The fusion proteins of many enveloped viruses are activated by a proteolytic priming step. The priming event utilizes a host cell protease to make a specific cleavage in the envelope (fusion) protein, often in the immediate vicinity of the fusion peptide. Exposure of the fusion peptide is a critical part of the virus entry process, and without proteolytic priming, virus infection cannot be initiated. While proteases are important drug targets for many diseases, the targeting of host cell proteases involved in virus entry has received little attention. Host cell proteases are usually under tight regulation, and the host has evolved highly specific inhibitors, which have high affinity for their natural protease. We propose to take advantage of such natural inhibitors as a host-targeted therapeutic approach for influenza and other respiratory viruses. In the case of viral infection, it is apparent that in many cases priming is not occurring via a single protease, but rather via a sub-set of related proteases expressed in a given tissue. Thus a single natural (or modified-natural) inhibitor with some degree of broad specificity is likely to be a viable anti-viral therapeutic. As several virus families likely share the same or overlapping activating proteases, a single inhibitor is also likely to be active against a sub-set of viruses in distinct families. We have recently developed a kunitz-type protease inhibitor (SPINT2 or HAI-2) as a lead therapeutic for treatment of epidemic and pandemic influenza, as well as other enveloped viruses including paramyxoviruses. Our focus in the project will be influenza A and B viruses, as well as human metapneumovirus and human parainfluenza virus 1. We propose to characterize our lead candidate inhibitor, both in vitro and in cell culture, including primary respiratory tract cells, and to develop engineered derivates with improved potency. Given that many viruses are activated by host cell proteases, we feel this strategy is a prudent one, with our eventual goal being to develop a system that can be used to treat multiple infectious agents.