The GAF-tandem domain of phosphodiesterase 5 as a potential drug target.

Classic PDE5 inhibitors interact with and block the catalytic site of PDE5. They have been clinically validated for treatment of erectile dysfunction as well as reduction of pulmonary arterial pressure, improvement of exercise capacity, quality of life, and arterial oxygenation in patients with secondary pulmonary hypertension. Minor side effects are visual disturbances, headache, migraine, back pain, and interaction with nitrates (hypotension). Some of those side effects presumably can be ameliorated by improving selectivity and pharmacokinetics; other side effects probably are target related due to inhibition of basic physiological processes. Target related side effects may be bypassed by using PDE5 inhibitors with a different mode of action: PDE5, like PDE2, PDE6, PDE10, and PDE11, is a multidomain protein with an N-terminal tandem GAF domain, which in case of PDE5, is allosterically activated by cGMP. Potential inhibitors acting at the PDE5 GAF domain would be expected to inhibit only pathophysiologically upregulated PDE5 activity, whereas basal activity of PDE5 would remain unaffected.Here, we summarize a high-throughput screening campaign to identify inhibitors of the regulatory GAF domain of human PDE5. To target the regulatory domain independently from the catalytic site, we used a chimeric reporter enzyme: The hPDE5 GAF-tandem domain functionally replaced the GAF domain in the cyanobacterial adenylyl cyclase CyaB1. We identified inhibitors that target the GAF domain and also inhibitors that target the bacterial cyclase.Compounds binding to the PDE5 GAF domain were reanalysed with native human PDE5 to demonstrate inhibition using capillary electrophoresis. This identified 16 compounds that act on the GAF domain of PDE5. Two compounds fulfilled the initial requirement to inhibit, exclusively, activated PDE5, but not basal PDE5 activity.

A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway.

The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin alpha protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin alpha binding and import activity.

Automated high throughput screening for serine kinase inhibitors using a LEADseeker scintillation proximity assay in the 1536-well format.

High-throughput screening in the 1536-well format has been largely restricted to solution-based and cell-based screens. In this article, we show the feasibility of a completely automated, robust scintillation proximity assay in the 1536-well format that is suitable to identify inhibitors for a serine/threonine kinase from a compound library. The introduction of [(33)P]phosphate into a biotinylated peptide substrate mirrors the activity of the kinase. The peptide is immobilized on streptavidin-coated LEADseeker imaging beads and [(33)P]phosphate incorporation is detected with the LEADseeker imaging system of Amersham Pharmacia Biotech. To improve the liquid handling procedures for imaging bead suspensions in the low microliter range, we developed a novel trough with an integrated stirring function. A comparison of the 1536-well assay to a 384-well assay revealed a comparable assay quality with Z' factors of about 0.7 for the 384-well format and 0.6 for the 1536-well format. In an automated screen of a random compound collection, 94.4% of the inhibitory compounds could be identified with both assay formats. Dose-response curves were performed for a selection of identified kinase inhibitors and revealed similar IC(50) values for both assay formats.