Screening in a cell-based assay for inhibitors of microglial nitric oxide production reveals calmodulin-regulated protein kinases as potential drug discovery targets

A high-throughput screening (HTS) assay for inhibitors of nitric oxide (NO) production by activated microglia was developed and used to compare the re lative activities of various anti-inflammatory compounds and cell-permeable protein kinase inhibitors. BV-2 cells, an immortalized Line that retains p henotypic features of microglia and produces NO in response to lipopolysacc haride (LPS), were used in the activation paradigm for the HTS assay. A cha racteristic feature of the compounds that were the most potent dose-depende nt inhibitors of NO production is their ability to modulate serine/threonin e protein kinases. The anti-inflammatory compound K252a, an inhibitor of ca lmodulin (CaM)-regulated protein kinases, had one of the highest potencies in the assay. Other classes of kinase inhibitors, including the protein kin ase A inhibitor H-89, the mitogen activated protein kinase inhibitors PD980 59 and SB203580, and the tyrosine kinase inhibitor genistein, were less pot ent and efficacious than K252a or the general serine/threonine/tyrosine kin ase inhibitor staurosporine. K252a suppresses production of the inducible n itric-oxide synthase (iNOS). The inhibitory effect of K252a is not due to c ell toxicity and does not correlate with inhibition of NF kappa B nuclear t ranslocation. The mechanism of action appears to involve inhibition of phos phorylation of the transcription factor CREB, a protein whose activity is m odulated by phosphorylation by CaM-dependent protein kinases. These data su ggest that signal transduction pathways mediated by CaM-dependent protein k inases warrant future study as potential drug discovery targets. (C) 1999 P ublished by Elsevier Science B.V. All rights reserved.