The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors.

A quantitative nonisotopic solution assay for gelatinases and inhibitors was developed using biotinylated gelatin as enzyme substrate. In this assay, residual biotinylated substrate is sandwiched between avidin-coated plates and streptavidin-peroxidase and is quantified by the peroxidase reaction. This assay was useful for measuring gelatinase activities and defining the activities of gelatinase inhibitors. When 23 tetracycline analogues were compared, significant differences in gelatinase B inhibition were found between various compounds. 4-epioxytetracycline base, 4-epichlortetracycline, meclocyclinesulfosalicylate, and unmodified metacycline and minocycline proved to be the most potent gelatinase B (EC 3.4.24.35) inhibitors. The gelatinase B inhibitory activity of tetracyclines was clearly dissociated from their antimicrobial activity. The effect of high-molecular-weight inhibitors, such as monoclonal antibodies, was also demonstrable in the microtiter plate assay. In view of the pathophysiological function of gelatinases, the definition of gelatinase inhibitors with known efficacy, safety, and side effects is crucial for the treatment of diseases such as rheumatoid arthritis and multiple sclerosis. Particular tetracyclines fulfil these criteria and the described assay is useful for defining other gelatinase-inhibiting lead compounds.