Thio- and oxoflavopiridols, cyclin-dependent kinase 1-selective inhibitors: synthesis and biological effects.

Flavopiridol analogues, thio- and oxoflavopiridols which contain a sulfur (16) or oxygen (18) atom linker between a chromone ring and the hydrophobic side chain, are selective cyclin-dependent kinase 1 (CDK1) inhibitors with an IC(50) of 110 and 130 nM. These analogues were prepared from key intermediate 7 by substituting the ethyl sulfoxide. Enantio pure intermediate piperidone 10 was obtained from the racemic piperidone 8 via a very efficient "dynamic kinetic resolution" in 76% yield. Hydrophobic side chains such as chlorophenyl or tert-butyl produced potent CDK1 inhibitory activity, while hydrophilic side chains such as pyrimidine or aniline caused a severe reduction in CDK inhibitory activity. These analogues are competitive inhibitors with respect to ATP, and therefore activity was dependent upon the CDK subunit without being affected by the cyclin subunit or protein substrate. Thio- and oxoflavopiridols 16 and 18 are not only selective within the CDK family but also discriminated between unrelated serine/threonine and tyrosine protein kinases. CDK1 selective thio- and oxoflavopiridol analogues inhibit the colony-forming ability of multiple human tumor cell lines and possess a unique antiproliferative profile in comparison to flavopiridol.

Identification of CDK4 sequences involved in cyclin D1 and p16 binding.

Activation of CDK4 is regulated, in part, by its association with a D-type cyclin. Conversely, CDK4 activity is inhibited when it is bound to the cyclin-dependent kinase inhibitor, p16(INK4A). To investigate the molecular basis of the interactions between CDK4 and cyclin D1 or p16(INK4A) we performed site-directed mutagenesis of CDK4. The interaction was examined using in vitro translated wild type and mutant CDK4 proteins and bacterially expressed cyclin D1 and p16 fusion proteins. As mutational analysis of CDC2 suggests that its cyclin binding domain is primarily located near its amino terminus, the majority of the mutations constructed in CDK4 were located near its amino terminus. In addition, CDK4 residues homologous to CDC2 sites involved in Suc1 binding were also mutated. Our analysis indicates that cyclin D1 and p16 binding sites are overlapping and located primarily near the amino terminus. All CDK4 mutations that resulted in decreased p16 binding capability also diminished cyclin D1 binding. In contrast, amino-terminal sequences were identified, including the PSTAIRE region, that are important for cyclin D1 binding but are not involved in p16 binding.

Agonist-induced desensitization and phosphorylation of human 5-HT1A receptor expressed in Sf9 insect cells.

The human 5-HT1A receptor was expressed in Sf9 insect cells to examine desensitization as manifested by agonist-induced uncoupling from G proteins and second messengers. New binding sites were detected after infection of cells with the 5-HT1A receptor-bearing baculovirus. 5-HT1A receptor agonists caused inhibition of cAMP accumulation that could be attenuated by specific receptor antagonists. Brief pretreatment with 5-HT resulted in (1) an uncoupling of receptor from G proteins as evidenced by a loss of high-affinity agonist binding sites and a diminished ability of the receptor to increase incorporation of AA-GTP into endogenous Go alpha-like G proteins, (2) a decreased ability of the receptor to inhibit cAMP accumulation, and (3) increased phosphorylation of the 5-HT1A receptor on serine and threonine residues. Phosphorylation occurred in the presence of a number of cyclic nucleotide dependent kinase inhibitors, and desensitization of the cAMP response occurred in the presence of H-7 and also in cells with prolonged exposure to PMA. Both phosphorylation and desensitization were markedly attenuated by 100 nM and 1 microM heparin and demonstrated similar time courses and concentration-response relationships. Those results demonstrate a close association between agonist-induced desensitization and phosphorylation of the 5-HT1A receptor in Sf9 cells through a pathway that mainly does not involve protein kinase A or C and might involve a G protein-linked receptor kinase.

Human 5-HT1A receptor expressed in insect cells activates endogenous G(o)-like G protein(s).

Insect cell expression systems are used to characterize signaling components such as G protein-coupled receptors. As such, one must know whether endogenous G proteins couple to non-native receptors. We examined G protein linkages after infection of Sporodoptera frugiperda (Sf9) cells with a baculovirus encoding the 5-HT1A receptor. Receptor expression was confirmed by immunoblot. Some of the receptors were functional, showing guanine nucleotide-sensitive binding to the specific agonist ligand [3H]8-hydroxy-2-(di-n-propylamino)-1,2,3,4-tetranaphthalene). Peak expression (approximately 150 fmol/mg of membrane protein) was attained approximately 72-96 h post-infection. 5-HT-increased covalent binding of [32P]GTP-azidoanilide to a 40 kDa band, which was identified as a G protein by nucleotide blocking, Mg2+ dependence, and immunoblot and immunoprecipitation studies. The band comigrated with 1) pertussis toxin substrate(s), and 2) a band recognized by two G(o) alpha antisera and one common to heterotrimeric G protein alpha-subunits, but not by sera specific for Gs alpha or G(i) alpha. Labeled species could be precipitated with a G(o) alpha antiserum. 5-HT-increased labeling of the band was prevented by preincubation with pertussis toxin. These studies suggest that the 5-HT1A receptor couples effectively to native insect cell G(o)-like proteins.

Elevation of cAMP is required for down-regulation, but not agonist-induced desensitization, of endogenous dopamine D1 receptors in opossum kidney cells. Studies in cells that stably express a rat cAMP phosphodiesterase (rPDE3) cDNA.

D1 dopamine receptors stimulate cAMP accumulation in opossum kidney (OK) cells, but this response is attenuated by pretreatment with dopamine. Dopamine pretreatment also causes a reduction in D1 dopamine receptor number. We transfected OK cells with a rat cAMP phosphodiesterase cDNA (rPDE3) in order to determine the contribution of elevations of cAMP to those two phenomena. Wild-type (WT) OK cells were compared to three clones (C, H, and N) which demonstrated stable expression of the rPDE3 phenotype and genotype, rPDE3 RNA expression was confirmed in clones C, H, and N (but not in WT-OK cells) by reverse transcriptase-polymerase chain reaction. A functional rPDE3 phenotype was demonstrated in that dopamine-responsive cAMP accumulation was absent in clones C, H, and N in intact cells, but could be restored by preincubation with cAMP phosphodiesterase inhibitors, or by using washed membranes from those clones. All three clones had increased cAMP phosphodiesterase activity when compared to WT-OK cells (approximately 100% increase), and blunted or absent dopamine (1 microM)-induced protein kinase A activation. After pretreatment with dopamine (1 microM) for 1 h, clones C, H, and N desensitized equally well as WT-OK cells (approximately 40-50% reduction in maximal increase in cAMP). In contrast, down-regulation of D1 dopamine receptors was blunted for clone C (20% receptor loss) and absent for clones H and N, when compared to a 45% loss of receptors for WT-OK cells. These findings suggest that in OK cells pretreated with 1 microM dopamine (i) cAMP accumulation is not necessary for dopamine-induced desensitization, but (ii) is necessary for down-regulation of D1 dopamine receptors, and (iii) that the down-regulation and desensitization processes may be differentially regulated.

Porcine granulosa cells do not express transforming growth factor-beta 2 (TGF-beta 2) messenger ribonucleic acid: molecular basis for their inability to produce TGF-beta activity comparable to that of rat granulosa cells.

In contrast to rat granulosa cells (GC), GC of the pig and cow produce very low levels of transforming growth factor-beta (TGF-beta)-like activity in vitro. Because cultured rat GC predominantly express TGF-beta 2 messenger RNA (mRNA) and secrete high levels of the protein, we hypothesized that TGF-beta 2 mRNA expression by porcine GC might be absent or diminished, thus providing a molecular explanation(s) for their relatively low levels of TGF-beta production. We tested this hypothesis by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay analysis. When analyzed by RT-PCR, porcine GC RNA from 1-3 mm follicles did not yield the expected 489 base pair (bp) TGF-beta 2 product but instead generated a smaller than anticipated 240 bp species; porcine testis RNA generated both the 240 and the anticipated 489 bp products. Sequencing these species indicated that the smaller form was not a novel TGF-beta 2 splice variant, and that the 489-bp product was porcine TGF-beta 2. This is the first reported nucleotide sequence for porcine TGF-beta 2; it is 90% and 91% identical to murine and human TGF-beta 2 sequences, respectively. Further RT-PCR analysis of porcine GC RNA resulted in the identification of bp products representing TGF-beta 1 and TGF-beta 3 mRNA. Enzyme-linked immunosorbent assay analysis of porcine GC conditioned medium confirmed the presence of TGF-beta 1 at very low levels. TGF-beta 2 was undetectable. Comparable analysis of GC from the diethylstilbestrol-treated prepubertal rat demonstrated the presence of TGF-beta 1 and TGF-beta 3 mRNA by RT-PCR and very low levels of the corresponding protein products in conditioned culture medium. Collectively, these results suggest that the inability of porcine GC to express TGF-beta 2 mRNA could explain the very low levels of TGF-beta activity secreted by these cells in vitro.