Discovery of ONO-8590580: A novel, potent and selective GABAA α5 negative allosteric modulator for the treatment of cognitive disorders.

The identification and SAR development of a series of negative allosteric modulators of the GABAA α5 receptor is described. This novel series of compounds was optimised to provide analogues with high GABAA α5 binding affinity, high α5 negative allosteric modulatory activity, good functional subtype selectivity and low microsomal turnover, culminating in identification of ONO-8590580.

Refinement of the dentinogenesis imperfecta type II locus to an interval of less than 2 centiMorgans at chromosome 4q21 and the creation of a yeast artificial chromosome contig of the critical region.

Dentinogenesis imperfecta type II is an autosomal-dominant disorder of dentin formation which has been mapped to the 6.6 centiMorgan D4S2691-D4S2692 interval at human chromosome 4q21. In the current investigation, the use of four short tandem repeat polymorphisms has allowed the critical region to be refined to an interval of less than 2 centiMorgans defined by recombination events in unrelated, affected individuals from two families both of which show independent evidence for linkage to chromosome 4q21. The creation of a yeast artificial chromosome contig of this newly defined interval has allowed us to demonstrate that the critical region encompasses approximately 2 Mb of DNA and that the dentin-specific gene, dentin sialoprotein, maps to this interval within 300 kb of dentin matrix acidic phosphoprotein 1 and bone sialoprotein. Moreover, dentin sialoprotein shows no recombination with the dentinogenesis imperfecta type II phenotype. Dentin sialoprotein is therefore a candidate for the dentinogenesis imperfecta type II locus.

Elucidation of the sequence and the genomic organization of the human dentin matrix acidic phosphoprotein 1 (DMP1) gene: exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II.

The dentin matrix acidic phosphoprotein 1 (DMP1) gene has been mapped to human chromosome 4q21 and shown to exhibit no recombination with the autosomal dominant disorder of dentin formation, dentinogenesis imperfecta type II. In the current study, sequencing of DMP1 cDNA and genomic clones has indicated that the human gene contains an open reading frame of 1539 bp, which predicts a highly acidic, serine-rich protein of 513 amino acids. Comparison of the human DMP1-coding sequence with that of the rat, mouse, and cow indicated that the predicted protein contains a conserved hydrophobic signal peptide sequence and an Arg-Gly-Asp cell attachment sequence. The gene is encoded by six exons, the splicing phase of which is type 0, the first exon containing solely 5' untranslated sequence. Sequencing of each of the coding exons in individuals affected by dentinogenesis imperfecta type II failed to reveal any disease-specific mutations, suggesting that mutations in DMP1 are not causative of this condition at least in the two families examined in this study.

Cloning and expression analysis of the bovine dentin matrix acidic phosphoprotein gene.

The dentin matrix acidic phosphoprotein gene has been mapped to human chromosome 4q21 and mouse chromosome 5q21. Expression studies have implicated a role for this gene in the mineralization of dentin. In the current investigation, a cDNA encoding bovine dentin matrix acidic phosphoprotein has been cloned and sequenced. A comparison of the bovine gene with its rat counterpart has indicated that the genes are conserved (67.4% identity; 79.5% similarity), particularly in the region of presumed functional elements such as the hydrophobic signal peptide sequence, the cell attachment Arg-Gly-Asp tripeptide, and numerous serine residues which are likely candidates for phosphorylation. Zoo blot analysis further indicated that a similar gene is found in all mammalian species tested, but not in chicks. However, Northern analysis has indicated that in the cow the message is detectable at high levels in fetal bovine brain and cultured long bone as well as in odontoblasts. These results support a potential role for dentin matrix acidic phosphoprotein in dentinogenesis.

Mapping of the human dentin matrix acidic phosphoprotein gene (DMP1) to the dentinogenesis imperfecta type II critical region at chromosome 4q21.

Dentinogenesis imperfecta type II (DGI1) is an autosomal dominant disorder of dentin formation, which has been mapped to human chromosome 4q12-q21. The region most likely to contain the DGI1 locus is a 3.2-cM region surrounding the osteopontin (SPP1) locus. Recently, a novel dentin-specific acidic phosphoprotein (dmp1) has been cloned in the rat and mapped to mouse chromosome 5q21. In the current investigation, we have isolated a cosmid containing the human DMP1 gene. The isolation of a short tandem repeat polymorphism at this locus has allowed us to map the DMP1 locus to human chromosome 4q21 and demonstrate that it is tightly linked to DGI1 in two families (Zmax = 11.01, theta = 0.001). The creation of a yeast artificial chromosome contig around SPP1 has further allowed us to demonstrate that DMP1 is located within 150 kb of the bone sialoprotein and 490 kb of the SPP1 loci, respectively. DMP1 is therefore a strong candidate for the DGI1 locus.