The different strategies for designing GPCR and kinase targeted libraries.

In recent years the trend in combinatorial library design has shifted to include target class focusing along with diversity and drug-likeness criteria. In this manuscript we review the computational tools available for target class library design and highlight the areas where they have proven useful in our work. The protein kinase family is used to illustrated structure-based target class focused library design, and the G-protein coupled receptor (GPCR) family is used to illustrate ligand-based target class focused library design. Most of the tools discussed are those designed for libraries targeted to a single protein and are simply applied "brute-force" to a large number of targets within the family. The tools that have proven to be the most useful in our work are those that can extract trends from the computational data such as docking and clustering or data mining large amounts of structure activity or high throughput screening data. Finally, areas where improvements are needed in the computational tools available for target class focusing are highlighted. These areas include tools to extract the relevant patterns from all available information for a family of targets, tools to efficiently apply models for all targets in the family rather than just a small subset, mining tools to extract the relevant information from the computational absorption, distribution, metabolism, excretion and toxicity (ADMET) and targeting data, and tools to allow interactive exploration of the virtual space around a library to facilitate the selection of the library that best suits the needs of the design team.

Homology modeling of the estrogen receptor subtype beta (ER-beta) and calculation of ligand binding affinities.

Estrogen is a steroid hormone playing critical roles in physiological processes such as sexual differentiation and development, female and male reproductive processes, and bone health. Numerous natural and synthetic environmental compounds have been shown capable of estrogenic effects. This area has been the focus of significant fundamental and applied research due both to the potential detrimental effects of these compounds upon normal physiological processes and to the potential beneficial effects of tissue-selective estrogen agonists/antagonists for the prevention and treatment of numerous diseases. Genomic effects of the active form of estrogen, 17beta-estradiol, are mediated through at least two members of the steroid hormone receptor superfamily, estrogen receptor subtype alpha (ER-alpha) and estrogen receptor subtype beta (ER-beta). At the time of this work, the X-ray crystal structure of the ER-alpha had been elucidated, however, coordinates of the ER-beta were not publicly available. Based upon the significant structural conservation across members of the steroid hormone receptor family, and the high sequence homology between ER-alpha and ER-beta (>60%), we have developed a homology model of the ER-beta structure. Using the crystal structure of ER-alpha and the homology model of ER-beta, we demonstrate a strong correlation between computed values of the binding-energy and published values of the observed relative binding affinity (RBA) for a variety of compounds for both receptors, as well as the ability to identify receptor subtype selective compounds. Furthermore, using the recently available crystal structure of ER-beta for comparison purposes, we show that not only is the predicted homology model structurally accurate, but that it can be used to assess ligand binding affinities.

The autonomous transactivation domain in helix H3 of the vitamin D receptor is required for transactivation and coactivator interaction.

A ligand-inducible transactivation function (AF-2) exists in the extreme carboxyl terminus of the vitamin D receptor (VDR) that is essential for 1alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-activated transcription and p160 coactivator interaction. Crystallographic data of related nuclear receptors suggest that binding of 1, 25-(OH)2D3 by VDR induces conformational changes in the ligand-binding domain (LBD), the most striking of which is a packing of the AF-2 helix onto the LBD adjacent to helices H3 and H4. In this study, a panel of VDR helix H3 mutants was generated, and residues in helix H3 that are important for ligand-activated transcription by the full-length VDR were identified. In particular, one mutant (VDR (Y236A)) exhibited normal ligand binding and heterodimerization with the retinoid X receptor (RXR) but was transcriptionally inactive. Yeast two-hybrid studies and in vitro protein interaction assays demonstrated that VDR (Y236A) was selectively impaired in interaction with AF-2-interacting coactivator proteins such as SRC-1 and GRIP-1. These data indicate an importance of helix H3 in the mechanism of VDR-mediated transcription, and they support the concept that helix H3 functions in concert with the AF-2 domain to form a transactivation surface for binding the p160 class of nuclear receptor coactivators.

Trauma-induced neurotoxicity in rat hippocampal neurons.

BACKGROUND AND PURPOSE: We have previously shown that traumatic injury of hippocampal cells triggers release of a soluble neurotoxin that can be transferred to an uninjured culture. The mechanism of this trauma-induced neurotoxicity is independent of glutamate receptor activation. We extended this observation to study the mechanism of this neurotoxicity. METHODS: Dissociated rat hippocampal neurons were traumatized by disrupting the culture by scratching the plate. The toxicity expressed by the injured culture was studied by transferring the medium to an uninjured culture and assessing the death rate by trypan blue exclusion. RESULTS: This neurotoxin is stable in the medium at room temperature for several hours and withstands boiling. The molecular weight is between 100 and 500. The release and the effect of this toxin seem to be independent of glutamate receptor activation. The toxicity is unaffected by removal of extracellular calcium. However, dantrolene dose-dependently blocked the toxicity in the recipient culture, suggesting that the release of intracellular stores of calcium is involved in the toxic effect. This release of calcium is likely to be followed by an activation of nitric oxide synthase because competitive nitric oxide synthase inhibitors attenuated this toxicity. Consistent with this result, cholecystokinin octapeptide significantly reduced cell death when combined with this toxic medium. CONCLUSIONS: Traumatic injury of dissociated cells can propagate neurotoxicity in uninjured cells by a soluble toxin released into the extracellular space. This toxin causes a rise in cytosolic calcium that activates nitric oxide synthase that can be blocked by cholecystokinin.

The vitamin D receptor interacts with general transcription factor IIB.

The vitamin D receptor (VDR) heterodimerizes with retinoid X receptors (RXR) on many vitamin D-responsive promoter elements, suggesting that this complex is the active factor in vitamin D-mediated transcription. However, the mechanism of transcriptional regulation following VDR-RXR binding to DNA is not well characterized. Using a yeast two-hybrid protein interaction assay, we demonstrate that VDR forms specific protein: protein contacts with the basal transcription factor TFIIB. Deletion analysis indicated that the carboxyl-terminal ligand binding domain of VDR interacted with a 43-residue amino-terminal domain in TFIIB. The interaction with TFIIB showed selectivity for the ligand binding domain of VDR as similar regions of RXR alpha or of retinoic acid receptor alpha did not couple with TFIIB. Binding assays with purified proteins showed a direct interaction between VDR and TFIIB in vitro. These data suggest a mechanism for VDR-dependent transcription in which protein contacts between VDR and TFIIB may impart regulatory information to the transcription preinitiation complex.