Our success in producing large quantities of crystallization-grade proteins led to a small-scale structural genomics project aiming to solve the three-dimensional structures of proteins involved in Type III secretion in Yersinia pestis, the causative agent of plague. Because the Type III secretion system (T3SS) is essential for virulence, the resulting structural information could be used to develop effective countermeasures for this potential agent of bioterrorism. We have already solved 13 novel structures and are in the process of solving more of them, including several protein-protein complexes. In one case, we have already begun the process of structure-assisted drug development. One of the cytotoxic effector proteins that Yersinia injects into mammalian cells via the T3SS, YopH, is a potent eukaryotic-like protein tyrosine phosphatase (PTPase). YopH dephosphorylates several proteins associated with the focal adhesion in eukaryotic cells, thereby enabling the bacterium to avoid phagocytosis and destruction by macrophages. In collaboration with Dr. Terrence Burke Jr. (Laboratory of Medicinal Chemistry, CCR) and Dr. Robert Ulrich (USAMRIID), we are attempting to develop small molecule inhibitors of YopH. Thus far we have identified several compounds that inhibit YopH with IC50 values in the low micromolar range, but it has proven very difficult to co-crystallize these compounds with the enzyme. However, we have determined a high-resolution structure (1.5 ) of the YopH PTPase in complex with a nonhydrolyzable hexapeptide substrate analog, providing us with yet another starting point for the development of inhibitors. Several new approaches are currently being employed (2010) in the search for YopH inhibitors. These include the construction of libraries of compounds in which various functional groups are tethered to a phosphotyrosyl mimetic, substrate-assisted screening (SAS), and soaking YopH crystals with cocktails of drug-like """"""""fragments"""""""" to identify weak binders for further optimization. We have recently expanded our range of targets for structural studies to include virulence factors from other potential agents of bioterrorism, including the variola major (smallpox) virus, Francisella tularensis, the causative agent of tularemia, and Venezuelan Equine Encephalitis Virus (VEEV). Crystal structures of potential molecular targets from all three of these sources have recently been determined. One target, the dual specificity H1 phosphatase encoded by smallpox virus, is currently the focus of another structure-based drug development project. We have recently obtained co-crystal structures of this molecular target in complex with two different small molecules from our fragment cocktails (2009). Both ligands are bound in the active site of the enzyme and may be promising leads for further optimization. The nsp2 protease encoded by VEEV is also a potential target for therapeutic antiviral agents. We have crystallized the catalytic domain of the protease and determined its structure (2009). However, for a variety of reasons, the crystals we obtained are less than ideal for a drug development project. Accordingly, efforts are underway to modify the amino acid sequence of the enzyme in order to improve the properties of the crystals.
