The desire to inhibit zinc-dependent matrix metalloproteinases (MMPs) has, over 
the course of the last 30 years, led to the development of a plethora of MMP 
inhibitors that bind directly to the active-site metal. With one exception, all 
of these drugs have failed in clinical trials, due to many factors, including an 
apparent lack of specificity for MMPs. To address the question of whether these 
inhibitors are selective for MMPs in a biological setting, a cell-based 
screening method is presented to compare the relative activities of zinc, heme 
iron, and non-heme iron enzymes in the presence of these compounds using the 
RAW264.7 macrophage cell line. We screened nine different zinc-binding groups 
(ZBGs), four established MMP inhibitors (MMPis), and two novel MMP inhibitors 
developed in our laboratory to determine their selectivities against five 
different metalloenzymes. Using this model, we identified two nitrogen donor 
compounds--2,2'-dipyridylamine (DPA) and triazacyclononane (TACN)--as the most 
selective ZBGs for zinc metalloenzyme inhibitor development. We also 
demonstrated that the model could predict known nonspecific interactions of some 
of the most commonly used MMPis, and could also give cross-reactivity 
information for newly developed MMPis. This work demonstrates the utility of 
cell-based assays in both the design and the screening of novel metalloenzyme 
inhibitors.