David C. Eustice

Mechanism of action of DuP 721: inhibition of an early event during initiation of protein synthesis.

The mode of action of DuP 721 was investigated. This compound was active primarily against gram-positive bacteria, including multiply resistant strains of staphylococci. Although inactive against wild-type Escherichia coli, DuP 721 did inhibit E. coli when the outer membrane was perturbed by genetic or chemical means. Pulse-labeling studies with E. coli PLB-3252, a membrane-defective strain, showed that DuP 721 inhibited amino acid incorporation into proteins. The 50% inhibitory concentration of DuP 721 for protein synthesis was 3.8 micrograms/ml, but it was greater than 64 micrograms/ml for RNA and DNA syntheses. The direct addition of DuP 721 to cell-free systems did not inhibit any of the reactions of protein synthesis from chain initiation through chain elongation with either synthetic or natural mRNA as template. However, cell extracts prepared from DuP 721 growth-arrested cells were defective in initiation-dependent polypeptide synthesis directed by MS2 bacteriophage RNA. These cell-free extracts were not defective in polypeptide elongation or in fMet-tRNA(fMet)-dependent polypeptide synthesis stimulated by poly(G.U). We conclude, therefore, that DuP 721 exerts its primary action at a step preceding the interaction of fMet-tRNA(fMet) and 30S ribosomal subunits with the initiator codon.

Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis.

Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 microM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin- and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C] lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.

The mechanism of action of DuP 721, a new antibacterial agent: Effects on macromolecular synthesis

Pulse labeling studies with Bacillus subtilis showed that DuP 721 inhibited protein synthesis. The IC50 of DuP 721 for protein synthesis was 0.25 micrograms/ml but it was greater than 32 micrograms/ml for RNA and DNA synthesis. In cell-free systems, DuP 721 concentrations up to 100 microM did not inhibit peptide chain elongation reactions under conditions where chloramphenicol, tetracycline and hygromycin B inhibited these reactions. Furthermore, Dup 721 did not cause phenotypic suppression of nonsense mutations suggesting that DuP 721 did not inhibit peptide chain termination. Thus, the mechanism of action of DuP 721 is at a target preceeding chain elongation.

Imidazo[1,2-a]pyrazine diaryl ureas: Inhibitors of the receptor tyrosine kinase EphB4

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent and Nexavar, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.

Rat osteotesticular phosphatase gene (Esp): genomic structure and chromosome location.

Abstract. Osteotesticular phosphatase (OST-PTPase) is a class III receptor-type tyrosine phosphatase (RPTPase). It has 10 tandem fibronectin III-like (FN-III) repeats in the extracellular region and two phosphatase domains in the intracellular region. The expression of the rat OST-PTPase gene, Esp, is restricted to osteoblasts and Sertoli cells, and the transcript level in osteoblasts is highly up-regulated by parathyroid hormone and cAMP treatment. We report here the cloning and characterization of the rat Esp gene, including a 2.9-kb 5′ flanking region sequence. Two potential binding sites for Osf2/Cbfa1, an osteoblast-specific transcription factor, are present in the promoter. Esp is composed of 35 exons, but spans merely 20 kb, making it the most compact RPTPase gene identified. Each FN-III repeat is encoded by a single exon flanked with phase 1 introns. Two phosphatase domains are encoded by 16 exons in a genomic organization similar to those in RPRPα, RPTPγ, and Ptprc genes. Esp was mapped by fluorescence in situ hybridization to rat chromosome 13q1. These results represent the first genomic structure of a mammalian class III RPTPase gene.

Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. Rats were subjected to a two-thirds hepatectomy followed 20 h later by i.p. injection of N-OH-AAF. 4 h after carcinogen injection, it was found that N-OH-AAF caused a dose-dependent inhibition of [3H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery beginning 28 h after carcinogen treatment. Radioimmunoassay of deoxyguanine-C8 adducts remaining in liver DNA indicated that the recovery began prior to detection of adduct removal. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF (Zurlo and Yager, 1984), were cultured under conditions that promote replicative DNA synthesis. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. [3H]Thymidine pulse times and subsequent chase times were adjusted to equalize amounts of DNA synthesis in control and UV-irradiated cells. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. Pulse-chase studies revealed that subsequent joining of nascent chains in UV-irradiated hepatocytes occurred at a rate comparable to or faster than controls and that this could be inhibited by caffeine. The results obtained from both the in vivo and in vitro studies show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with our previous studies demonstrating the ability of UV-irradiated hepatocytes to carry out enhanced reactivation of UV-irradiated herpes virus lend support to the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis.