A mutant Escherichia coli tyrosyl-tRNA synthetase utilizes the unnatural amino acid azatyrosine more efficiently than tyrosine

Alloproteins, proteins that contain unnatural amino acids, have immense potential in biotechnology and medicine. Although various approaches for alloprotein production exist, there is no satisfactory method to produce large quantities of alloproteins containing unnatural amino acids in specific positions. The tyrosine analogue azatyrosine, l-beta-(5-hydroxy-2-pyridyl)-alanine, can convert the ras-transformed phenotype to normal phenotype, presumably by its incorporation into cellular proteins. This provided the stimulus for isolation of a mutant tyrosyl-tRNA synthetase (TyrRS) capable of charging azatyrosine to tRNA. A plasmid library of randomly mutated Escherichia coli tyrS (encoding TyrRS) was made by polymerase chain reaction techniques. The desired TyrRS mutants were selected by screening for in vivo azatyrosine incorporation of E. coli cells transformed with the mutant tyrS plasmids. One of the clones thus isolated, R-6-A-7, showed a 17-fold higher in vivo activity for azatyrosine incorporation than wild-type TyrRS. The mutant tyrS gene contained a single point mutation resulting in replacement of phenylalanine by serine at position 130 in the protein. Structural modeling revealed that position 130 is located close to Asp(182), which directly interacts with tyrosyladenylate. Kinetic analysis of aminoacyl-tRNA formation by the wild-type and mutated F130S TyrRS enzymes showed that the specificity for azatyrosine, measured by the ratios of k(cat)/K(m) for tyrosine and the analogue, increased from 17 to 36 as a result of the F130S mutation. Thus, the high discrimination against azatyrosine is significantly reduced in the mutant enzyme. These results suggest that utilization of F130S TyrRS for in vivo protein biosynthesis may lead to efficient production of azatyrosine-containing alloproteins.     

Inhibitory activity of cyanidin-3-rutinoside on alpha-glucosidase

Cyanidin-3-rutinoside, a natural anthocyanin, inhibited alpha-glucosidase from baker's yeast in dose-responsive manner. The IC50 value was 19.7 microM +/- 0.24 microM, compared with the IC50 value of voglibose (IC50 = 23.4 +/- 0.30 microM). Cyanidin-3-rutinoside was found to be a non-competitive inhibitor for yeast alpha-glucosidase with a Ki value in the range of 1.31-1.56 x 10(-5)M. These results indicated that cyanidin-3-rutinoside could be classed as a new alpha-glucosidase inhibitor.     

c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts display more decondensed nucleosomal organization than normal fibroblasts

We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras-transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene-transfected cells, but to a lesser extent than in the mutant ras-transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.     

Tropomyosin-2 cDNA lacking the 3' untranslated region riboregulator induces growth inhibition of v-Ki-ras-transformed fibroblasts.

The levels of high molecular weight isoforms of tropomyosin (TM) are markedly reduced in ras-transformed cells. Previous studies have demonstrated that the forced expression of tropomyosin-1 (TM-1) induces reversion of the transformed phenotype of ras-transformed fibroblasts. The effects of the related isoform TM-2 on transformation are less clear. To assess the effects of forced expression of the TM-2 protein on ras-induced tumorigenicity, we introduced a TM-2 cDNA lacking the 3' untranslated region riboregulator into ras-transformed NIH 3T3 fibroblasts. TM-2 expression resulted in a flatter cell morphology and restoration of stress fibers. TM-2 expression also significantly reduced growth rates in low serum, soft agar, and nude mice. The reduced growth rates were associated with a prolongation of G0-G1. To identify the mechanism of TM-2-induced growth inhibition, we analyzed the effects of TM-2 reexpression of ERK and c-jun N-terminal kinase (JNK) activities. Levels of ERK phosphorylation and activity in TM-2-transfected tumor cells were comparable to those in mock-transfected tumor cells. JNK activity was only modestly increased in ras-transformed cells relative to untransformed NIH 3T3 cells and only slightly reduced as result of forced TM-2 expression. We conclude that the partially restored expression of the TM-2 protein induces growth inhibition of ras-transformed NIH 3T3 cells without influencing ERK or JNK activities. Furthermore, the 3' untranslated region riboregulator of the alpha-tropomyosin gene is not needed for the inhibition of ras-induced growth.     

Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells arises from increased choline kinase activity, not from phosphatidylcholine breakdown.

The cellular concentration of phosphocholine has been reported to be significantly elevated in Ha-ras-transformed NIH 3T3 cells, but not in v-sis transformants (J. C. Lacal, J. Moscat, and S. A. Aaronson, Nature [London] 330:269-271, 1987). It was suggested that the phosphocholine arises from constitutive hydrolysis of phosphatidylcholine by phospholipase C, an activity that would also account for the elevated 1,2-diacylglycerol found in ras-transformed cells. I have demonstrated that the increased phosphocholine arises through the induction of choline kinase activity. No increased breakdown of phosphatidylcholine was observed in ras-transformed cells. The elevation in diacylglycerol is therefore unlikely to be a consequence of phosphatidylinositol or phosphatidylcholine turnover.     

Synthesis and anticancer activity of 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (AZT)

A series of novel N-alkyl 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (6-15) was synthesized by means of phosphonylation of 3'-azido-3'-deoxythymidine (4) with P-chloromethylphosphonic ditriazolide (3) followed by a reaction with the appropriate amine. The synthesized phosphonamidates 6-15 were evaluated for their cytotoxic activity in two human cancer cell lines: oral (KB) and breast (MCF-7) using the sulforhodamine B (SRB) assay. The highest activity in KB human cancer cells was displayed by phosphonamidate 8 (IC(50)=5.8 μg/mL), however, this compound was less potent than the parent AZT (IC(50)=3.1 μg/mL). Phosphonamidate 10 showed only moderate activity (IC(50)=12.1 μg/mL) whereas the other phosphonamidates proved inactive. Similarly, the highest activity in MCF-7 human cancer cells was displayed by phosphonamidate 8 (IC(50)=3.7 μg/mL) but it proved somewhat less active than AZT (IC(50)=2.6 μg/mL). Some activity was also displayed by phosphonamidate 10 (IC(50)=12.8 μg/mL) but the other phosphonamidates were found inactive. Hydrolysis studies indicate that the synthesized phosphonamidates are likely to act as prodrugs of the parent nucleoside (AZT). Transport measurements showed that the most active phosphonamidates (8 and 10) were able to permeate across the intestinal epithelium in vitro. The apparent permeability coefficients determined in Caco-2 cell monolayers indicated that these compounds could be moderately absorbed in humans.     

Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.

Two major species of diacylglycerol kinase (type I and type II) were separated from brain cytosol and from NIH-3T3 or ras-transformed 3T3 cells by heparin-agarose chromatography. Multiple species of diacylglycerol kinase were also detected by non-denaturing isoelectric focusing. The two peaks of activity were of similar size, both co-eluted at approximately 95 kDa from a Superose f.p.l.c. column. Type II enzyme (pI 8.0) was more active when substrate was presented in a deoxycholate/phosphatidylserine undefined environment, as opposed to an octyl glucoside/phosphatidylserine micellar environment. Type II activity was also enhanced by the presence of phosphatidylcholine as cofactor. Type I enzyme (pI 4.0) was more active in the presence of either phosphatidylserine or phosphatidylinositol. Type I and II enzymes had different ATP affinities. Both enzymes showed a preference for diacylglycerol substrates with saturated acyl chains of 10-12 carbon atoms. The cytosolic enzyme activity was able to bind to diacylglycerol-enriched membranes in NIH-3T3 fibroblasts, and this translocation was unaffected in ras-transformed 3T3 cells. These results demonstrate the presence of multiple diacylglycerol kinases in brain cytosol and NIH-3T3 and ras-transformed 3T3 cells. The enzymes differ in cofactor, ATP and substrate requirements. These results can explain some of the contradictions between previous studies of cytosolic diacylglycerol kinase activity, and suggest the presence of a family of such kinases that are differentially regulated by phospholipid cofactors.     

Non-nucleoside HIV-1 reverse-transcriptase inhibitors. Part 10. Synthesis and anti-HIV activity of 5-alkyl-6-(1-naphthylmethyl)pyrimidin-4(3H)-ones with a mono- or disubstituted 2-amino function as novel 'dihydro-alkoxy-benzyl-oxopyrimidine' (DABO) analogues

A series of structurally related 2,5-disubstituted 6-(1-naphthylmethyl)-pyrimidin-4(3H)-ones, compounds 6a-6r, were synthesized and evaluated for their in vitro anti-HIV activities in MT-4 cells. Most of the new compounds investigated showed moderate-to-good activities against wild-type HIV-1, with IC(50) values in the range 5.64-0.21 microM. Compound 6d was the most potent congener (IC(50)=0.21 microM, SI=724) in inhibiting HIV-1 replication, which is ca. 25 times more effective than the reference compound 2',3'-dideoxyinosine (DDI). Preliminary structure-activity relationship (SAR) studies revealed that both modulation of the amino function at C(2) and of the alkyl group at C(5) of the pyrimidine ring are crucial for high anti-HIV-1 activity.     

Inhibition of tyrosine phenol-lyase from Citrobacter freundii by 2-azatyrosine and 3-azatyrosine.

The interactions of 2-azatyrosine and 3-azatyrosine with tyrosine phenol-lyase (TPL) from Citrobacter freundii have been examined. 2-Aza-DL-tyrosine and 3-aza-DL-tyrosine were synthesized by standard methods of amino acid synthesis, while the L-isomers were prepared from 3-hydroxypyridine and 2-hydroxypyridine, respectively, with TPL (Watkins, E. B., and Phillips, R. S. (2001) Bioorg. Med. Chem. Lett. 11, 2099-2100). 3-Azatyrosine was examined as a potential transition state analogue inhibitor of TPL. Both compounds were found to be competitive inhibitors of TPL, with K(i) values of 3.4 mM and 135 microM for 3- and 2-aza-L-tyrosine, respectively. Thus, 3-azatyrosine does not act as a transition state analogue, possibly due to the lack of tetrahedral geometry at C-1. However, 2-aza-L-tyrosine is the most potent competitive inhibitor of TPL found to date. The K(i) value of 2-aza-L-tyrosine is half that of 2-aza-DL-tyrosine, indicating that the D-enantiomer is inactive as an inhibitor. Neither azatyrosine isomer was shown to be a substrate for beta-elimination, based on coupled assays with lactate dehydrogenase and on HPLC measurements. Both isomers of azatyrosine form equilibrium mixtures of external aldimine and quinonoid intermediates when they bind to TPL. However, 2-azatyrosine reacts about 10-fold faster to form a quinonoid intermediate than does 3-azatyrosine. Since 2-azatyrosine is in the zwitterion or phenolate ion form at all the pH values examined, the strong binding of this compound suggests that L-tyrosine may be bound to the active site of TPL as the phenolate anion.     

Design, synthesis, and biological evaluation of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides as cyclooxygenase isozyme inhibitors

A group of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides, possessing either a F or a substituted-phenyl ring substituent (4-F, 2,4-F2, 4-SO2Me, 4-OCHMe2) attached to its C-4 or C-6 position, was prepared using a palladium-catalyzed Suzuki cross-coupling reaction for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. Although N-acetyl-3-carboxymethyl-6-fluorobenzenesulfonamide [14, COX-1 IC50 = 2.26 microM; COX-2 IC50 = 0.012 microM; COX-2 selectivity index (SI) = 188] and N-acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c, COX-1 IC50 >100 microM; COX-2 IC50 = 0.15 microM; COX-2 SI >667) exhibited potent in vitro COX-2 inhibitory activity and high COX-2 selectivity, both compounds were inactive anti-inflammatory agents in a carrageenan-induced rat paw edema assay. In contrast, the less potent and less selective COX-2 inhibitors N-acetyl-2-carboxymethyl-4-fluorobenzenesulfonamide (12, COX-1 IC50 = 4.25 microM; COX-2 IC50 = 0.978 microM; COX-2 SI = 4.3), N-acetyl-2-carboxymethyl-4-(2,4-difluorophenyl)benzenesulfonamide (17c, COX-1 IC50 = 1.02 microM; COX-2 IC50 = 1.00 microM; COX-2 SI = 1.02), and N-acetyl-3-carboxymethyl-6-(4-methanesulfonylphenyl)benzenesulfonamide (20e, COX-1 IC50 = 0.109 microM; COX-2 IC50 = 1.14 microM; COX-2 SI = 0.095) exhibited moderate anti-inflammatory activity where a 75 mg/kg oral dose reduced inflammation 26%, 14%, and 20%, respectively, at 3 h postdrug administration relative to the reference drug aspirin where a 50 mg/kg oral dose reduced inflammation by 25% at 3 h postdrug administration.     

A novel macrocyclic lactone with insecticidal bioactivity from Streptomyces microflavus neau3

A novel macrocyclic lactone (1) was isolated from the fermentation broth of Streptomycesmicroflavus neau3, and the structure was elucidated by extensive spectroscopic analysis. Compound 1 showed high acaricidal activity against adult mites (IC(50)=11.1 μg mL(-1)), and nematocidal activity against Caenorhabditis elegans (IC(50)=17.4 μg mL(-1)), especially the acaricidal activity against mite eggs with an IC(50) of 37.1 μg mL(-1), which was relative higher than that of the commercial acaricide and nematocide milbemycins A(3)/A(4).     

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.     

The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors

The competitive antagonist d-tubocurarine (curare) has greater potency at mouse than at human 5-hydroxytryptamine 3A (5-HT3A) receptors, despite 84% amino acid sequence identity between the receptors. Within the ligand binding domain of this receptor are six loops (A-F). A previous report demonstrated that loop C of the 5-HT3A receptor contributed to differential potency between the receptors [Hope, A. G. et al. (1999) Mol. Pharmacol. 55, 1037-1043]. The present study tested the hypothesis that loop F plays a significant role in conferring interspecies curare potency differences. Wild-type, chimeric, and point mutant 5-HT3A receptors were expressed in Xenopus oocytes, and two-electrode voltage clamp electrophysiological recordings were performed. Our data suggest that loops C and F contribute to curare potency, given that the curare IC50's (concentration of drug that produces 50% inhibition of the response) for chimeric human receptors with substitutions of mouse residues in loop C (40.07 +/- 2.52 nM) or loop F (131.8 +/- 5.95 nM) were intermediate between those for the mouse (12.99 +/- 0.77 nM) and human (1817 +/- 92.36 nM) wild-type receptors. Two human point mutant receptors containing mouse receptor substitutions in loop F (H-K195E or H-V202I) had significantly lower curare IC50's than that of the human receptor. The human double mutant receptor, H-K195E,V202I, had the same curare IC50 (133.8 +/- 6.38 nM) as that of the human receptor containing all six loop F mouse substitutions. These results demonstrate that two loop F residues make a significant contribution in determining curare potency at the 5-HT3A receptor.     

Synthesis and antileishmanial activity of 3-imidazolylalkylindoles. Part I

The present study was designed to investigate conazoles as new antileishmanial agents. Several 3-imidazolylalkyl-indoles were prepared under mild reaction conditions and pharmacomodulation at N1 and C5 of the indole ring and at the level of the alkyl chain (R) was carried out starting from the corresponding 3-formylindoles 7-10. All target imidazolyl compounds 38-52 were evaluated in vitro against Leishmania mexicana promastigotes; ketoconazole, amphotericin B and meglumine antimoniate were used as references. Eight out of fifteen compounds (40,43,44,47,48, 50, 51 and 52) exerted similar activity to ketoconazole, with IC50 values in the range of 2.10-3.30 microg/mL. However the most potent compound, 1-(2-bromobenzyl)-3-(1H-imidazol-1-ylmethyl)-1H-indole (38), exhibited IC50 value (0.011+/-0.003 microg/mL) 270-fold lower than that of ketoconazole. Four compounds (38, 43, 50 and 52) were also tested against intracellular amastigotes of L. mexicana; compound 38 exhibited the highest activity with an IC50 value of 0.018+/-0.004 microg/mL.     

Synthesis, biological evaluation, and molecular docking of N-{3-[3-(9-methyl-9H-carbazol-3-yl)-acryloyl]-phenyl}-benzamide/amide derivatives as xanthine oxidase and tyrosinase inhibitors

Claisen-Schmidt condensation of 3-formyl-9-methylcarbazole with various amides of 3-aminoacetophenone afforded N-{3-[3-(9-methyl-9H-carbazol-3-yl)-acryloyl]-phenyl}-benzamide/amide derivatives. All compounds were investigated for their in vitro xanthine oxidase (XO), tyrosinase and melanin production inhibitory activity. Most of the target compounds had more potent XO inhibitory activity than the standard drug (IC(50)=4.3-5.6μM). Interestingly, compound 7q bearing cyclopropyl ring was found to be the most potent inhibitor of XO (IC(50)=4.3μM). Molecular modelling study gave an insight into its binding modes with XO. Compounds 7a, 7d, 7e, 7g, and 7k were found to be potent inhibitors of tyrosinase (IC(50)=14.01-17.52μM). These results suggest the possible use of these compounds for the design and development of novel XO and tyrosinase inhibitors.     

2,4-Disubstituted piperidines as selective CC chemokine receptor 3 (CCR3) antagonists: synthesis and selectivity

Linear unselective CCR3 antagonist leads with IC(50) values in the 200 nM range were converted into low nM binding compounds selective at CCR3 by moving the piperidine nitrogen substituent to the carbon at the 2-position of the ring. Substitution of the piperidine nitrogen with simple alkyl and acyl groups was found to improve the selectivity of this new compound class. In particular, N-{3-[(2S, 4R)-1-(propyl)-4-(4-fluorobenzyl)piperidinyl]propyl}-N'-(3-acetylphenyl)urea exhibited single digit nanomolar IC(50) values for CCR3 with >100-fold selectivity against an extensive counter screen panel.     

Discovery of N-propylurea 3-benzylpiperidines as selective CC chemokine receptor-3 (CCR3) antagonists

The discovery of novel and selective small molecule antagonists of the CC Chemokine Receptor-3 (CCR3) is presented. Simple conversion from a 4- to 3-benzylpiperidine gave improved selectivity for CCR3 over the serotonin 5HT(2A) receptor. Chiral resolution and exploration of mono- and disubstitution of the N-propylurea resulted in several 3-benzylpiperidine N-propylureas with CCR3 binding IC(50)s under 5 nM. Data from in vitro calcium mobilization and chemotaxis assays for these compounds ranged from high picomolar to low nanomolar EC(50)s and correlated well with antagonist binding IC(50)s.     

Discovery and structure-activity relationships of urea derivatives as potent and novel CCR3 antagonists

The synthesis and structure-activity relationships of ureas as CCR3 antagonists are described. Optimization starting with lead compound 2 (IC(50)=190 nM) derived from initial screening hit compound 1 (IC(50)=600 nM) led to the identification of (S)-N-((1R,3S,5S)-8-((6-fluoronaphthalen-2-yl)methyl)-8-azabicyclo[3.2.1]octan-3-yl)-N-(2-nitrophenyl)pyrrolidine-1,2-dicarboxamide 27 (IC(50)=4.9 nM) as a potent CCR3 antagonist.     

Cytotoxic mechanism of XK469: resistance of topoisomerase IIbeta knockout cells and inhibition of topoisomerase I

Topoisomerase IIbeta knockout mouse cells (beta-/-) were found to have only slight resistance to m-AMSA, a dual topoisomerase IIalpha-IIbeta poison, as compared to wild-type cells (beta+/+) during 1 h or 3 day exposures to the drug. In contrast, the beta-/- cells were greater than threefold resistant to XK469, a selective topoisomerase IIbeta poison during three day drug exposures (beta+/+ IC(50) = 175 microM, beta-/- IC(50) = 581 microM). Short term (1 h) exposure to XK469 was not cytotoxic to either beta-/- or beta+/+ cells, suggesting that anticancer therapy with XK469 may be more efficacious if systemic levels can be prolonged. During studies on topoisomerase activity in nuclear extracts of the beta+/+ and beta-/- cells, we found evidence that XK469 is a weak topoisomerase I catalytic inhibitor. The high IC(50) for topoisomerase I inhibition (2 mM) suggests that topoisomerase I is not a significant target for XK469 cytotoxicity.