Oxazolidinones mechanism of action: inhibition of the first peptide bond formation.

Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA templates but have no significant effect on poly(A)-dependent translation. Here we show that various oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70 S ribosomes using initiator-tRNA or N-protected CCA-Phe as a P-site substrate and puromycin as an A-site substrate. Steady-state kinetic analysis shows that oxazolidinones display a competitive inhibition pattern with respect to both the P-site and A-site substrates. This is consistent with a rapid equilibrium, ordered mechanism of the peptidyltransferase reaction, wherein binding of the A-site substrate can occur only after complex formation between peptidyltransferase and the P-site substrate. We propose that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiator fMet-tRNA(i)(Met) to the ribosomal peptidyltransferase P-site, which is vacant only prior to the formation of the first peptide bond.

4-Aryl-2-anilinopyrimidines as corticotropin-releasing hormone (CRH) antagonists.

A series of 4-aryl-2-(N-ethylanilino)pyrimidines has been synthesized as corticotropin-releasing hormone (CRH) inhibitors. The effect of substitution on each aromatic ring on receptor binding was investigated.

Non-peptide corticotropin-releasing hormone antagonists: syntheses and structure-activity relationships of 2-anilinopyrimidines and -triazines.

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist alpha-helical CRH(9-41) (Ki = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated adenylate cyclase activity in the same tissue, but it was less potent than alpha-helical CRH(9-41) (IC50 = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1 receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH1 Ki = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines.

The synthesis and CRF receptor binding affinities of several new series of N-aryltriazolo- and -imidazopyrimidines and -pyridines are described. These cyclized systems were prepared from appropriately substituted diaminopyrimidines or -pyridines by nitrous acid, orthoester, or acyl halide treatment. Variations of amino (ether) pendants and aromatic substituents have defined the structure-activity relationships of these series and resulted in the identification of a variety of high-affinity agents (Ki's < 10 nM). On the basis of this property and lipophilicity differences, six of these compounds (4d,i,n,x, 8k, 9a) were initially chosen for rat pharmacokinetic (PK) studies. Good oral bioavailability, high plasma levels, and duration of four of these compounds (4d,i,n,x) prompted further PK studies in the dog following both iv and oral routes of administration. Results from this work indicated 4i,x had properties we believe necessary for a potential therapeutic agent, and 4i1 has been selected for further pharmacological studies that will be reported in due course.

MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products.

In search of antiinflammatory drugs with a new mechanism of action, U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with an IC50 of 0.07 microM for MEK 1 and 0.06 microM for MEK 2. U0126 can undergo isomerization and cyclization reactions to form a variety of products, both chemically and in vivo, all of which exhibit less affinity for MEK and lower inhibition of AP-1 activity than parent, U0126.

Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries.

Antisense oligonucleotides can vary significantly and unpredictably in their ability to inhibit protein synthesis. Libraries of chimeric oligonucleotides and RNase H were used to cleave and thereby locate sites on human multidrug resistance-1 RNA transcripts that are relatively accessible to oligonucleotide hybridization. In cell culture, antisense sequences designed to target these sites were significantly more active than oligonucleotides selected at random. This methodology should be generally useful for identification of potent antisense sequences. Correlation between oligonucleotide activity in the cell culture assay and in an in vitro RNase H assay supports the proposed role of the enzyme in the mechanism of antisense suppression in the cell.

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. II. 3',5'-blocked oligonucleotides.

The pharmacokinetics and metabolism of four radiolabeled phosphodiester oligonucleotides with 3'- and 5'-blocked ends were studied in mice and compared with previously studied, unblocked, all-phosphodiester and all-phosphorothioate oligonucleotides. The radiolabel was a tritiated methyl group enzymatically attached at an internal cytidine. The ends of the blocked phosphodiester oligonucleotides were protected by cyclization or by incorporation of either phosphorothioate or methylphosphonate linkages. Although these modifications protected the blocked oligonucleotides from degradation by exonucleases present in mouse serum, degradation initiated by endonucleases was 50% complete in 0.5-5 hr. After intravenous injection, the blocked oligonucleotides were much less stable than the all-phosphorothioate oligonucleotide and only marginally more stable than the previously studied, unblocked phosphodiester oligonucleotide. Even a "chimeric" blocked oligonucleotide with 16 phosphorothioate linkages and eight contiguous phosphodiester linkages was rapidly degraded. Despite the favorable serum binding, tissue accumulation, and stability observed with phosphorothioate oligonucleotides, these properties did not provide the chimeric oligonucleotide access to a compartment where its phosphodiester linkages were stable. In other respects, the blocked and chimeric phosphodiester oligonucleotides also resembled the unblocked phosphodiester oligonucleotide; radiolabel was cleared rapidly from the blood, there was little evidence of tissue accumulation, high performance liquid chromatographic analysis of tissue extracts showed extremely rapid degradation to mononucleotides, and only mononucleotide metabolites were present in urine. In summary, blocked phosphodiester oligonucleotides are rapidly attacked by endonucleases present in mice. Unless this problem is less serious in primates, such blocked oligonucleotides will be relatively unattractive candidates for drug development.

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate.

Biodistribution and metabolism of oligonucleotides were determined using a 3H-labeled 20-nucleotide phosphodiester and its phosphorothioate analog. The oligonucleotides were radiolabeled by 3H-methylation of an internal deoxyctidine with HhaI methylase and S- [3H]adenosylmethionine. Biodistribution studies were conducted after intravenous injection of 6 mg/kg (5 muCi) oligonucleotide. Metabolism of the oligonucleotides was determined by paired-ion high performance liquid chromatography. After phosphodiester injections, radiolabel rapidly cleared the blood. Relative initial concentrations were as follows: kidney > blood > heart > liver > lung > spleen. Radiolabel in spleen peaked at 1 hr and remained elevated for 24 hr. At 2 hr the concentration in all organs, except spleen, was equal to that in blood. High performance liquid chromatographic analysis of the kidney, liver, and spleen extracts and urine indicated extremely rapid metabolism to monomer. Results of studies after the injection of phosphorothioate oligonucleotide differed from those using the phosphodiester. Despite its rapid clearance from blood, phosphorothioate accumulated rapidly in all tissues, especially the kidney. Kidney uptake increased over time, remaining very high for 24 hr. Ratios of organ to blood concentrations at 2 hr for all organs were 5:1 or greater. Kidney and liver ratios were 84:1 and 20:1, respectively. Analysis of the kidney and liver extracts and urine indicated that slow metabolism occurred. These data suggest that phosphodiester oligonucleotides would have limited therapeutic utility. The stability and organ distribution of the phosphorothioate oligonucleotide imply that such oligonucleotides may have therapeutic potential.

7,8-Dihydro-8-oxoadenine as a replacement for cytosine in the third strand of triple helices. Triplex formation without hypochromicity.

Oligonucleotides containing thymine and cytosine (or 5-methylcytosine) bases are known to bind to specific homopurine sequences in double-stranded DNA by means of T.AT and C+.GC base triplets. Cytosine in the third strand of such triple helices can be completely replaced by 7,8-dihydro-8-oxoadenine a base which should not require protonation to form base triplets. Experiments using native PAGE and inhibition of triplex-directed photo-cross-linking demonstrate that triplexes with 7,8-dihydro-8-oxoadenine in the third strand are as stable at pH 6.0 as triplexes with 5-methylcytosine. The stability of triplexes with 7,8-dihydro-8-oxoadenine, unlike those with 5-methylcytosine, is not substantially diminished upon raising the pH to 7.4. Surprisingly, triplex formation with an oligonucleotide containing only thymine and 7,8-dihydro-8-oxoadenine was not associated with significant hypochromicity and could not be detected in conventional thermal denaturation experiments.

Denaturing HPLC purification of tritylated oligonucleotides using tetraethylammonium hydroxide.

Purification of oligonucleotides by HPLC is limited by association between failure sequences and full-length oligonucleotide. We describe a protocol for denaturing purification of 5'-dimethoxytritylated oligonucleotides that ensures that separation of tritylated and non-tritylated species will not be complicated by strand association. Fully denaturing conditions are produced by the use of tetraethylammonium hydroxide, which is a basic reagent with ion-pairing properties similar to triethylammonium acetate. The method also includes two other convenient features: a) the option of loading the crude oligonucleotide without removing concentrated ammonium hydroxide and b) detritylation on the column with separation of dimethoxytrityl alcohol.

Detection of single base differences using biotinylated nucleotides with very long linker arms.

A simple primer extension method for detecting nucleotide differences is based on the substitution of mobility-shifting analogs for natural nucleotides (1). This technique can detect any single-base difference that might occur including previously unknown mutations or polymorphisms. Two technical limitations of the original procedure have now been addressed. First, switching to Thermococcus litoralis DNA polymerase has eliminated variability believed to be due to the addition of an extra, non-templated base to the 3' end of DNA by Taq DNA polymerase. Second, with the analogs used in the original study, the mobility shift induced by a single base change can usually be resolved only in DNA segments 200 nt or smaller. This size limitation has been overcome by synthesizing biotinylated nucleotides with extraordinarily long linker arms (36 atom backbone). Using these new analogs and conventional sequencing gels (0.4 mm thick), mutations in the human beta-hexosaminidase alpha and CYP2D6 genes have been detected in DNA segments up to 300 nt in length. By using very thin (0.15 mm) gels, single-base polymorphisms in the human APOE gene have been detected in 500-nt segments.

Cytotoxicity of synthetic racemic ptilocaulin: a novel cyclic guanidine.

(+)-Ptilocaulin, a novel cyclic guanidine extracted from the Caribbean sponge Ptilocaulis aff. P. Spiculifer, is reported to have broad spectrum antimicrobial activity in vitro as well as in vitro activity against L1210 murine leukemia. To more fully evaluate this compound as an anticancer agent, the in vitro cell growth inhibitory potencies of synthetic racemic ptilocaulin and ten clinical anticancer drugs were determined and compared in 16 different normal and transformed human and murine cell populations. Potency, expressed as the 50% inhibitory concentration (IC50), was determined by a tetrazolium reduction (MTT) assay. Ptilocaulin showed a fairly broad spectrum of in vitro activity against colon and mammary adenocarcinomas, melanomas, leukemias, transformed fibroblasts and normal lymphoid cells (IC50s 0.05- greater than 10 micrograms/ml). This activity was comparable to that of many of the clinical drugs, including vinca alkyloids, antibiotics, alkylators and antimetabolites. Cell viability was affected only after a 72 hr exposure to the compound. In a clonogenic assay, cytocidal effects were observed after 24-72 hr exposures to 10 x IC50 concentrations of ptilocaulin, as evidenced by failure of cells to resume growth after removal of the compound. Cytostatic effects were observed at less than or equal to IC50 concentrations, as evidenced by resumption of growth to near-control levels after removal of the compound. Ptilocaulin was toxic at 50 and 25 mg/kg in an in vivo L1210 tumor model and was ineffective at lower concentrations (T/Cs 100-112%). In vivo studies in a more sensitive tumor system are recommended but are limited by the lack of availability of sufficient quantities of the compound.

A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides.

A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different chemically tuned succinylfluorescein dye distinguished by its fluorescent emission is described. Avian myeloblastosis virus reverse transcriptase is used in a modified dideoxy DNA sequencing protocol to produce a complete set of fluorescence-tagged fragments in one reaction mixture. These DNA fragments are resolved by polyacrylamide gel electrophoresis in one sequencing lane and are identified by a fluorescence detection system specifically matched to the emission characteristics of this dye set. A scanning system allows multiple samples to be run simultaneously and computer-based automatic base sequence identifications to be made. The sequence analysis of M13 phage DNA made with this system is described.