SMN protein is required throughout life to prevent spinal muscular atrophy disease progression.

Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient

Discovery of novel small molecule inhibitors of VEGF expression in tumor cells using a cell-based high throughput screening platform.

Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal homeostatic state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5\u27 and 3\u27 untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies

The minor gentamicin complex component, X2, is a potent premature stop codon readthrough molecule with therapeutic potential.

Nonsense mutations, resulting in a premature stop codon in the open reading frame of mRNAs are responsible for thousands of inherited diseases. Readthrough of premature stop codons by small molecule drugs has emerged as a promising therapeutic approach to treat disorders resulting from premature termination of translation. The aminoglycoside antibiotics are a class of molecule known to promote readthrough at premature termination codons. Gentamicin consists of a mixture of major and minor aminoglycoside components. Here, we investigated the readthrough activities of the individual components and show that each of the four major gentamicin complex components representing 92-99% of the complex each had similar potency and activity to that of the complex itself. In contrast, a minor component (gentamicin X2) was found to be the most potent and active readthrough component in the gentamicin complex. The known oto- and nephrotoxicity associated with aminoglycosides preclude long-term use as readthrough agents. Thus, we evaluated the components of the gentamicin complex as well as the so-called "designer" aminoglycoside, NB124, for in vitro and in vivo safety. In cells, we observed that gentamicin X2 had a safety/readthrough ratio (cytotoxicity/readthrough potency) superior to that of gentamicin, G418 or NB124. In rodents, we observed that gentamicin X2 showed a safety profile that was superior to G418 overall including reduced nephrotoxicity. These results support further investigation of gentamicin X2 as a therapeutic readthrough agent

Discovery of Novel Small Molecule Inhibitors of VEGF Expression in Tumor Cells Using a Cell-Based High Throughput Screening Platform

<div><p>Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal “homeostatic” state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5’ and 3’ untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS^™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies.</p></div

Structure and biological activity of a representative HTS hit, PTC-858.

<p>(<b>A</b>) Chemical structure of PTC-858 (6-bromo-1-(1H-pyrrol-1-yl)-2,3,4,9-tetrahydro-1H-carbazole). (<b>B</b>) Inhibition of VEGF production in HeLa cells by PTC-858. Results are expressed as percent inhibition of hypoxia-induced VEGF production relative to vehicle-treated controls from a representative study. Cytotoxicity was performed in parallel to the VEGF ELISA using a Celltiter Glo assay kit (Promega). (<b>C</b>) Western blot analysis of effects of PTC-858 on membrane bound VEGF expression in HT-1080 cells. The same amount of protein for each cell lysate was loaded for western blot analysis. β-actin was used as an internal loading control. (<b>D</b>) PTC-858 preferentially inhibits reporter gene expression under the control of VEGF UTRs compared to the control UTRs derived from the vector. The stable cell lines B9 and B12 used in this study were generated in HEK293 cells transfected with the constructs shown in the diagrams on the top of the graphs. The activity of luciferase was measured with the substrate Bright-Glow (Promega). (<b>E</b>) PTC-858 has no dose-dependent effects on VEGF mRNA expression. Endogenous VEGF and β-actin message levels in the HeLa cells were determined using real-time PCR (pre-designed primer sets purchased from Applied Biosystems). Results shown in the graph from a typical study were normalized to the internal control of β-actin. Assay was done in triplicate and average VEGF mRNA levels from DMSO treated samples were arbitrarily set at 1.</p

Discovery and Optimization of Indolyl-Containing 4‑Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure–activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved <i>in vitro</i> antibacterial activity against fluoroquinolone resistant strains. Compounds <b>6o</b> and <b>6v</b> are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds <b>6o</b> and <b>6v</b> showed improved MIC₉₀ values against <i>Escherichia coli</i> (0.5–1 μg/mL) and <i>Acinetobacter baumannii</i> (8–16 μg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of <i>E. coli</i>

Selectivity and cytotoxicity of two HTS hits.

<p>Selectivity and cytotoxicity of two HTS hits.</p