This continuing project is focused on developing new approaches, methods, and strategies for the discovery of metalloprotein inhibitors. Metalloproteins are an important class of medicinal targets that are relevant to treating numerous diseases including but not limited to cancer, arthritis, and bacterial infections. Most therapeutics that inhibit metalloproteins employ a metal-binding pharmacophore (MBP) to bind to the active site metal ion. Despite the importance of these MBPs, few studies have focused on methods to identify, optimize, and understand what MBPs are best for a given metalloprotein target of interest. In the last research period (9/2011 - 7/2014, ~34 months at the time of this renewal submission), this program generated several new findings relevant to metalloprotein inhibitors, including: a) screening of an MBP isostere library against several metalloproteins targets and developing sub libraries of hit compounds; b) obtaining thermodynamic and structural data on MBPs in an archetypal metalloprotein (carbonic anhydrase); c) modeling of these interactions using computational approaches in order to understand the molecular basis for our findings; and d) assessing the on-target selectivity of several metalloprotein inhibitors. The previous project period was very productive, yielding ~15 manuscripts, one book chapter, several patent disclosures, and contributing to the formation of a biotechnology startup company. In ongoing studies, we propose to greatly expand our current efforts, as well as explore other areas of investigation relevant to the discovery, development, and optimization of metalloprotein inhibitors. In Specific Aim 1, we will develop new MBP libraries that are rationally tailored toward certain classes of metalloproteins (e.g. those with dinuclear active sites containing two adjacent metal ions). In Specific Aim 2, we will expand our efforts on the structural biology and computational modeling of MBP binding, including an examination of how changes in metal ion active site composition alter MBP affinity. In Specific Aim 3, we will study the selectivity of metalloprotein inhibitors for their target enzymes. We will evaluate the activit of metalloprotein inhibitors in the presence of competing metalloproteins and investigate how treatment with metalloenzyme inhibitors perturbs metal ion homeostasis in living cells. Finally, we will continue to aid investigators around the world by screening our libraries against new metalloprotein targets and by developing sub libraries that take these efforts from hit-to-lead.