Characterization of a Drosophila Ortholog of the Cdc7 Kinase: A ROLE FOR Cdc7 IN ENDOREPLICATION INDEPENDENT OF CHIFFON*

Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G₁/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.     

Guanidino Acids Act as ρ1 GABA<Subscript>C</Subscript> Receptor Antagonists

GABA_C receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective agents for this class of receptors. Guanidino analogs related to glycine, β-alanine and taurine were evaluated at human ρ₁GABA_C receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining were inactive. (S)-2-Guanidinopropionic acid (IC₅₀ = 2.2 μM) and guanidinoacetic acid (IC₅₀ = 5.4 μM; K _B = 7.75 μM [pK _B = 5.11 ± 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the β-alanine and GABA guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced activity indicating that steric effects may impact on activity. The results of this study contribute to the structure–activity-relationship profile required in developing novel therapeutic agents.     

Synthesis of new six- and seven-membered 1-N-iminosugars as promising glycosidase inhibitors

New six- and seven-membered 1-N-iminosugars were prepared from d-glucose by the stereoselective Michael addition of nitromethane to d-glucose derived α,β-unsaturated ester A followed by one pot reduction of nitro/ester functionality and subsequent amine protection to get N-Cbz protected aminol 6. Hydrolysis of 1,2-acetonide and reductive aminocyclization gave seven membered 1-N-iminosugar 5b. While, hydrolysis of 1,2-acetonide followed by NaIO(4) oxidative cleavage and hydrogenation using 10% Pd(OH)(2)/C, H(2) gave six membered 1-N-iminosugar 4a; the hydrogenation using 10% Pd/C-H(2) however, gave N-methyl substituted 1-N-iminosugar 4b. The hydrochloride salts of 4a/4b and 5b were found to be specific α-galactosidase and moderate α-glucosidae inhibitors, respectively, in micro molar range.     

The discovery of novel isoflavone pan peroxisome proliferator-activated receptor agonists

Twenty three dual PPARα and γ molecules of natural product origin, previously reported by our group, were further investigated for pan PPAR transactivation against PPARδ. The in vitro cell toxicity profile, as well as, in silico study of the most active molecules within this new class of pan PPAR agonists are also described. 3′,5′ Dimethoxy-7 hydroxyisoflavone 6, Ψ-baptigenin 7, 4′ fluoro-7 hydroxyisoflavone 8, and 3′ methoxy-7 hydroxyisoflavone 9 were identified as the most potent molecules studied within the set compared to the commercially available pan PPAR agonist, bezafibrate 1. These novel active molecules may thus be useful as future leads in PPAR-related disorders, including type II diabetes mellitus and metabolic syndrome.