Druggable targets and targeted drugs: enhancing the development of new therapeutics.

The advent of functional genomics, proteomics, chemi-informatics, and other systems-based scientific approaches have raised expectations of novel targets for drug discovery and design. Similarly, advances in materials sciences and biomolecular chemistries raised the prospects of highly targeted therapeutics that maximize efficacy while minimizing systemic toxicity. In spite of these advances, the gross measure of approvable drug output is declining, with only 17 new chemical entities approved by the FDA in 2007. This is in the face of high levels of R&D expenditures in both public and private sectors, and suggests that new, integrative approaches are needed in order to maximally exploit the rapidly expanding knowledge of potential drug and disease targets. The convergence of novel druggable targets with new chemical entities that can be specifically targeted to disease-causing sites and genes represents one means of creating highly efficacious and specific therapies. The approaches that are needed to facilitate such convergence include merging computational methods, systems biology, and gene-linked categorization of diseases with the use of appropriate drug delivery vehicles.

Elucidation of stannin function using microarray analysis: implications for cell cycle control.

Stannin (Snn) is a highly conserved, vertebrate protein whose cellular function is unclear. We have recently demonstrated in human umbilical vein endothelial cells (HUVECs) that Snn gene expression is significantly induced by tumor necrosis factor-alpha (TNF-alpha) in a protein kinase C-epsilon (PKC-epsilon)-dependent manner. In HUVEC, TNF-alpha stimulation of HUVECs results in altered gene expression, and a slowing or halting of cell growth. An initial set of experiments established that Snn knockdown via siRNA, prior to TNF-alpha treatment, resulted in a significant inhibition of HUVEC growth compared to TNF-alpha treatment alone. In order to assess how Snn may be involved in TNF-alpha signaling in HUVEC growth arrest, we performed microarray analysis of TNF-alpha-stimulated HUVECs with and without Snn knockdown via siRNA. The primary comparison made was between TNF-alpha-stimulated HUVECs and TNF-alpha-exposed HUVECs that had Snn knocked down via Snn-specific siRNAs. Ninety-six genes were differentially expressed between these two conditions. Of particular interest was the significant upregulation of several genes associated with control of cell growth and/or the cell cycle, including interleukin-4, p29, WT1/PRKC, HRas-like suppressor, and MDM4. These genes act upon cyclin D1 and/or p53, both of which are key regulators of the G1 phase of the cell cycle. Functional studies further supported the role of Snn in cell growth, as cell cycle analysis using flow cytometry shows a significant increase of G1 cell cycle arrest in HUVECs with Snn knockdown in response to TNF-alpha treatment. Together these studies suggest a functional role of Snn in regulation of TNF-alpha-induced signaling associated with HUVEC growth arrest.

The protein stannin binds 14-3-3zeta and modulates mitogen-activated protein kinase signaling.

The molecular mechanisms underlying the selective toxicity of trimethyltin (TMT) remain unclear. Stannin (Snn), a protein preferentially expressed in TMT-sensitive cells, provides a direct link to the molecular basis for TMT toxicity. Recent evidence demonstrated that Snn peptides bind and de-alkylate TMT to dimethyltin (DMT); Snn may mediate both TMT and DMT toxicity. In this study, we demonstrate that Snn co-immunoprecipitates with a scaffolding protein 14-3-3, specifically with 14-3-3zeta isotype. Consistent with this, a detailed amino acid sequence analysis shows that Snn contains a putative 14-3-3 protein-binding site located within its hydrophilic loop. In addition, we present the evidence that Snn overexpression results in reduced extracellular regulated kinase activation and increased p38 activation. In contrast, the activity of c-Jun N-terminal kinase did not change following Snn overexpression. This is the first evidence that demonstrates a direct interaction between Snn and MAPK signaling molecules. Together, these findings indicate a role of Snn in modulation of MAPK signaling pathways through its interactions with 14-3-3zeta.

Protein kinase C epsilon regulates tumor necrosis factor-alpha-induced stannin gene expression.

Stannin (Snn) is a highly conserved vertebrate protein that has been closely linked to trimethyltin (TMT) toxicity. We have previously demonstrated that Snn is required for TMT-induced cell death. Others have shown that TMT exposure results in tumor necrosis factor-alpha (TNFalpha) production and that TNFalpha treatment induces Snn gene expression in human umbilical vein endothelial cells (HUVECs). In this study, we investigated a signaling mechanism by which Snn gene expression is regulated by TMT and demonstrated that TNFalpha stimulates Snn gene expression in a protein kinase C epsilon-dependent manner in HUVECs in response to TMT exposure. Supporting this, we show that TMT-induced toxicity is significantly blocked by pretreatment with an anti-TNFalpha antibody in HUVECs. Using a quantitative real-time polymerase chain reaction assay, we also show that the level of Snn gene expression is significantly increased in HUVECs in response to either TMT or TNFalpha treatment. This TNFalpha-induced Snn gene expression is blocked when HUVECs were pretreated with bisindolylmaleimide I, an inhibitor of protein kinase C (PKC). In contrast, when HUVECs were treated with phorbol 12-myristate 13-acetate, a PKC activator, we observed a significant increase in Snn gene expression. Using isotype-specific siRNA against PKC, we further show that knockdown of PKC epsilon, but not PKC delta or PKC zeta, significantly blocked TNFalpha-induced Snn gene expression. Together, these results indicate that TNFalpha-induced, PKC epsilon-dependent Snn expression may be a critical factor in TMT-induced cytotoxicity.

Stannin, a protein that localizes to the mitochondria and sensitizes NIH-3T3 cells to trimethyltin and dimethyltin toxicity.

Stannin (Snn) is a highly conserved, 88-amino acid protein that may mediate the selective toxicity of organotins. Snn is localized in tissues with known sensitivity to trimethyltin (TMT), including the central nervous system, immune system, spleen, kidney and lung. Cells in culture that do not express Snn show considerable resistance to TMT toxicity. In vitro, Snn peptide can bind TMT in a 1:1 ratio and can de-alkylate TMT to dimethyltin (DMT). We now show that transfection with Snn sensitized TMT-resistant NIH-3T3 mouse fibroblasts to both TMT and DMT cytotoxicity. Triple label confocal microscopy of Snn-transfected cells and Percoll gradient purification of mitochondria showed Snn localized to the mitochondria and other membrane structures. The mitochondrial localization of Snn, coupled with its ability to bind and dealkylate organotin compounds, indicates a possible mechanism by which selective alkyltin toxicity might be mediated.

Domoic acid enhances Bcl-2-calcineurin-inositol-1,4,5-trisphosphate receptor interactions and delayed neuronal death in rat brain slices.

Mechanisms of neuronal death following neuronal damage due to domoic acid are not completely defined. Bcl-2, a survival protein, protects neurons from ischemia and excitotoxin-induced damage. We previously demonstrated that Bcl-2 shuttles calcineurin to its substrates and may regulate calcium release from internal stores during neuronal ischemia. We now confirm that during excitotoxicity induced by domoic acid, calcineurin-Bcl-2 and calcineurin-1,4,5-inositol-trisphosphate receptor (IP3-R) interactions increase. Furthermore, we now show that calcineurin-IP3-R interactions are mediated by Bcl-2 in brain slices following short-term treatment with domoic acid (10 microM). Domoic acid induced late neuronal death and caspase-3-like activity in organotypic cortical and hippocampal cultures. These experiments further define the mechanisms by which neurons respond to excitotoxic insults, and suggest that interactions between calcineurin and its target proteins may influence cellular responses to injury.

Abnormal cerebellar signaling induces dystonia in mice.

Dystonia is a relatively common neurological syndrome characterized by twisting movements or sustained abnormal postures. Although the basal ganglia have been implicated in the expression of dystonia, recent evidence suggests that abnormal cerebellar function is also involved. In these studies, a novel mouse model was developed to study the role of the cerebellum in dystonia. Microinjection of low doses of kainic acid into the cerebellar vermis of mice elicited reliable and reproducible dystonic postures of the trunk and limbs. The severity of the dystonia increased linearly with kainate dose. Kainate-induced dystonia was blocked by the glutamatergic antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide and reproduced by domoic acid microinjection, suggesting that the induction of dystonia is dependent on glutamatergic activation in this model. The abnormal movements were not associated with kainate-induced seizures, because EEG recordings showed no epileptiform activity during the dystonic events. Neuronal activation, as assessed by in situ hybridization for c-fos, revealed c-fos mRNA expression in the cerebellum, locus ceruleus, and red nucleus. In contrast, regions associated with epileptic seizures, such as the hippocampus, did not exhibit increased c-fos expression after cerebellar kainate injection. Furthermore, in transgenic mice lacking Purkinje cells, significantly less dystonia was induced after kainic acid injection, implicating Purkinje cells and the cerebellar cortex in this model of dystonia. Together, these data suggest that abnormal cerebellar signaling produces dystonia and that the cerebellum should be considered along with the basal ganglia in the pathophysiology of this movement disorder.