Yersinia pestis, the causative agent of plague, is considered to be one of the most likely instruments of bioterrorism. Like many Gram-negative bacterial pathogens, Y. pestis utilizes a type III (contact dependent) secretion system (TTSS) to inject cytotoxic effector proteins directly into eukaryotic cells where they interfere with signaling pathways that regulate inflammation and cytoskeleton dynamics, thereby enabling the bacteria to avoid engulfment and destruction by macrophages and other professional phagocytes. Our objective is to facilitate the development of anti-plague therapeutics by attempting to solve the three-dimensional structures of key virulence factors associated with the TTSS in Y. pestis, which has been termed the Yop (Yersinia outer protein) virulon. Because these """"""""molecular terrorists"""""""" play a critical role in pathogenesis, it is likely to be difficult if not impossible to engineer weaponized variants of Y. pestis that are resistant to antivirulence drugs but that can still cause disease. ? ? The TTSS is comprised of approximately 50 different proteins, and a null mutation in almost any of them abrogates virulence. Accordingly, nearly all of these proteins are potential molecular targets for anti-plague therapeutics. Ours is a genome-driven, bottom-up approach to target selection. Believing that the drug discovery process works most effectively when structural biology and screening approaches can be used in concert, our strategy is first to determine which potential targets are amenable to structure-based approaches and then prioritize them on the basis of what is currently known about their biological functions and mechanisms of action. Twelve novel protein structures have been determined to date: YopM, a cytotoxic agent of unknown function; YopE, a GTPase activating protein that specifically targets RhoA, Rac1 and Cdc42; SycE, the cognate secretion chaperone for YopE; the N-terminal domain of YopH, a phosphotyrosine-dependent protein-protein interaction module; LcrV (V antigen), a protective antigen and key regulator of type III secretion; the type III secretion chaperone SycH in complex with a fragment of the regulatory protein YscM2; a ternary complex between the regulatory protein YopN and its heterodimeric secretion chaperone SycN/YscB; a binary complex between YopN and another regulatory protein TyeA; YscE, a soluble protein of unknown function; and the protease resistant core domain of YopR, another virulence factor of unknown function.? ? Historically, enzymes have been the most successful therapeutic targets for structure-based drug design. Protein Tyrosine Phosphatases (PTPases) are signaling enzymes that participate in the regulation of numerous cellular functions, including growth, mitogenesis, motility, cell-cell interactions, metabolism, gene transcription, and the immune response. Because the aberrant action of PTPases has been linked to debilitating diseases like cancer, diabetes, osteoporosis, and immune dysfunctions, the development of PTPase inhibitors is a very active area of research in the pharmaceutical industry. Indeed, highly potent and specific inhibitors of some mammalian PTPases have already been described, helping to establish proof-of-principal for the concept. One of the cytotoxic effector proteins that Y. pestis injects into mammalian cells via the TTSS, YopH, is a potent eukaryotic-like PTPase. YopH dephosphorylates several proteins associated with the focal adhesion in eukaryotic cells. Because the PTPase activity of YopH is essential for virulence and the enzyme crystallizes readily, we view it as a particularly promising target for therapeutic intervention. Accordingly, we are collaborating with Dr. Terrence Burke (Laboratory of Medicinal Chemistry, NCI), a chemist with considerable expertise in the field of PTPase inhibitor design, and Dr. Robert Ulrich (USAMRIID) to develop inhibitors of the YopH PTPase.
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