Chemical library screening identifies a small molecule that downregulates SOD1 transcription for drugs to treat amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (fALS) accounts for 10% of ALS cases, and about 25% of fALS cases are due to mutations in superoxide dismutase 1 (SOD1). Mutant SOD1-mediated ALS is caused by a gain of toxic function of the mutant protein, and the SOD1 level in nonneuronal neighbors, including astrocytes, determines the progression of ALS (non-cell-autonomous toxicity). Therefore, the authors hypothesized that small molecules that reduce SOD1 protein levels in astrocytes might slow the progression of mutant SOD1-mediated ALS. They developed and optimized a cell-based, high-throughput assay to identify low molecular weight compounds that decrease SOD1 expression transcriptionally in human astrocyte-derived cells. Screening of a chemical library of 9600 compounds with the assay identified two hit compounds that selectively and partially downregulate SOD1 expression in a dose-dependent manner, without any detectable cellular toxicity. Western blot analysis showed that one hit compound significantly decreased the level of endogenous SOD1 protein in H4 cells, with no reduction in expression of ß-actin. The assay developed here provides a powerful strategy for discovering novel lead molecules for treating familial SOD1-mediated ALS.

Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL.

OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.

A new synbiotic, Lactobacillus casei subsp. casei together with dextran, reduces murine and human allergic reaction.

We studied the development of atopic dermatitis-like skin lesions in NC/Nga mice and the allergic symptoms and blood patterns of healthy volunteers during the cedar (Cryptomeria japonica) pollen season in Japan following oral administration of a new synbiotic, Lactobacillus casei subsp. casei together with dextran. The combination of L. casei subsp. casei and dextran significantly decreased clinical skin severity scores and total immunoglobulin E levels in sera of NC/Nga mice that had developed picryl chloride-induced and Dermatophagoides pteronyssinus crude extract-swabbed atopic dermatitis-like skin lesions. During the most common Japanese cedar pollen season, synbiotic L. casei subsp. casei and dextran in humans led to no significant changes in total nasal and ocular symptom scores, in the levels of cedar pollen-specific immunoglobulin E, interferon-gamma and thymus and activation regulated chemokine or in the number of eosinophils in sera, whereas the placebo group showed a tendency for increased levels of cedar pollen-specific immunoglobulin E, thymus and activation regulated chemokine and number of eosinophils, and a decrease in interferon-gamma levels. Thus, the oral administration of synbiotic L. casei subsp. casei together with dextran appears to be an effective supplement for the prevention and treatment of allergic reactions.