1-(5-Carboxyindol-1-yl)propan-2-ones as inhibitors of human cytosolic phospholipase A2α: Synthesis and properties of bioisosteric benzimidazole,benzotriazole and indazole analogues

The indole ring systems of the cytosolic phospholipase A₂α (cPLA₂α) inhibitor 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (2) and the isomeric 6-carboxylic acid (3) were replaced by benzimidazole, benzotriazole and indazole scaffolds, respectively. The effect of the structural variations on cPLA₂α inhibitory potency, metabolic stability and solubility was studied. The lead 2 and the indazole-5-carboxylic acid 28 were the metabolically most stable compounds in an assay with rat liver microsomes, while the benzimidazole-5-carboxylic acid derivative 13 possessed the best water solubility (22 μg/mL at pH 7.4). The indazole-5-carboxylic acid 28 revealed the highest cPLA₂α inhibitory potency of the compounds in this series. With an IC₅₀-value of 0.005 μM it was about sevenfold more active than the lead 2.     

Characterization of an Escherichia coli Mutant Deficient in Dihydrolipoyl Dehydrogenase Activity

A mutant of Escherichia coli deficient in dihydrolipoyl dehydrogenase (DHL) activity has been isolated and its characteristics have been studied. The activities of the pyruvic dehydrogenase (PDC) and alpha-ketoglutaric dehydrogenase complexes (KDC) are not present in extracts of the mutant unless purified dihydrolipoyl dehydrogenase is added. Experiments with antiserum to DHL have shown that cross-reacting material exists in mutant extracts. This suggests that the dihydrolipoyl dehydrogenase mutation (dhl(-)) is a missense structural mutation. The mutation maps very close to, if not adjacent to, the ace loci, and is not linked to the suc loci. This means the dhl locus is grouped with the genes for the other components of the PDC and not with the genes for KDC. The mutation is also transducible into prototrophic strains, demonstrating that no prior mutation is necessary for the DHL activity deficiency to exist. This evidence is consistent with the idea that there is only one gene for DHL and is supported by previous biochemical studies which have shown that DHL preparations from either enzyme complex are electrophoretically and immunochemically indistinguishable. Possible mechanisms for the genetic and metabolic control of DHL, PDC, and KDC are discussed.     

Control of simian virus 40 gene expression at the levels of RNA synthesis and processing: thermally induced changes in the ratio of the simian virus 40 early mRNA''s and proteins

Examination of the simian virus 40 early mRNA's from infected AGMK or CV-1 cells showed that the ratio of large T- to small t-antigen mRNA's increased with an increased incubation temperature. In tsA58 mutant-infected cells, an increased incubation temperature resulted in the overproduction of early RNAs'; however, the ratio of the early mRNA's was the same, at any temperature, in both wild-type- and tsA58-infected cells. Thus, the thermally induced alteration in the early mRNA ratios was apparently not affected by the tsA mutation or by the overproduction of early RNA in tsA mutant-infected cells. Time course studies at various temperatures showed that, although the ratio of large T- to small t-antigen mRNA's increased with temperature, at any one temperature it was consistent from early to late times of infection. Furthermore, the ratio of the early mRNA's adjusted in temperature shift experiments. Thus, the ratio of the early mRNA's appeared to be intrinsic to the thermodynamic environment of the cell. The thermally induced alterations in the early mRNA's were reflected at the protein level by parallel changes in the ratio of large T- to small t-antigens. These data suggest a level of gene expression control which may function at the stage of splicing.     

The cap and the 3' splice site similarly affect polyadenylation efficiency.

The 5' cap of a mammalian pre-mRNA has been shown to interact with splicing components at the adjacent 5' splice site for processing of the first exon and the removal of the first intron (E. Izaurralde, J. Lewis, C. McGuigan, M. Jankowska, E. Darzynkiewicz, and I.W. Mattaj, Cell 78:657-668, 1994). Likewise, it has been shown that processing of the last exon and removal of the last intron involve interaction between splicing components at the 3' splice site and the polyadenylation complex at the polyadenylation signal (M. Niwa, S. D. Rose, and S.M. Berget, Genes Dev. 4:1552-1559, 1990; M. Niwa and S. M. Berget, Genes Dev. 5:2086-2095, 1991). These findings suggest that the cap provides a function in first exon processing which is similar to the function of the 3' splice site at last exon processing. To determine whether caps and 3' splice sites function similarly, we compared the effects of the cap and the 3' splice site on the in vitro utilization of the simian virus 40 late polyadenylation signal. We show that the presence of a m7GpppG cap, but not a cap analog, can positively affect the efficiency of polyadenylation of a polyadenylation-only substrate. Cap analogs do not stimulate polyadenylation because they fail to bind titratable cap-binding factors. The failure of cap analogs to stimulate polyadenylation can be overcome if a 3' splice site is present upstream of the polyadenylation signal. These data indicate that factors interacting with the cap or the 3' splice site function similarly to affect polyadenylation signal, along with m7GpppG cap, is inhibitory to polyadenylation. This finding suggests that the interaction between the cap-binding complexes and splicing components at the 5' splice site may form a complex which is inhibitory to further processing if splicing of an adjacent intron is not achieved.     

Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.     

The snRNP-free U1A (SF-A) complex(es): identification of the largest subunit as PSF, the polypyrimidine-tract binding protein-associated splicing factor.

We have previously shown that a specific monoclonal antibody prepared against the U1A protein, MAb 12E12, is unique in its ability to recognize a form of U1A which is not associated with the U1snRNP. This unique form of U1A, termed snRNP-free U1A or SF-A, was found to be complexed with a novel set of non-snRNP proteins (O'Connor et al., 1997, RNA 3:1444-1455). Here we demonstrate that the largest protein in these SF-A complex(es), p105, is the polypyrimidine-tract binding protein-associated factor (PSF), an auxiliary splicing factor. We show that PSF copurifies and co-immunoprecipitates with SF-A from 293T cell nucleoplasm and that it interacts with SF-A in vitro. In addition, we show that MAb 12E12 inhibits both splicing and polyadenylation in an in vitro coupled splicing and polyadenylation reaction. This suggests that SF-A and/or the SF-A complex(es) perform an important function in both processing reactions and possibly in last exon definition.