Evaluation of cyclooxygenase-2 inhibitors using pulsed ultrafiltration mass spectrometry

Since selective inhibition of the inducible form of cyclooxygenase (COX-2) might retain all the benefits of classical nonsteroidal antiinflammatory ag ents while avoiding the major side effects associated with inhibition of th e constitutive isoform COX-1, COX-2 has become an important target for the discovery and development of new antiinflammatory drugs. To aid in the disc overy and characterization of such selective inhibitors, we have applied a mass spectrometry-based screening technique, pulsed ultrafiltration mass sp ectrometry, using COX-2 as the target. In a blind study, 18 samples enriche d with one or more inhibitors of COX-2 were evaluated. The matrixes for the test samples consisted of DMSO, r DMSO solutions of a plant extract, or a bacterial fermentation broth extract. The composition of the samples was un known during the assays, as were the concentrations of the COX-2 inhibitors . A soluble recombinant form of human COX-2 was incubated with each sample, and then an aliquot of each mixture was injected into the stirred ultrafil tration chamber fitted with a 30 000 MW cutoff ultrafiltration membrane. Af ter the unbound and weakly bound compounds were washed away, the ligand-rec eptor complexes were disrupted using an acidified 10% methanol solution. Th e released ligands were trapped on a Cls cartridge and then identified usin g liquid chromatography-negative ion electrospray mass spectrometry with th e trapping cartridge as the HPLC column. Neither the plant matrix nor the f ermentation broth extract were found to interfere with the assay. Two or th ree ligands for COX-2 were identified in each sample, which included polar and nonpolar compounds and inhibitors with IC50 values ranging from 100 mu M to 10 nM.