(1-Benzylindole-3-yl)alkanoic acids; novel nonsteroidal inhibitors of steroid 5 alpha-reductase (I)

A novel series of indole-3-alkanoic acids with varied N-benzyl substituents were synthesized as nonsteroidal inhibitors of steroid 5 alpha-reductase. The structure-activity relationships in this series were studied and the optimum carboxylic acid side chain was butyric acid. Furthermore, compounds with a diaryl substituent at the 1-position of the indole ring displayed strong inhibitory activities in vitro. Amongst these derivatives, 4-[1-(6,6-dimethyl-6H-dibenzo[b,d]pyran-3-yl)methylindol-3-yl]b uty ric acid (FR119680) displayed very high inhibitory activity in vitro against rat prostatic 5 alpha-reductase (IC50 = 5.0 nM) and good in vivo activity in the castrated young rat model.     

Potent inhibitors of acyl-CoA:cholesterol acyltransferase. 2. Structure-activity relationships of novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amides

Novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives 1 were synthesized and tested for their ability to inhibit rabbit small intestinal ACAT (acyl-CoA:cholesterol acyltransferase) and lower serum total cholesterol in cholesterol-fed rats. Among the synthesized compounds, N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives showed potent ACAT inhibitory activity. The synthesis and structure-activity relationships of these compounds are described. A methyl group at position 6 of the 2,3-dihydrobenzofuran moiety was important for potent ACAT inhibitory activity. In the series of N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl) amides, lipophilicity of the acyl moiety was necessary for the potent ACAT inhibitory activity. The highly lipophilic acid amides N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)-2,2- dimethyldodecanamide (10) and 6-(4-chlorophenoxy)-N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-y l)-2,2-dimethyloctanamide (50) showed potent activity. Introduction of a dimethylamino group at position 5 of the 2,3-dihydrobenzofuran moiety resulted in highly potent activity. The most potent compound, N-[5-(dimethylamino)-2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl ]-2,2-dimethyldodecanamide (13, TEI-6620), showed highly potent ACAT inhibitory activity (rabbit small intestine IC50 = 0.020 microM, rabbit liver IC50 = 0.009 microM), foam cell formation inhibitory activity (rat peritoneal macrophage IC50 = 0.030 microM), extremely potent serum cholesterol-lowering activity in cholesterol-fed rats (71% at a dose of 0.3 mg/kg/day po), and good bioavailability in fed dogs (Cmax = 2.68 microg/mL at 1 h, 10 mg/kg po).     

PNU 157706, a novel dual type I and II 5alpha-reductase inhibitor

PNU 157706 is a novel dual inhibitor of 5alpha-reductase (5alpha-R), the enzyme responsible for the conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT). Tested on a crude preparation of human or rat prostatic 5alpha-R, PNU 157706 caused enzyme inhibition with IC50 values of 20 and 34 nM, respectively, compared to the values of 32 and 58 nM shown by finasteride. Furthermore, PNU 157706 was highly potent in inhibiting human recombinant 5alpha-R type I and II isozymes, showing IC50 values of 3.9 and 1.8 nM and, therefore, it was several folds more potent than finasteride (IC50 values of 313 and 11.3 nM), particularly on the type I isozyme. PNU 157706 was shown to have no binding affinity for the rat prostate androgen receptor (RBA 0.009% that of DHT). In adult male rats, a single oral dose of 10 mg/kg of PNU 157706 caused a marked and longer lasting reduction of prostatic DHT than did finasteride (at 24 h inhibition by 89 and 47%, respectively). In prepubertal, T- or DHT-implanted castrated rats, PNU 157706, given orally for 7 days at the dose of 10 mg/kg/day, markedly reduced ventral prostate weight in T- but not in DHT-implanted animals, thus showing to be devoid of any anti-androgen activity. In adult rats treated orally for 28 days, PNU 157706 resulted markedly more potent (16-fold) than finasteride in reducing prostate weight, the ED50 values being 0.12 and 1.9 mg/kg/day, respectively. These results indicate that PNU 157706 is a promising, potent inhibitor of both type II and I human 5alpha-R with a very marked antiprostatic effect in the rat.     

Endocrine properties of the testosterone 5 alpha-reductase inhibitor turosteride (FCE 26073)

Turosteride was tested in a series of studies for its effect on 5 alpha-reductase and for its possible influence on other steroidogenic enzymes and on steroid receptors. The compound was found to inhibit human and rat prostatic 5 alpha-reductases with IC50 values of 55 and 53 nM, respectively, whereas it caused a less marked inhibition of the dog enzyme (IC50 2.2 microM). Turosteride showed no relevant effect on rat adrenal C20,22-desmolase (IC50 254 microM) and human placental aromatase (IC50 > 100 microM), and only at relatively high concentrations it caused inhibition of human placental 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) (IC50 2.5 microM). Turosteride was found to be a selective 5 alpha-reductase inhibitor showing no noteworthy binding to receptors for androgens (relative binding affinity, RBA, 0.004%), estrogens (< or = 0.005%), progesterone (< 0.005%), glucocorticoids (< 0.01%) and mineralocorticoids (< 0.03%). Its biochemical profile was similar to that of finasteride, whereas 4-MA (17 beta-N,N-diethyl-carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) was confirmed to be a non-selective 5 alpha-reductase inhibitor, showing a degree of binding affinity to the androgen receptor (RBA 0.1%) and a marked inhibition of 3 beta-HSD-I (IC50 32 nM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, turosteride reduced the ventral prostate and seminal vesicle growth promoting effect of TP, with IC50 values of approximately 5 and 6.7 mg/kg/day, whereas levator ani weight was unchanged. In comparison, 4-MA was approx. 3-fold less potent than turosteride in reducing the prostate and seminal vesicle weights and caused a marked reduction of levator ani weight, thus showing its unselectivity.     

Studies on 5-lipoxygenase inhibitors. II. Discovery, optical resolution and enantioselective synthesis of FR110302, a highly potent non-redox type 5-lipoxygenase inhibitor

A novel series of 2,2-dialkyl-5-(2-quinolylmethoxy)-1,2,3, 4-tetrahydro-1-naphthols was synthesized and evaluated as 5-lipoxygenase (5-LO) inhibitors. Systematic optimization led to identification of several highly potent non-redox type 5-LO inhibitors with nanomolar IC50s as racemic mixtures. Optical resolution of racemate 50 indicated that its 5-LO inhibitory activity was enantiospecific and due to the (+)-enantiomer. An efficient synthetic route to the (+)-enantiomers via asymmetric reduction of tetralone intermediates was established. The best compound, (+)-2,2-dibutyl-5-(2-quinolylmethoxy)-1,2,3,4-tetrahydro-1-naphtho l (FR110302, (+)-50), showed potent inhibitory activity against leukotriene (LT) biosynthesis by intact neutrophiles in rats (IC50 4.9 nM) and in humans (IC50 40 nM). Furthermore oral administration of FR110302 significantly inhibited neutrophil migration in the rat air pouch model at 1 mg/kg.     

Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase

Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole).     

19-nor-10-azasteroids: a novel class of inhibitors for human steroid 5alpha-reductases 1 and 2

Steroid 5alpha-reductase is a system of two isozymes (5alphaR-1 and 5alphaR-2) which catalyzes the NADPH-dependent reduction of testosterone to dihydrotestosterone in many androgen sensitive tissues and which is related to several human endocrine diseases such as benign prostatic hyperplasia (BPH), prostatic cancer, acne, alopecia, pattern baldness in men and hirsutism in women. The discovery of new potent and selective 5alphaR inhibitors is thus of great interest for pharmaceutical treatment of these diseases. The synthesis of a novel class of inhibitors for human 5alphaR-1 and 5alphaR-2, having the 19-nor-10-azasteroid skeleton, is described. The inhibitory potency of the 19-nor-10-azasteroids was determined in homogenates of human hypertrophic prostates toward 5alphaR-2 and in DU-145 human prostatic adenocarcinoma cells toward 5alphaR-1, in comparison with finasteride (IC50 = 3 nM for 5alphaR-2 and approximately 42 nM for 5alphaR-1), a drug which is currently used for BPH treatment. The inhibition potency was dependent on the type of substituent at position 17 and on the presence and position of the unsaturation in the A and C rings. delta9(11)-19-Nor-10-azaandrost-4-ene-3,17-dione (or 10-azaestra-4,9(11)-diene-3,17-dione) (4a) and 19-nor-10-azaandrost-4-ene-3,17-dione (5) were weak inhibitors of 5alphaR-2 (IC50 = 4.6 and 4.4 microM, respectively) but more potent inhibitors of 5alphaR-1 (IC50 = 263 and 299 nM, respectively), whereas 19-nor-10-aza-5alpha-androstane-3,17-dione (7) was inactive for both the isoenzymes. The best result was achieved with the 9:1 mixture of delta9(11)- and delta8(9)-17beta-(N-tert-butylcarbamoyl)-19-nor-10-aza-4- androsten-3-one (10a,b) which was a good inhibitor of 5alphaR-1 and 5alphaR-2 (IC50 = 127 and 122 nM, respectively), with a potency very close to that of finasteride. The results of ab initio calculations suggest that the inhibition potency of 19-nor-10-azasteroids could be directly related to the nucleophilicity of the carbonyl group in the 3-position.     

Structure-activity relationships for 1-phenylbenzimidazoles as selective ATP site inhibitors of the platelet-derived growth factor receptor

1-Phenylbenzimidazoles are shown to be a new class of ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR). Structure-activity relationships (SARs) are narrow, with closely related heterocycles being inactive. A systematic study of substituted 1-phenylbenzimidazoles showed clear SARs. Substituents at the 4'- and 3'-positions of the phenyl ring are tolerated but do not significantly improve activity, while substituents at the 2'-position abolish it. Substituents in the 2-, 4-, and 7-positions of the benzimidazole ring (with the exception of 4-OH) also abolish activity. Most substituents at the 5- and 6-positions maintain or increase activity, with the 5-OH, 5-OMe, 5-COMe, and 5-CO2Me analogues being >10-fold more potent than the parent 1-phenylbenzimidazole. The 5-OMe analogue was both the most potent inhibitor, and showed the highest selectivity (50-fold) between PDGFR and FGFR isolated enzymes, and also a moderately effective inhibitor (IC50 = 1.9 microM) of PDGF-stimulated PDGFR autophosphorylation in rat aorta smooth muscle cells.     

Lineage commitment of HLA-DR/CD38-defined progenitor cell subpopulations in bone marrow and mobilized peripheral blood assessed by four-color immunofluorescence

ONO-9302 [epristeride; (-)-17beta-(tert-butylcarbamoyl)androsta-3,5-diene-3-carboxy lic acid] is a novel inhibitor of steroid 5alpha-reductase. We studied in vitro and in vivo effects of ONO-9302 on the rat prostatic tissue in comparison with those of the anti-androgen allylestrenol. ONO-9302 inhibited the rat prostatic enzyme with an IC50 value of 11 nM, whereas allylestrenol was about 80,000-fold less potent. The growth of ventral prostate, which was induced by the subcutaneous injection of testosterone propionate in the castrated rats, was significantly reduced by ONO-9302 at oral doses of 1-100 mg/kg/day. Allylestrenol showed a significant effect only at a dose of 100 mg/kg/day. In mature male rats, ONO-9302 significantly reduced the ventral prostate weight at doses of 10-100 mg/kg/day and decreased prostatic 5alpha-dihydrotestosterone (DHT) content associated with a rise in testosterone (T) content at doses of 0.1-100 mg/kg/day. Plasma hormone levels (i.e., T, DHT, luteinizing hormone (LH) and follicle stimulating hormone (FSH)) were not altered significantly. Allylestrenol significantly reduced the ventral prostate weight at doses of 10-100 mg/kg/day. However, unlike ONO-9302, allylestrenol reduced both the prostatic DHT and T contents and also lowered plasma T, DHT, LH and FSH levels at a dose of 30 mg/kg/day. These results suggest that ONO-9302 reduces the prostatic growth by inhibiting the conversion of T to DHT in the prostate without lowering blood T level unlike anti-androgen drugs.     

The effect of lead selection on traditional and heart rate-adjusted ST segment analysis in the detection of coronary artery disease during exercise testing

1. The present studies were designed to measure the affinity of UP 269-6, a newly developed angiotensin AT1 receptor antagonist, for vascular AT1 receptors from normotensive and hypertensive rats and to investigate in vitro, its effects on angiotensin II (AII)-induced hyperplasia and hypertrophy of vascular smooth muscle cells (VSMC). In addition the in vivo effects of UP 269-6 on neointimal proliferation in a carotid artery balloon injury in normotensive rats were also investigated. 2. UP 269-6 selectively inhibited [125I]-Sar1-Ile8-AII binding to vascular AT1 receptors present on VSMC derived from normotensive Wistar rat and from SHR (Ki = 16.6 +/- 3.6 nM and 7.5 +/- 2.0 nM, respectively). In comparison, losartan and its metabolite, EXP 3174, inhibited [125I]-Sar1-Ile8-AII binding to vascular AT1 receptors derived from both cell models with Ki values slightly lower (losartan) and higher (EXP 3174), respectively, than that of UP 269-6. 3. AII (1 microM) induced a weak and variable hyperplastic response (4 to 32% increase in cell number) in Wistar rat VSMC after 96 h. 4. AII (1 microM) induced a time-dependent increase in cell number in VSMC from SHR. UP 269-6 inhibited concentration-dependently this effect with an IC50 value of 159 +/- 58 nM. Losartan was clearly less potent and EXP 3174 showed nearly the same inhibitory potency, compared to UP 269-6. UP 269-6 (1 microM) inhibited nearly completely the action of AII. 5. AII (500 nM) caused maximal stimulation of protein synthesis in Wistar rat VSMC (117 +/- 36%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 28 +/- 6 nM, 3504 +/- 892 nM and 21 +/- 3 nM, respectively. 6. AII (50 nM) induced maximal stimulation of protein synthesis in SHR VSMC (237 +/- 67%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 16 +/- 3 nM, 282 +/- 122 nM and 3.3 +/- 1.0 nM, respectively. 7. UP 269-6 (75 mg kg-1 day-1) administered orally in the diet for 20 days induced a 38% reduction in neointimal area and a 36% reduction in neointima/media ratio associated with the intimal thickening induced by carotid artery balloon injury. 8. In conclusion, UP 269-6 was shown to be a potent antiproliferative agent both in vitro on AII-induced hyperplasia and hypertrophy of VSMC derived from normotensive and hypertensive rats, and in vivo upon intimal thickening induced by carotid artery balloon injury in the rat.     

Axially chiral N-benzyl-N,7-dimethyl-5-phenyl-1, 7-naphthyridine-6-carboxamide derivatives as tachykinin NK1 receptor antagonists: determination of the absolute stereochemical requirements

A potent and orally active NK1 antagonist, trans-N-[3, 5-bis(trifluoromethyl)benzyl]-7,8-dihydro-N, 7-dimethyl-5-(4-methylphenyl)-8-oxo-1,7-naphthyridine-6-carboxamide (1t), was shown to exist as a mixture of separable and stable (R)- and (S)-atropisomers (1t-A and 1t-B) originating from the restricted rotation around the -C(6)-C(=O)- bond; the antagonistic activities of 1t-A were ca. 6-13-fold higher than those of 1t-B. Analogues of 1t (3), which have (S)- and (R)-methyl groups at the benzylic methylene portion of 1t, were prepared and separated into the diastereomeric atropisomers, 3a-A, 3a-B and 3b-A, 3b-B, in enantiomerically pure forms. Among the four isomers of 3, the (aR, S)-enantiomer (3a-A) exhibited the most potent antagonistic activities with an IC50 value of 0.80 nM (in vitro inhibition of [125I]BH-SP binding in human IM-9 cells) and ED50 values of 9.3 micrograms/kg (iv) and 67.7 micrograms/kg (po) (in vivo inhibition of capsaicin-induced plasma extravasation in guinea pig trachea), while the activity of the (aS,R)-enantiomer (3b-B) was the weakest with an IC50 value of 620 nM. The structure-activity relationships in this series of antagonists indicate that the (R)-configuration at the axial bond and the stacking (or stacking-like) conformation between the two phenyl rings as shown in 1t-A and 3a-A are essential for high-affinity binding and suggest that the amide moiety functions as a hydrogen bond acceptor in the interaction with the receptor.     

Selective peptidic and peptidomimetic inhibitors of Candida albicans myristoylCoA: protein N-myristoyltransferase: a new approach to antifungal therapy

MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins. Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.     

Effect of a coffee lipid (cafestol) on cholesterol metabolism in human skin fibroblasts

The receptor (R) for epidermal growth factor (EGF) is expressed at high levels on human breast cancer cells and associates with ErbB2, ErbB3, and Src proto-oncogene family protein tyrosine kinases (PTKs) to form membrane-associated PTK complexes with pivotal signaling functions. Recombinant human EGF was conjugated to the soybean-derived PTK inhibitor genistein (Gen) to construct an EGF-R-directed cytotoxic agent with PTK inhibitory activity. The EGF-Gen conjugate was capable of binding to and entering EGF-R-positive MDA-MB-231 and BT-20 breast cancer cells (but not EGF-R-negative NALM-6 or HL-60 leukemia cells) via its EGF moiety, and it effectively competed with unconjugated EGF for target EGF-R molecules in ligand binding assays. EGF-Gen inhibited the EGF-R tyrosine kinase in breast cancer cells at nanomolar concentrations, whereas the IC50 for unconjugated Gen was >10 microM. Notably, EGF-Gen triggered a rapid apoptotic cell death in MDA-MB-231 as well as BT-20 breast cancer cells at nanomolar concentrations. The EGF-Gen-induced apoptosis was EGF-R-specific because cells treated with the control granulocyte-colony stimulating factor-Gen conjugate did not become apoptotic. Apoptosis was dependent both on the PTK inhibitory function of Gen and the targeting function of EGF, because cells treated with unconjugated Gen plus unconjugated EGF did not undergo apoptosis. The IC50s of EGF-Gen versus unconjugated Gen against MDA-MB-231 and BT-20 cells in clonogenic assays were 30 +/- 3 nM versus 120 +/- 18 microM (P < 0.001) and 30 +/- 10 nM versus 112 +/- 17 microM (P < 0.001), respectively. Thus, the EGF-Gen conjugate is a >100-fold more potent inhibitor of EGF-R tyrosine kinase activity in intact breast cancer cells than unconjugated Gen and a >100-fold more potent cytotoxic agent against EGF-R+ human breast cancer cells than unconjugated Gen. Taken together, these results indicate that the EGF-R-associated PTK complexes have vital antiapoptotic functions in human breast cancer cells and may therefore be used as therapeutic targets.     

Potent growth inhibitory activity of zidovudine on cultured human breast cancer cells and rat mammary tumors

Originally designed as an antitumor agent, zidovudine (AZT) has exhibited only marginal tumor growth inhibitory activity. Recently, three abstracts have described positive clinical outcomes for a small number of patients with advanced breast cancer treated with weekly infusions of either methotrexate or cisplatin and AZT. Consequently, we conducted a preclinical study of the anti-breast cancer and anti-mammary tumor activity of AZT. Here we have demonstrated that AZT, alone, has a preferential in vitro and in vivo effect on breast and mammary cancer cells. It is 1000 times as potent as an inhibitor of the in vitro growth of the human breast cancer cell line MCF-7 (IC50 = 10 +/- 5 nM) than of the growth of the T-cell leukemia cell line CEM (IC50 = 14 +/- 2 microM). A novel mechanism for this preferential effect on growth is indicated by the 3-4-fold increase in production of phosphorylated AZT (mono-, di-, and triphosphate) in MCF-7 relative to CEM. We extended these in vitro observations to in vivo studies in rats and found that AZT is a potent in vivo inhibitor of the growth of methylnitrosourea-induced rat mammary tumors without any apparent toxic effects on internal organs. These preclinical results demonstrate, for the first time, that AZT has significant anti-breast cancer activity and strongly suggest that the clinical usefulness of this drug is worthy of investigation.     

Fluorescence detection of calcium-binding proteins with quinoline Ca-indicator quin2

Positively charged cyclic peptides (three to seven amino acids) have been tested for their inhibitory effects on Na+/Ca2+ exchange in the cardiac sarcolemma vesicles. The lead structure of Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) has been systematically modified for identification of important pharmacophores. In cyclic peptides (intramolecular S-S bond, the carboxyl terminal is locked with amide (CONH2), and positive charge is retained by one or two arginines, ornithines, or lysines. Thirty-five different cyclic peptides show IC50 values in the range of 2-800 microM, suggesting that some specific structure-activity relationships may determine the inhibitory effects. Shortening of the FRCRCFa length to four amino acids decreases the inhibitory potency by 10-80-fold. The substitution of Arg2 or Arg4 in FRCRCFa with lysine or ornithine decreases the inhibitory potency by 5-12-fold, suggesting that both arginines are beneficial for inhibition. The substitution of Phe1 in FRCRCFa by 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid produces a potent inhibitor (IC50 = 2-4 microM). The N-myristoylated FRCRCFa exhibits an inhibitory potency (IC50 = 8-10 microM) similar to that of the parent FRCRCFa peptide, thereby arousing a new possibility for the development of a cell-permeable blocker of the Na+/Ca2+ exchanger, D-Arg4 or D-Cys5 substitutions in FRCRCFa do not alter the inhibitory effect, whereas the L-to-D substitutions of other amino acids in FRCRCFa reduce the inhibitory potency by 4-5-fold. Thus, the L-to-D substitutions of Arg4 and/or Cys5 have a potential to increase the peptide stability to proteolytic degradation. The insertion of proline outside of the ring of FRCRCFa diminishes the inhibitory potency by 3-6-fold, whereas proline introduction into the ring decreases the inhibitory potency by 16-20-fold. The replacement of Cys3 and Cys5 in FRCRCFa with beta, beta-dimethylcystein has no significant effect on the inhibitory potency, suggesting that the S-S bond is not exposed to the interface of the peptide/receptor interaction. In conclusion, the current data support a proposal that the conformationally constrained Arg-Cys-Arg-Cys structure is obligatory for inhibition of Na+/Ca2+ exchange, whereas hydrophobic additions at the carboxyl and amino ends have limited effects in increasing the inhibitory potency.     

Pore residues critical for mu-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis

We have studied mu-conotoxin (mu-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amino acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to mu-CTX compared to wild-type rSkM1 channel (IC50 = 17.5 +/- 2.8 nM). E758C and D1241C were less sensitive to mu-CTX block (IC50 = 220 +/- 39 nM and 112 +/- 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced mu-CTX sensitivity (IC50 = 1.9 +/- 0.1, 4.9 +/- 0.9, and 5.5 +/- 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to mu-CTX block compared to WT (p < 0.05). Application of the negatively charged, sulfhydryl-reactive compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the toxin sensitivity of D1241C (IC50 = 46.3 +/- 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 +/- 21.8 nM). On the other hand, the positively charged methanethiosulfonate-ethylammonium (MTSEA) completely abolished the mu-CTX sensitivity of E758C (IC50 > 1 microM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan-to-cysteine mutants partially or fully restored the wild-type mu-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 +/- 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 +/- 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for mu-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.     

Discovery of TBC11251, a potent, long acting, orally active endothelin receptor-A selective antagonist

Previously we reported the discovery of amidothiophenesulfonamides as endothelin receptor-A antagonists with high potency and selectivity. Replacement of an amide group in this class of compounds with an acetyl group maintained the in vitro binding affinity and in vivo activity while providing a compound with oral bioavailability and longer duration of action. The optimal compound discovered during these studies, 15q (TBC11251), binds competitively to human ETA receptors with a Ki of 0.43 +/- 0.03 nM and an IC50 of 1.4 nM (IC50 for ETB = 9800 nM). This compound inhibits ET-1-induced stimulation of phosphoinositide turnover with a Ki of 0.686 nM and a pA2 of 8.0. The compound has a serum half-life in the rat and the dog of 6-7 h and 60-100% oral bioavailability. This compound is one of the most selective ETA antagonists reported and therefore is suitable for additional pharmacological and clinical investigation of the role of ETA receptors in diseases.     

Prognostic significance of tumour markers in endometrial cancer

BACKGROUND: Steroid 5 alpha-reductase is implicated in the pathogenesis of benign prostatic hyperplasia (BPH). We studied the in vitro and in vivo effects of FR146687, a new inhibitor of 5 alpha-reductase. METHODS: Two isozymes of rat and human 5 alpha-reductases were expressed in 293 cells. In vivo effects of drugs were evaluated on rat and dog prostates. Castrated immature rats were injected with testosterone propionate (TP) or 5 alpha-dihydrotestosterone propionate (DHTP) to induce growth of the ventral prostates. Testosterone and 5 alpha-dihydrotestosterone (DHT) contents in rat and dog prostates were measured by gas chromatography-mass spectrophotometry (GC-MS). RESULTS: FR146687 showed noncompetitive inhibition in both isozymes and no inhibitory effects on other steroid oxidoreductases. In mature rats and castrated immature rats treated with TP, FR146687 dose-dependently reduced ventral prostate and seminal vesicle weight at doses above 0.1 mg/kg, while castrated immature rats treated with DHTP were not affected by FR146687. FR146687 showed more potent reduction of rat prostates than finasteride. DHT concentration in the prostates was significantly reduced when FR146687 was administered to rats and beagles. CONCLUSIONS: FR146687 is a dual inhibitor for 5 alpha-reductase isozymes and significantly reduced the growth and DHT content in the prostate.