We have exploited the existence of a second copy of the human SMN gene (SMN2) to develop a high-throughput screening strategy to identify potential small molecule therapeutics for the genetic disease spinal muscular atrophy (SMA), which is caused by the loss of the SMN1 gene. Our screening process was designed to identify synthetic compounds that increase the total amount of full-length SMN messenger RNA and protein arising from the SMN2 gene, thereby suppressing the deleterious effects of losing SMN1. A cell-based bioassay was generated that detects SMN2 promoter activity, on which greater than 550,000 compounds was tested. This resulted in the identification of 17 distinct compounds with confirmed biological activity on the cellular primary assay, belonging to nine different structural families. Six of the nine scaffolds were chosen on the basis of their drug-like features to be tested for their ability to modulate SMN gene expression in SMA patient-derived fibroblasts. Five of the six compound classes altered SMN mRNA levels or mRNA splicing patterns in SMA patient-derived fibroblasts. Two of the compound classes, a quinazoline compound series and an indole compound, also increased SMN protein levels and nuclear gem/Cajal body numbers in patient-derived cells. In addition, these two distinct scaffolds showed additive effects when used in combination, suggesting that they may act on different molecular targets. The work described here has provided the foundation for a successful medicinal chemistry effort to further advance these compounds as potential small molecule therapeutics for SMA.

We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. which rely on PCR.

FYVE-type zinc finger-containing phosphoinositide kinase (PIKfyve) catalyzes the formation of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) from phosphatidylinositol 3-phosphate (PI(3)P). PIKfyve has been implicated in multiple cellular processes, and its role in the regulation of toll-like receptor (TLR) pathways and the production of proinflammatory cytokines has sparked interest in developing small-molecule PIKfyve inhibitors as potential therapeutics to treat autoimmune and inflammatory diseases. We developed three orthogonal assays to identify and qualify small-molecule inhibitors of PIKfyve: (1) a purified component microfluidic enzyme assay that measures the conversion of fluorescently labeled PI(3)P to PI(3,5)P by purified recombinant full-length human 6His-PIKfyve (rPIKfyve); (2) an intracellular protein stabilization assay using the kinase domain of PIKfyve expressed in HEK293 cells; and (3) a cell-based functional assay that measures the production of interleukin (IL)-12p70 in human peripheral blood mononuclear cells stimulated with TLR agonists lipopolysaccharide and R848. We determined apparent K values for both ATP and labeled PI(3)P in the rPIKfyve enzyme assay and evaluated the enzyme's ability to use phosphatidylinositol as a substrate. We also tested four reference compounds in the three assays and showed that together these assays provide a platform that is suitable to select promising inhibitors having appropriate functional activity and confirmed cellular target engagement to advance into preclinical models of inflammation.

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Background: The PI3K pathway is a target of significant interest for cancer drug development due to its prominent role in cancer cell growth and survival. However, PI3K pathway inhibition alone has limited efficacy in the presence of oncogenic Ras mutations, which are found in approximately one-third of all human tumors. In preclinical models, efficacy in Ras-mutated tumors has required the inhibition of both PI3K and other Ras-activated pathways. One key function of these pathways is to regulate eIF-4E, a central factor in cap-dependent translation of critical proteins involved in growth and survival (e.g., c-myc and Mcl-1), and anti-tumor activity has been associated with the selective inhibition of translation of these proteins. Simultaneous inhibition of PI3K and other Ras-activated pathways that converge on eIF-4E-mediated protein translation represents an attractive strategy for oncology drug development. Methods and Results: Using rational drug design, we identified a novel chemical series of small molecules that possess potent activity against PI3K and additional Ras-regulated kinases implicated in protein translation. The anti-tumor activity of these multiplex PI3K inhibitors was assessed against a panel of 30 human tumor cell lines comprised of various genetic backgrounds and histotypes. Multiplex PI3K inhibitors demonstrated broad and potent anti-proliferative activity in all cell lines tested (EC50: 10 nM - 500 nM) and induced cell death in Ras-mutated cell lines (e.g., PANC-1, HCT116 and A549). Mechanistically, these multiplex PI3K inhibitors induced caspase activity, inhibited the phosphorylation of AKT, 4E-BP1, ribosomal S6 and eIF-4E, and inhibited the expression of c-myc and Mcl-1 proteins. In contrast, PI3K-selective inhibitors were partially cytostatic in Ras-mutated tumor cell lines, inhibiting cell proliferation by at most 60-90%. PI3K-selective inhibitors also failed to induce significant increases in caspase activity, inhibit eIF-4E phosphorylation, or inhibit c-myc and Mcl-1 expression. Conclusion: We have discovered novel multiplex PI3K inhibitors that demonstrate potent anti-tumor activity in Ras-mutant tumors via inhibition of eIF-4E-mediated protein translation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4491.