Mats Ormö

Discovery of novel potent and highly selective glycogen synthase kinase-3β (GSK3β) inhibitors for Alzheimer's disease: design, synthesis, and characterization of pyrazines.

Glycogen synthase kinase-3β, also called tau phosphorylating kinase, is a proline-directed serine/threonine kinase which was originally identified due to its role in glycogen metabolism. Active forms of GSK3β localize to pretangle pathology including dystrophic neuritis and neurofibrillary tangles in Alzheimers disease (AD) brain. By using a high throughput screening (HTS) approach to search for new chemical series and cocrystallization of key analogues to guide the optimization and synthesis of our pyrazine series, we have developed highly potent and selective inhibitors showing cellular efficacy and blood-brain barrier penetrance. The inhibitors are suitable for in vivo efficacy testing and may serve as a new treatment strategy for Alzheimers disease.

An ultrafiltration assay for nucleotide binding to ribonucleotide reductase

Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C.

Correlation between active form and dimeric structure of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart.

The active form of purified mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated by crosslinking with dimethylsuberimidate and SDS-PAGE, with or without pretreatment with the inactivating detergent Triton X-100. In the absence of detergent, crosslinked isomers of the dimeric form of 208–235 kDa were obtained. Addition of detergent led to the simultaneous loss of the dimers and the bulk of the activity. Removal of the detergent led to a partial restoration of both activity and the dimeric forms. The results suggest that the active form is a dimer, and that the detergent-dependent conversion to the largely inactive monomer is reversible. It is proposed that the mechanism of inactivation of transhydrogenase by Triton X-100 involves a disruption of essential hydrophobic interactions between the membrane-spanning regions of the monomers.

Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli

The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues. In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis. The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol. The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site. Our classification of redox-active cysteines differs from the location of the active site cysteines in E. coli ribonucleotide reductase suggested previously (Lin, A.-N. I., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905-6909).