Utilization of Nitrophenylphosphates and Oxime-Based Ligation for the Development of Nanomolar Affinity Inhibitors of the Yersinia Pestis Outer Protein H (YopH) Phosphatase

摘要

Our current study reports the first KM optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (KM = 80 μM was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime-ligation. A co-crystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employed for the design of furanyl-based oxime derivatives. By this process, a potent (IC50 = 190 nM) and non-promiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a non-cytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH.