Nonsteroidal selective glucocorticoid modulators: the effect of C-5 alkyl substitution on the transcriptional activation/repression profile of 2,5-dihydro-10-methoxy-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines.

The preparation and characterization of a series of selective glucocorticoid receptor modulators are described. The preliminary structure-activity relationship of nonaromatic C-5 substitution on the tetracyclic quinoline core showed a preference for small lipophilic side chains. Proper substitution at this position maintained the transcriptional repression of proinflammatory transcription factors while diminishing the transcriptional activation activity of the ligand/glucocorticoid receptor complex. The optimal compounds described in this study were the allyl analogue 18 and cyclopentyl analogue 32. These candidates showed slightly less potent, highly efficacious E-selectin repression with significantly reduced levels of glucocorticoid response element activation in reporter gene assays vs prednisolone. Allyl analogue 18 was evaluated in vivo. An oral dose of 18 showed an ED(50) = 1.7 mg/kg as compared to 1.2 mg/kg for prednisolone in the Sephadex-induced pulmonary eosinophilia model and an ED(50) = 15 mg/kg vs 4 mg/kg for prednisolone in the carrageenan-induced paw edema model.

Drug discovery and the intracellular receptor family.

Drug discovery using intracellular receptors (IRs) as targets presents its own set of unique complications and advantages. The natural ligands for these receptors are, in many cases, already used as drugs. To effectively exploit these targets, newer molecules must have either increased receptor selectivity or increased tissue or gene selectivity to reduce side effects. The search for these molecules will yield new therapeutics as well as new insights into the mechanism of action of these receptors and their ligands.

Structure and function of the small subunit of TFIIF (RAP30) from Drosophila melanogaster.

To study the mechanism of basal transcription by RNA polymerase II, a cDNA encoding the Drosophila homologue of the small subunit of TFIIF (also referred to as TFIIF30, RAP30, factor 5b, and gamma) was isolated. The Drosophila TFIIF30 gene is located at region 86C on the right arm of the third chromosome. The protein encoded by the cDNA, termed dTFIIF30, was synthesized in Escherichia coli and purified to greater than 95% homogeneity. In reconstituted transcription reactions with purified basal factors, the specific activity of dTFIIF30 was identical to that of its human homologue. Moreover, a carboxyl-terminal fragment, designated dF30(119-276), which contains the carboxyl-terminal 158 amino acid residues of dTFIIF30, was found to possess approximately 50% of the transcriptional activity as full-length dTFIIF30. The interaction of dTFIIF30 with the large subunit of TFIIF (also referred to as TFIIF74, RAP74, factor 5a, and beta) was investigated by glycerol gradient sedimentation analyses. In these experiments, dTFIIF30, but not dF30(119-276), assembled into a stable heteromeric complex with TFIIF74. These results, combined with those of previous work on TFIIF, support a model for TFIIF30 function in which the carboxylterminal region constitutes a functional domain that can interact with RNA polymerase II to mediate basal transcription, whereas the amino terminus comprises a domain that interacts with TFIIF74.

In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.

In eukaryotes, initiation of mRNA synthesis is a multistep process that is carried out by RNA polymerase II and auxiliary factors that are commonly referred to as basal or general factors. In this study accurate initiation of transcription was reconstituted with purified, Escherichia coli-synthesized TFIIB, TBP (the TATA box-binding polypeptide of the TFIID complex), and the 30-kD subunit of TFIIF (also known as RAP30), along with purified, native RNA polymerase II from Drosophila embryos, calf thymus, or HeLa cells. This minimal set of factors was able to transcribe a subset of the promoters tested. The addition of both subunits of TFIIE and the 74-kD subunit of TFIIF increased the efficiency of transcription by a factor of 2 to 4. In contrast, the inclusion of a crude TFIID fraction from Drosophila embryos in place of recombinant TBP resulted in a strong dependence on TFIIE. By gel mobility-shift analysis, TFIIB, TBP, RAP30, and polymerase were able to assemble into DB and DBPolF30 complexes with transcriptionally competent (wild type or initiator mutant), but not with transcriptionally inactive (TATA and TATA/initiator mutant), versions of the Drosophila Adh promoter. Thus, it appears that RNA polymerase II is able to initiate transcription subsequent to assembly of the DBPolF30 complex, which is a minitranscription complex that represents the central core of the RNA polymerase II transcriptional machinery.

Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos.

We describe the preparation and biochemical properties of a soluble nuclear fraction derived from Drosophila embryos. This extract, which can be easily prepared in 2.5 hr, is capable of accurate and efficient RNA polymerase II transcription of a variety of diverse genes from both Drosophila and mammals. With the relatively strong promoter of the Drosophila Krüppel gene, it is possible to achieve 20% template usage in a single round of transcription, which is considerably higher than the template usage of approximately 3% seen with standard nuclear extracts. Further, although U small nuclear RNA genes are refractory to transcription with HeLa transcription extracts, the soluble nuclear fraction transcribes a U1 small nuclear RNA gene from Drosophila. Moreover, transcriptional activation by sequence-specific activators can be attained in vitro with the soluble nuclear fraction. The overall transcriptional efficiency appears limited to 0.45 transcript per template of DNA per 30 min, but the mechanism of limitation is not known. The soluble nuclear fraction, which was developed to recreate the environment within the nucleus, should be useful when high efficiencies of RNA polymerase II transcription are desired.

Fractionation of the general RNA polymerase II transcription factors from Drosophila embryos.

We have subdivided the components of the basic RNA polymerase II machinery from Drosophila embryos into three fractions and RNA polymerase II. The RNA polymerase II was 90% homogeneous and possessed the IIa form of the largest subunit. By substitution of these factors with their mammalian homologues in reconstituted transcription reactions, we have determined that the three Drosophila fractions contain transcription factor (TF)IIB, TFIIE/F, and TFIID. In addition, the fraction with TFIID contains another essential activity, which we have tentatively designated as TFIIZ. There was no apparent requirement for TFIIA. The reconstituted transcription factors accurately transcribe both Drosophila and mammalian genes. This fractionated system should serve as a source of general transcription factors for the study of RNA polymerase II transcription in Drosophila as well as other eukaryotes.