Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Expression, activity, and function of phosphodiesterases in the mature and immature ductus arteriosus.

A patent ductus arteriosus is due in large part to increased sensitivity of the premature ductus to PGE2. After PGE2 stimulation, cAMP concentrations are higher in the immature than in the mature ductus. cAMP concentrations depend on the rates of adenyl cyclase production and phosphodiesterase (PDE)-mediated degradation. We used ductus from immature (n = 25) and mature (n = 21) fetal sheep to investigate whether a developmental increase in PDE activity could explain the diminished cAMP accumulation that follows PGE2 stimulation in the mature ductus. With advancing gestation, mRNA expression of the smooth muscle PDE isoforms (PDE1A, 1B, 1C, 3A, 3B, 4D, and 5A) increased in the ductus as did their hydrolytic activities. Selective inhibitors of PDE1, PDE3, and PDE4 relaxed the mature and immature ductus in the presence of inhibitors of prostaglandin and nitric oxide production. The mature ductus required higher concentrations of each of the PDE inhibitors to inhibit its tension to the same extent as in the immature ductus. There were no developmental changes in PDE expression in the fetal aorta. In conclusion, we observed a developmental increase in cAMP and cGMP PDE activity that contributes to the decreased sensitivity of the late-gestation ductus arteriosus to vasodilators like PGE2.

Importance of cAMP-response element-binding protein in regulation of expression of the murine cyclic nucleotide phosphodiesterase 3B (Pde3b) gene in differentiating 3T3-L1 preadipocytes.

Incubation of 3T3-L1 preadipocytes with isobutylmethylxanthine (IBMX), dexamethasone, and insulin, alone or in combination, demonstrated that IBMX, which increased cAMP-response element-binding protein (CREB) phosphorylation, was the predominant regulator of Pde3b expression. Real time PCR and immunoblotting indicated that in 3T3-L1 preadipocytes, IBMX-stimulated induction of Pde3b mRNA and protein was markedly inhibited by dominant-negative CREB proteins. By transfecting preadipocytes, differentiating preadipocytes, and HEK293A cells with luciferase reporter vectors containing different fragments of the 5'-flanking region of the Pde3b gene, we identified a distal promoter that contained canonical cis-acting cAMP-response elements (CRE) and a proximal, GC-rich promoter region, which contained atypical CRE. Mutation of the CRE sequences dramatically reduced distal promoter activity; H89 inhibited IBMX-stimulated CREB phosphorylation and proximal and distal promoter activities. Distal promoter activity was stimulated by IBMX and phorbol ester (PMA) in Raw264.7 monocytes, but only by IBMX in 3T3-L1 preadipocytes. Chromatin immunoprecipitation analyses with specific antibodies against CREB, phospho-CREB, and CBP/p300 (CREB-binding protein) showed that these proteins associated with both distal and proximal promoters and that interaction of phospho-CREB, the active form of CREB, with both Pde3b promoter regions was increased in IBMX-treated preadipocytes. These results indicate that CRE in distal and proximal promoter regions and activation of CREB proteins play a crucial role in transcriptional regulation of Pde3b expression during preadipocyte differentiation.

Identification of promoter elements in 5'-flanking region of murine cyclic nucleotide phosphodiesterase 3B gene.

We describe techniques for identifying functional promoter elements in the 5'-flanking region of the murine cyclic nucleotide phosphodiesterase 3B (mPDE3B) gene. The 5'-flanking region of the mPDE3B gene was cloned and sequenced, and putative transcription factor binding sites were identified with computational tools. A series of reporter plasmids containing the luciferase gene fused to different fragments of the 5'-flanking region of the mPDE3B gene was constructed and used to transfect 3T3-L1 fibroblasts or differentiating adipocytes. Reporter gene assays showed that there are two promoter regions in the 5'-flanking region in the mPDE3B gene: a distal region located approx 4 kb upstream of the translation initiation site that contains cAMP-response element (CRE) cis-acting elements, and a proximal region that is GC rich and lacks TATA sequences. The distal promoter region induced much higher luciferase activity than did the proximal one. Mutation of the CRE sequences or reversal of the orientation of the CRE-containing region abolished promoter activity of the distal region. Electrophoretic mobility shift assay analysis indicated that binding to CRE elements was greater in nuclear extracts from differentiating adipocytes than from fibroblasts. Mapping of transcription initiation sites suggested that the distal promoter region might function as an enhancer, whereas the proximal promoter drives transcription of the mPDE3B gene.

Effects of the human immunodeficiency virus-protease inhibitor, ritonavir, on basal and catecholamine-stimulated lipolysis.

Several of the aspartic acid protease inhibitors used to treat HIV infection increase basal lipolysis in adipocytes, but the cellular mechanisms leading to this augmentation are not well understood. We therefore studied the effects of chronic exposure to the HIV protease inhibitor, ritonavir, on the lipolytic cascade in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with ritonavir for 14 d (during and after differentiation) enhanced basal, isoproterenol (Iso)-stimulated, and cAMP analog-stimulated lipolysis. Enhancement of lipolysis was observed after Iso at concentrations between 0.1 and 10 mum. Despite a significant decrease in cyclic nucleotide phosphodiesterase (PDE)3B activity and protein levels, there were no changes in Iso-stimulated intracellular cAMP, protein kinase A (PKA) expression, or PKA activity. Ritonavir-augmented lipolysis was also observed under conditions that reversed the effect on PDE3B activity via preincubation with 1 mum (-)-N(6)-(2-phenylisopropyl)adenosine. In ritonavir-treated cells, protein expression of the lipid droplet-protective protein, perilipin, was significantly decreased, whereas there was no change in hormone-sensitive lipase. Activation of ERK1/2 by Iso did not play a role in the augmentation. We conclude that ritonavir decreases PDE3B and perilipin protein expression and affects both basal and catecholamine-stimulated lipolysis in 3T3-L1 adipocytes primarily through actions at sites downstream of PKA.

Cyclic adenosine 3',5'-monophosphate regulation of corticotropin-releasing hormone promoter activity in AtT-20 cells and in a transformed hypothalamic cell line.

The regulation of CRH promoter activity by cAMP was studied in two cell lines, the pituitary corticotroph cell line AtT-20 and the immortalized hypothalamic cell line 4B, which expresses CRH and vasopressin. In 4B cells transfected with a CRH promoter-luciferase construct, the adenylyl cyclase stimulator, forskolin, increased luciferase activity in parallel with increases in intracellular cAMP. In 4B cells, however, the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiated forskolin-stimulated cAMP without affecting further increases in luciferase activity. In AtT-20 cells, forskolin plus isobutylmethylxanthine elevated cAMP only slightly, but increased luciferase activity to levels similar to those observed in 4B cells. AtT-20 cells were also unresponsive to 8-bromo-cAMP, due in part to higher phosphodiesterase (PDE) activities. Although both cells contained PDE1, -3, and -4, inhibition of either PDE4 or PDE1 potentiated luciferase activity stimulated by submaximal forskolin concentrations in 4B cells, while only simultaneous inhibition of PDE3 and PDE4 was effective in AtT-20 cells. The data show that minor elevations in intracellular cAMP are sufficient for full stimulation of CRH promoter activity regardless of the cell line. Furthermore, poor CRH promoter activation in AtT-20 cells appears to result from deficient cAMP production and rapid cAMP degradation by PDE.

Reduced phosphodiesterase 3 activity and phosphodiesterase 3A level in synthetic vascular smooth muscle cells: implications for use of phosphodiesterase 3 inhibitors in cardiovascular tissues.

Vascular smooth muscle cells (VSMC) in situ function to control contraction and are said to express a contractile phenotype. However, during development or in response to vascular damage, VSMC proliferate and express a more synthetic phenotype. A survey of literature values for contractile and synthetic VSMC phosphodiesterase (PDE) 3 and PDE4 activities identified a marked difference in the PDE3 and PDE4 activities of these cells. In this study, a comparison of PDE3 and PDE4 activities in contractile and synthetic VSMC demonstrates that a reduced PDE3/PDE4 activity ratio in synthetic VSMC correlates with a reduced PDE3 activity and is associated with marked reductions in PDE3A mRNA and protein levels. Because we show that similar reductions in PDE3 activity and PDE3A levels occur upon culture of human aortic VSMC and that this phenomenon associates with the phenotypic switch that occurs to VSMC in response to vascular damage, our findings are presented in the context that PDE3 inhibition might be expected to selectively alter functions of contractile VSMC.