using rat liver microsomes as a source of the esterase activity. Therefore these esters are lead compounds in the development of inhibitors of androgen biosynthesis for the treatment of hormone-dependent prostatic cancer.

Inhibition of aromatase expression by a psoralen-linked triplex-forming oligonucleotide targeted to a coding sequence.

The cytochrome P450 enzyme aromatase (P450arom) is an important target in breast cancer treatment. We have designed a 20-base pyrimidine oligodeoxynucleotide (ODN) which forms a sequence-specific triple helix (triplex) with a purine-rich tract in the P450arom coding sequence. The psoralen-linked ODN (Pso20T) formed photo-induced cross-linked products with target double-stranded DNA. Cross-linked adducts formed in vitro between ODNs and P450arom expression constructs were used to transfect COS and human MCF-7 breast cancer cells. Levels of aromatase transcripts and enzyme activity were significantly lower in cultures transfected with Pso20T-treated cDNA relative to controls. Pso20T had a lesser inhibitory effect on aromatase expression from a mutant P450arom construct, consistent with predicted effects of the mutations on triplex formation. These results are compatible with triplex-mediated interruption of transcription within intact cells.

Comparison between inhibition of protein kinase C and antagonism of calmodulin by tamoxifen analogues.

A variety of analogues of tamoxifen were tested for inhibition of protein kinase C (PKC) activity in MCF-7 breast cancer cells. These results were compared with the calmodulin antagonism exhibited by the analogues as measured by inhibition of calmodulin-dependent cyclic AMP phosphodiesterase. The same structural features that enhanced PKC inhibition also led to an increase in calmodulin antagonism, namely 4-iodination and elongation of the basic side-chain. The most potent analogue has a 4-iodine substituent and eight carbon atoms in its basic side-chain with IC50 values of 38 microM for PKC inhibition and 0.3 microM for calmodulin antagonism, which compares with 92 and 6.8 microM, respectively, for tamoxifen. Some selectivity was achieved with a ring-fused analogue that retained the potency of tamoxifen as a PKC inhibitor, but lacked calmodulin antagonism.

Novel steroidal inhibitors of human cytochrome P45017 alpha (17 alpha-hydroxylase-C17,20-lyase): potential agents for the treatment of prostatic cancer.

Steroidal compounds having a 17-(3-pyridyl) substituent together with a 16,17-double bond have been synthesized, using a palladium-catalyzed cross-coupling reaction of a 17-enol triflate with diethyl(3-pyridyl)borane, which are potent inhibitors of human testicular 17 alpha-hydroxylase-C17,20-lyase. The requirement for these structural features is stringent: compounds having 2-pyridyl (9), 4-pyridyl (10), or 2-pyridylmethyl (11) substituents instead of the 3-pyridyl substituent were either poor inhibitors or noninhibitory. Reduction of the 16,17-double bond to give 17 beta-pyridyl derivatives diminished potency with 3-pyridyl substitution (3-->27; IC50 for lyase, 2.9-->23 nM) but increased it with a 4-pyridyl substituent present (10-->28; IC50 1 microM-->53 nM). In contrast, a variety of substitution patterns in rings A-C of the steroid skeleton afforded inhibitors having potencies similar to those most closely related structurally to the natural substrates pregnenolone and progesterone, respectively 17-(3-pyridyl)androsta-5,16-dien-3 beta-ol (3, Kiapp < 1 nM; IC50 for lyase, 2.9 nM) and 17-(3-pyridyl)androsta-4,16-dien-3-one (15; IC50 for lyase, 2.1 nM). Thus compounds having variously aromatic ring A (18), saturated rings A/B (21, 22), and oxygenated ring C (26) exhibited IC50 values for lyase (1.8-3.0 nM) falling within a 2-fold range. The most potent compounds are candidates for development as drugs for the treatment of hormone-dependent prostatic carcinoma.

The deuterium isotope effect for the alpha-hydroxylation of tamoxifen by rat liver microsomes accounts for the reduced genotoxicity of [D5-ethyl]tamoxifen.

This study describes the application of on line HPLC-electrospray ionization MS in the structural determination of the metabolites formed following incubation with rat liver microsomes of an equimolar mixture of the anticancer drug tamoxifen and its [D5-ethyl]-analogue. The ratio of ca 3:1 between unlabelled and D4-labelled alpha-hydroxytamoxifen, indicating a large isotope effect for this metabolic process, accounted for the previously observed lower yield of DNA adducts formed in the livers of rats treated with D5-tamoxifen compared with unlabelled drug. The loss of one deuterium atom on metabolism discriminated hydroxyethylated metabolites from others and enabled two further such metabolites to be detected, namely alpha-hydroxytamoxifen N-oxide and alpha-hydroxy-N-desmethyltamoxifen of which the latter is novel. Furthermore, the use of [alpha-D2-ethyl]- and [beta-D3-ethyl] tamoxifens discriminated alpha- from beta-hydroxylated metabolites and proved that the metabolites described here were alpha-hydroxylated. In contrast to the alpha-hydroxylated metabolites, the other metabolites identified, namely tamoxifen N-oxide, N-desmethyltamoxifen, 4-hydroxytamoxifen and their deuterated counterparts were not depleted in the deuterated components. The use of on line HPLC-electrospray ionization MS combined with isotopic labelling is a powerful technique for probing the structures of metabolites, and, applied to tamoxifen, has provided further evidence that alpha-hydroxylation is an important pathway for the conversion of the drug into a DNA-reactive metabolite.

Rationally designed analogues of tamoxifen with improved calmodulin antagonism.

Computerized molecular modeling studies on the interactions of the antiestrogen tamoxifen (1) and its analogues bound to the calcium-binding protein calmodulin have guided the rational design of more potent antagonists. Compounds with either three or four methylene units in the basic side chain or slim lipophilic 4-substituents were expected to be more potent. All compounds were tested for antagonism of the calmodulin-dependent activity of cAMP phosphodiesterase and for binding affinity to the estrogen receptor from rat uteri. Some compounds were assayed for cytotoxicity against MCF-7 breast tumor cells in vitro. Introduction of lipophilic 4-substituents was accomplished by using palladium(0)-catalyzed coupling reactions with a 4-iodinated precursor. Both the 4-ethynyl (16 and 17) and 4-butyl (18 and 19) compounds were more potent calmodulin antagonists than tamoxifen. Extension of the basic aminoethoxy side chain of 4-iodotamoxifen (3) and idoxifene (2) ((E)-1-[4-[2-(N-pyrrolidino)ethoxy]phenyl]-1-(4-iodophenyl)-2-phen yl-1- butene) by one or two methylene units resulted in modest gains in calmodulin antagonism (10-13). All the compounds assayed retained estrogen receptor binding characteristics. The compound possessing the optimal combination of calmodulin antagonism and estrogen receptor binding was 12 ((E)-1-[4-[3-(N-pyrrolidino)propoxy]phenyl]-1-(4-iodophenyl)-2-phe nyl-1 - butene) (IC50 = 1.1 microM, RBA = 23). Correlation between calmodulin antagonism and cytotoxicity was demonstrated for selected compounds.

Evidence that tamoxifen binds to calmodulin in a conformation different to that when binding to estrogen receptors, through structure-activity study on ring-fused analogues.

A ring-fused analogue of tamoxifen, which had previously been shown to have practically identical estrogen receptor (ER) affinity and antitumour potency against estrogen responsive cells as tamoxifen, failed to inhibit calmodulin-dependent cyclic AMP phosphodiesterase. The substitution of an extra methyl group into the ring-fused analogue, at a position which the ethyl group of tamoxifen can occupy in one of its conformations, restored the calmodulin inhibition. Also, the replacement of the tamoxifen ethyl group by methyl diminishes calmodulin inhibition. Direct interaction of these tamoxifen analogues with calmodulin was demonstrated through the use of the fluorescent probe, 2-p-toluidinyl-naphthalene-6-sulfonic acid (TNS). These findings lead to the conclusion that tamoxifen binds to calmodulin in a conformation not accessible to the fused analogue and therefore likely to be different to that which binds to the ER. Also, the results on the ring-fused analogues indicate that the calmodulin binding cannot be essential for antitumour activity.

Effects of a new antioestrogen, idoxifene, on cisplatin- and doxorubicin-sensitive and -resistant human ovarian carcinoma cell lines.

Pyrrolidino-4-iodotamoxifen (idoxifene) is a new non-steroidal antioestrogen currently undergoing phase I clinical evaluation. Using idoxifene and tamoxifen and two additional analogues of tamoxifen (3-hydroxytamoxifen and 4-iodotamoxifen) and the imidazole-based calmodulin inhibitor, calmidazolium, a strong positive correlation (r2 > 0.95) was observed between cytotoxicity and inhibition of calmodulin-dependent cyclic AMP phosphodiesterase (e.g. mean IC50 across four human ovarian carcinoma cell lines of 4.5 microM for idoxifene and 6.3 microM for tamoxifen). Using two parent human ovarian carcinoma cell lines (41M and CH1; both oestrogen receptor negative) in which acquired resistance to doxorubicin or cisplatin has been generated, we have determined the ability of idoxifene to overcome resistance in these lines. At a non-toxic concentration of 2 microM, idoxifene appeared at least as effective as the clinically used multidrug resistance modifiers verapamil and tamoxifen in overcoming doxorubicin resistance in two acquired resistant cell lines shown to overexpress the P-170 efflux glycoprotein. Non-cross-resistance between cisplatin and idoxifene was observed in two acquired resistant cell lines possessing contrasting mechanisms of resistance to cisplatin (41McisR6 reduced drug transport and CH1cisR6 resistance mediated at the level of DNA). In one of four cell lines (CH1), synergism between idoxifene and cisplatin was observed by median effect analysis. However, with the 41M and its 6-fold cisplatin-resistant variant, antagonism was observed. These observations made by median effect analysis appeared to be unrelated to platinum uptake or removal of platinum-induced DNA interstrand cross-links. These in vitro data suggest that idoxifene may be usefully combined with doxorubicin in the clinical setting, but caution should be exercised in combining it with cisplatin in the treatment of certain tumours.

Lack of inhibition of placental estrone sulfatase and aromatase enzymes by vitamin D3 and its analogs.

The aromatase and estrone sulfatase enzymes are important sources of biologically active estrogens in postmenopausal women with breast cancer. Promising initial results in the treatment of endocrine-responsive breast cancer have been exhibited by 1 alpha 25-dihydroxyvitamin D3 and the synthetic vitamin D analogues MC903 and EB1089. However, these compounds together with vitamin D3 and vitamin D3 sulfate did not inhibit the human placental aromatase enzyme when assayed up to 20 microns. Only vitamin D3 sulfate and 1 alpha 25-dihydroxyvitamin D inhibited the estrone sulfatase activity in human placental microsomes, albeit at high concentration (32 and 37% inhibition, respectively with 50 microns each inhibitor). It is unlikely that inhibition of aromatase or estrone sulfatase enzymes contribute to the inhibitory effect of this group of compounds on breast cancer cells in vivo.

Intratumoral oestrone sulphatase activity as a prognostic marker in human breast carcinoma.

Oestrone sulphatase is an important source of local synthesis of biologically active oestrogens in human breast cancer. The oestrone sulphatase enzyme in the particulate fraction of human breast carcinoma was characterised. The Km was 8.91 microM, and the Vmax was 0.022 nmol min-1 mg-1. Oestrone sulphatase activity was detected in 93 of 104 human breast carcinoma samples (89%), and mean activity was 0.041 nmol min-1 mg-1 (range 0-0.399 nmol min-1 mg-1). There was no significant correlation between intratumoral oestrone sulphatase activity and oestrogen receptor status, or with any other prognostic factors. Intratumoral enzyme levels were not associated with time to recurrence or with overall survival time. It thus appears that, although a useful source of intratumoral oestrogens, oestrone sulphatase activity is not of prognostic significance in breast carcinoma.

Evaluation of some 4-fluoro- and 4-cyano derivatives of delta 4,3-ketosteroids as inhibitors of testosterone 5 alpha-reductase.

Some 4-fluorinated analogues of 3-oxo-delta 4 steroids and 4-cyano derivatives of progesterone and androstenedione were evaluated as inhibitors of steroid 5 alpha-reductase activity. Inhibitors of this enzyme may be useful in treating prostatic cancer. 4-Fluoroandrostenedione was a modest inhibitor of the rat enzyme (IC50 = 4.08 microM), while 4-cyanoprogesterone was a potent inhibitor of both the rat and human enzymes (IC50 values = 0.045 microM and 0.050 microM respectively). These two steroids were tested in vivo for activity against androgen sensitive organs in WHT mice. 4-Fluoroandrostenedione caused increases in organ weights, suggesting it is an androgen agonist, while the 4-cyano compound displayed modest androgen ablation. Therefore substitutions at the 4-position may produce compounds of therapeutic use in treating prostate cancer.