Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.

Adverse drug effects are often associated with pathological changes in tissue. An accurate depiction of the undesired affected area, possibly supported by mechanistic data, is important to classify the effects with regard to relevance for human patients. MALDI imaging MS represents a new analytical tool to directly provide the spatial distribution and the relative abundance of proteins in tissue. Here we evaluate this technique to investigate potential toxicity biomarkers in kidneys of rats that were administered gentamicin, a well known nephrotoxicant. Differential analysis of the mass spectrum profiles revealed a spectral feature at 12,959 Da that strongly correlates with histopathology alterations of the kidney. We unambiguously identified this spectral feature as transthyretin (Ser 28 -Gln 146 ) using an innovative combination of tissue microextraction and fractionation by reverse-phase liquid chromatography followed by a top-down tandem mass spectrometric approach. Our findings clearly demonstrate the emerging role of imaging MS in the discovery of toxicity biomarkers and in obtaining mechanistic insights concerning toxicity mechanisms. Proteomic strategies have been exploited to generate differential protein expression that can be correlated with environmental disease or toxicant exposure. In particular, improvements in two-dimensional gel electrophoresis and liquid chromatography coupled to mass spectrometry and the use of other emerging techniques have demonstrated the usefulness of a proteomic approach in toxicology (1-3). The early detection of disease markers and the rapid screening of experimental compounds either for toxicity or protective efficacy are of particular interest in this field (4). Imaging mass spectrometry (IMS)1 is a technique for the direct analysis of peptides and proteins from thin tissue sections using conventional MALDI-TOF mass spectrometers while preserving the abundance and spatial distribution of each analyte (5). The usefulness of this technique has been demonstrated in experimental models and in clinical settings (6, 7) as in the accurate and sensitive classification of nonsmall cell lung cancer in tumor biopsies (8).In this study we further demonstrated the potential applications of an IMS strategy in the study of a well known nephrotoxicant, the aminoglycoside antibiotic gentamicin (9), in the rat kidney. Aminoglycoside antibiotics are widely used, but nephrotoxicity (10) is a clear risk and occurs in 10 -20% (11) of treated patients. Gentamicin-induced nephrotoxicity is seldom fatal and is usually reversible but often results in long hospital stays. Thus, there is a great interest in finding potential markers for the toxicity event and to further elucidate the toxicity mechanism.We first established our IMS strategy by determining the differential protein expression within the main areas of the rat kidney (cortex, medulla, and papilla) to ensure that we could accurately differentiate the main substructures of this organ. We then investigated whether the kidney lesions sec...

Although both erlotinib and gefitinib target the EGF receptor (EGFR), erlotinib is effective in patients with EGFR wild-type or mutated tumors, whereas gefitinib is only beneficial for patients with activating mutations. To determine whether these differences in clinical outcomes can be attributed to their respective protein interaction profiles, a label-free, quantitative chemical proteomics study was conducted. Using this method, 24 proteins were highlighted in the binding profiles of erlotinib and gefitinib. Unlike gefinitib, erlotinib displaced the ternary complex formed by integrin-linked kinase (ILK), a-parvin, and PINCH (IPP). The docking of erlotinib in the three-dimensional structure of ILK showed that erlotinib has the ability to bind to the ATPbinding site, whereas gefitinib is unlikely to bind with high affinity. As the IPP complex has been shown to be involved in epithelial-to-mesenchymal transition (EMT) and erlotinib sensitivity has been correlated with EMT status, we used a cellular model of inducible transition and observed that erlotinib prevented EMT in a more efficient way than gefitinib by acting on E-cadherin expression as well as on IPP levels. A retrospective analysis of the MERIT trial indicated that, besides a high level of E-cadherin, a low level of ILK could be linked to clinical benefit with erlotinib. In conclusion, we propose that, in an EGFR wild-type context, erlotinib may have a complementary mode of action by inhibiting IPP complex activities, resulting in the slowing down of the metastatic process of epithelial tumors. Mol Cancer Ther; 12(4); 520-9. Ó2013 AACR.

Adult hippocampal neurogenesis is a remarkable form of brain plasticity through which new neurons are generated throughout life. Despite its important roles in cognition and emotion and its modulation in various preclinical disease models, the functional importance of adult hippocampal neurogenesis in human health has not been revealed because of a lack of tools for monitoring adult neurogenesis in vivo. Therefore, we performed an unbiased proteomics screen to identify novel proteins expressed during neuronal differentiation using a human neural stem cell model, and we identified the proteoglycan Glypican-2 (Gpc2) as a putative secreted marker of immature neurons. Exogenous Gpc2 binds to FGF2 and inhibits FGF2-induced neural progenitor cell proliferation. Gpc2 is enriched in neurogenic regions of the adult brain. Its expression is increased by physiological stimuli that increase hippocampal neurogenesis and decreased in transgenic models in which neurogenesis is selectively ablated. Changes in neurogenesis also result in changes in Gpc2 protein level in cerebrospinal fluid (CSF). Gpc2 is detectable in adult human CSF, and first pilot experiments with a longitudinal cohort indicate a decrease over time. Thus, Gpc2 may serve as a potential marker to monitor adult neurogenesis in both animal and human physiology and disease, warranting future studies.