ABSTRACT
The inhibition of the cytosolic isoenzyme BCAT that is expressed specifically in neuronal tissue is likely to be useful for the treatment of neurodegenerative and other neurological disorders where glutamatergic mechanisms are implicated. Compound 2 exhibited an IC50 of 0.8 microM in the hBCATc assays; it is an active and selective inhibitor. Inhibitor 2 also blocked calcium influx into neuronal cells following inhibition of glutamate uptake, and demonstrated neuroprotective efficacy in vivo. SAR, pharmacology, and the crystal structure of hBCATc with inhibitor 2 are described.
Subject(s)
Benzofurans/chemical synthesis , Benzofurans/therapeutic use , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/therapeutic use , Neurodegenerative Diseases/drug therapy , Sulfonamides/chemical synthesis , Sulfonamides/therapeutic use , Transaminases/antagonists & inhibitors , Animals , Benzofurans/chemistry , Calcium/antagonists & inhibitors , Calcium/metabolism , Cells, Cultured , Crystallography, X-Ray , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Glutamic Acid/drug effects , Glutamic Acid/metabolism , Humans , In Vitro Techniques , Models, Molecular , Molecular Structure , Neurons/cytology , Neurons/drug effects , Rats , Rats, Inbred Lew , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.
