Innate immune responses are important for initiation of homeostatic inflammatory responses to pathogens as well as sterile injury. They also initiate activation of the adaptive immune system for long term protection against pathogens. However, inflammation can itself be pathologic. In recent years a number of inflammatory syndromes have been traced to inappropriate activation of the Nod-like Receptor (NLR) branch of the innate immune system. In some instances these derive from pathogen initiated activation, in others it is sterile injury, and in yet others it is due to genetic errors in the receptors themselves. As yet there are no small molecules that inhibit NLR activation or signaling. In this proposal we describe plans to develop assays that will permit high throughput screening for inhibitors of NLR initiated signaling. NLR initiated signaling depends on interaction with downstream adaptors. Many of these interactions occur through so-called CARD domains which mediate receptor - adaptor interaction. CARD domains have been widely studied both structurally and functionally. These studies suggest that they are stable, compact, conserved structures. Conveniently, structuralstudiesshowthattheApaf1-CARD in complex with the Procaspase 9-CARD has N and C termini of both protein's chains that are accessible to solvent and in fairly close proximity, suggesting that these termini could be modified without loss of capacity to interact. In previous work we have exploited a protein fragment complementation system based on the enzyme b-lactamase (Bla). Bla can be split into two fragments which will not spontaneously recombine to form active Bla, but when these fragments are brought into proximity after being fused to two other interacting proteins, the fragments will recombine to form active Bla. In this work we propose to fuse the fragments of Bla to different, interacting CARD domains, such as the Apaf1 and Procaspase 9 CARD domains, express the two chimeric proteins in the same cell, and test for Bla function. Our preliminary results show that this does occur with Apaf1-CARD and Procaspase 9-CARD. We will then develop stable cell lines expressing the chimeras and use them to identify inhibitors of the interaction in small molecule libraries that are either commercially available or available through the NIH Molecular Library Screening Center Network. The inhibitors will be initially characterized for their ability to inhibit the CARD- CARD interactions and then for their ability to inhibit signaling initiated by the respective NLR.
Inflammation is a homeostatic response to infection as well as tissue injury but when it occurs inappropriately it can cause pathologic damage. This proposal describes development of a system to identify new inhibitors of inflammation that may ultimately be useful for drug development as well as basic research.
