Inhibition of KDM1A activity restores adult neurogenesis and improves hippocampal memory in a mouse model of Kabuki syndrome.

Kabuki syndrome (KS) is a rare cause of intellectual disability primarily caused by loss-of-function mutations in lysine-specific methyltransferase 2D (KMT2D), which normally adds methyl marks to lysine 4 on histone 3. Previous studies have shown that a mouse model of KS (Kmt2d +/ßGeo ) demonstrates disruption of adult neurogenesis and hippocampal memory. Proof-of-principle studies have shown postnatal rescue of neurological dysfunction following treatments that promote chromatin opening; however, these strategies are non-specific and do not directly address the primary defect of histone methylation. Since lysine-specific demethylase 1A (LSD1/KDM1A) normally removes the H3K4 methyl marks added by KMT2D, we hypothesized that inhibition of KDM1A demethylase activity may ameliorate molecular and phenotypic defects stemming from KMT2D loss. To test this hypothesis, we evaluated a recently developed KDM1A inhibitor (TAK-418) in Kmt2d +/ßGeo mice. We found that orally administered TAK-418 increases the numbers of newly born doublecortin (DCX)+ cells and processes in the hippocampus in a dose-dependent manner. We also observed TAK-418-dependent rescue of histone modification defects in hippocampus both by western blot and chromatin immunoprecipitation sequencing (ChIP-seq). Treatment rescues gene expression abnormalities including those of immediate early genes such as FBJ osteosarcoma oncogene (Fos) and FBJ osteosarcoma oncogene homolog B (Fosb). After 2 weeks of TAK-418, Kmt2d +/ßGeo mice demonstrated normalization of hippocampal memory defects. In summary, our data suggest that KDM1A inhibition is a plausible treatment strategy for KS and support the hypothesis that the epigenetic dysregulation secondary to KMT2D dysfunction plays a major role in the postnatal neurological disease phenotype in KS.

T-448, a specific inhibitor of LSD1 enzyme activity, improves learning function without causing thrombocytopenia in mice.

Dysregulation of histone H3 lysine 4 (H3K4) methylation has been implicated in the pathogenesis of several neurodevelopmental disorders. Targeting lysine-specific demethylase 1 (LSD1), an H3K4 demethylase, is therefore a promising approach to treat these disorders. However, LSD1 forms complexes with cofactors including growth factor independent 1B (GFI1B), a critical regulator of hematopoietic differentiation. Known tranylcypromine-based irreversible LSD1 inhibitors bind to coenzyme flavin adenine dinucleotide (FAD) and disrupt the LSD1-GFI1B complex, which is associated with hematotoxicity such as thrombocytopenia, representing a major hurdle in the development of LSD1 inhibitors as therapeutic agents. To discover LSD1 inhibitors with potent epigenetic modulation and lower risk of hematotoxicity, we screened small molecules that enhance H3K4 methylation by the inhibition of LSD1 enzyme activity in primary cultured rat neurons but have little impact on LSD1-GFI1B complex in human TF-1a erythroblasts. Here we report the discovery of a specific inhibitor of LSD1 enzyme activity, T-448 (3-((1S,2R)-2-(cyclobutylamino)cyclopropyl)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzamide fumarate). T-448 has minimal impact on the LSD1-GFI1B complex and a superior hematological safety profile in mice via the generation of a compact formyl-FAD adduct. T-448 increased brain H3K4 methylation and partially restored learning function in mice with NMDA receptor hypofunction. T-448-type LSD1 inhibitors with improved safety profiles may provide unique therapeutic approaches for central nervous system disorders associated with epigenetic dysregulation.

Purification of human papillomavirus-like particles expressed in silkworm using a Bombyx mori nucleopolyhedrovirus bacmid expression system.

A three-stage chromatography protocol for the purification of human papillomavirus-like particles (HPV-LPs) from the silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system was developed. For host cell DNA separation, anion exchange chromatography was used after screening for a suitable stationary phase. Using the two separation principles of cation exchange chromatography and metal affinity of ceramic hydroxyapatite (CHT) as a second stage, the amount of baculovirus in the sample was reduced to less than the detection limit of qPCR. The CHT separation was optimized with respect to the elution buffer used; 150-600 mM sodium phosphate, pH 7.2, resulted in the highest recovery of HPV-LPs. Using heparin chromatography, it was possible to reduce the sample volume and to thus highly concentrate the target protein during the separation of contaminating proteins. During the second purification stage, over 99.3% of the DNA was removed, and no infectious baculoviruses remained. After concentration by heparin column chromatography, over 99.9% of the DNA and protein had been removed. The purity achieved by this method exceeds that obtained by DDDDK-tag-based affinity chromatography and sucrose gradient ultracentrifugation, which were used as comparative purification methods. The 3-stage purification of HPV-LPs from silkworm fat bodies described here was a proof of concept and is a scalable method, but the overall yield remains to be improved.