Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide stereoisomers as novel positive allosteric modulators of sigma-1 receptor.

Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that (4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of sigma-1 receptor than were their optical antipodes.

TFIIH is an elongation factor of RNA polymerase I.

TFIIH is a multisubunit factor essential for transcription initiation and promoter escape of RNA polymerase II and for the opening of damaged DNA double strands in nucleotide excision repair (NER). In this study, we have analyzed at which step of the transcription cycle TFIIH is essential for transcription by RNA polymerase I. We demonstrate that TFIIH associates with the rDNA promoter and gene-internal sequences and leaves the rDNA promoter in a complex with RNA polymerase I after start of transcription. Moreover, mutations in the TFIIH subunits XPB and XPD found in Cockayne syndrome impair the interaction of TFIIH with the rDNA, but do not influence initiation complex formation or promoter escape of RNA polymerase I, but preclude the productivity of the enzyme by reducing transcription elongation in vivo and in vitro. Our results implicate that reduced RNA polymerase I transcription elongation and ribosomal stress could be one factor contributing to the Cockayne syndrome phenotype.

A novel activity enhances promoter escape of RNA polymerase I.

We have characterized a novel transcriptional activity from HeLa cells that is required for ribosomal gene transcription by RNA polymerase I. This activity has a native molecular mass of 16 kDa and does not bind to conventional chromatographic resins. Single-round and immobilized-template experiments revealed that initiation complex formation is independent of the novel activity. Functional studies showed that it stimulates the transition from initiation to elongation, promoter escape. Thus the novel activity does not resemble the mouse initiation/elongation factor TIF-IC but is a true novel entity.

Truncated Cockayne syndrome B protein represses elongation by RNA polymerase I.

Mutations in the Cockayne syndrome B (CSB) gene result in the human form of Cockayne syndrome. CSB protein has been shown to be a component of RNA polymerase I (Pol I) transcription. In this study, we have analyzed at which step of the transcription cycle CSB influences in vitro transcription by RNA Pol I. We demonstrate that CSB stimulates elongation of RNA Pol I in an ATP-independent manner. Moreover, CSB can be cross-linked to the rDNA promoter and gene-internal sequences. Partial deletion mutants of CSB strongly repress Pol I in vitro transcription, indicating an inhibitory function of truncated CSB. In addition, evidence that mutant CSB inhibits the elongation step of Pol I transcription is presented. Lack of CSB expression does not impair Pol I transcription, showing that CSB is not essential for ribosomal transcription. Our results implicate that repressed Pol I transcription could be one factor contributing to the Cockayne syndrome phenotype.

The transcriptional response to distinct growth factors is impaired in Werner syndrome cells.

The Werner syndrome protein (WRN) is mutated in Werner syndrome (WS) and plays a role in telomere maintenance, DNA repair and transcription. WS represents a premature aging syndrome with severe growth retardation. Here we show that WRN is critically required to mediate the stimulatory effect of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-b) and epidermal growth factor (EGF) on the activity of RNA polymerase I (Pol I). Recombinant WRN specifically reconstitutes RNA polymerase I transcription in extracts from Werner syndrome fibroblasts in vitro. In addition, we identified a critical role for WRN during promoter clearance of Pol I transcription, but not in elongation. Notably, WRN was isolated in a complex with Pol I and was crosslinked to the unmethylated, active proportion of rDNA genes in quiescent cells suggesting a so far unknown role for WRN in epigenetic regulation. This together with alterations in Pol I transcription provide a novel mechanism possibly underlying at least in part the severe growth retardation and premature aging in Werner syndrome patients.

Comparative study of CYP2B induction in the liver of rats and mice by different compounds.

Male Wistar rats and C57BL mice were treated by phenobarbital (PB), 2,4,6-triphenyldioxane-1,3 (TPD) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). The CYP2B specific activities (PROD and BROD) were determined in the animal livers. PB administration significantly increased levels of PROD- and BROD-activity in the rat and mouse livers, whereas TPD induced CYP2B activities only in rat liver and TCPOBOP--only in mouse liver. The result of Western-blot analysis showed that PB and TPD increased CYP2B protein content in rat liver, PB and TCPOBOP--in mouse liver. Results of multiplex RT-PCR showed that the increase in CYP2B enzymatic activities reflected at least in part an increased mRNA levels. Thus, our results provide evidence to support the conclusion that the species-dependent differences of CYP2B induction occur because of differences of transcriptional activation of CYP2B genes.