Development of HTS for discovery of IL-25 inhibitors

About This Grant

ABSTRACT Members of the IL-17 family (IL-17A-F) and its cognate receptors (IL-17RA-RE) mediate physiologically important immune responses, however, they can also drive inflammatory diseases, such as allergic asthma in case of IL-25. Despite its emerging clinical need, no drugs against IL-25 or its signaling pathway are currently clinically available. Our long-term goal is to identify small molecule inhibitors of the IL-25 pathway to probe and prevent IL-25-mediated diseases. The goal of this exploratory R21 application is to establish and validate a high throughput screening (HTS) system as well as relevant orthogonal assays that will enable successful IL-25 small molecule drug discovery using HTS-based hit identification and a defined hit advancement strategy. One major barrier for small molecule drug (SMD) development targeting IL-25 (and other members of the IL-17 family) is that the defined part of the signaling cascade relies on a series of hierarchically acting protein interactions that are inherently difficult for targeted drug development: IL-25 initiates signaling via binding and dimerization of IL-17 receptor RA- and RB-chains, which leads to recruitment/ dimerization of the common IL- 17R adaptor ACT1, followed by recruitment/ dimerization of TRAF6. IL-25 and IL-17RA/RB define the IL-25- specific signaling pathway, while ACT1 defines the common IL-17R family pathway. TRAF6, in turn, defines the boundary between various IL-17R family members and common, IL-17R-non-specific downstream pathways. Thus, signaling events up-stream of ACT1 represent a ‘drug target window’ that defines IL-25-specific inhibitors. Signaling events between ACT1 and TRAF6 represent a ‘drug target window’ that defines common inhibitors of the IL-17 family, including IL-17A as major driver of Th17 immune responses and inflammatory diseases. To overcome limitations related to targeted drug development, we developed a unique, cellular high throughput screening (HTS) platform that retains the advantages of phenotypic screening, i.e. the testing of complex responses in the natural environment of cells and, at the same time, eliminates non-specific compounds, thereby mitigating the major disadvantage of phenotypic screening. In this application, we propose (i) to validate already established reporter cell lines on robots in HTS/ 386 well- format and perform a small 3.6k pilot screen to test and calibrate our screening tool and (ii) to establish and validate a set of orthogonal assays that are suitable to advance IL-25-inhibitory compounds in a full-scale drug discovery campaign. We expect that establishment of these key components of HTS-based drug discovery (along with medicinal chemistry, pharmacology and clinical support by collaborators) will put us in an ideal position for future work to (i) identify specific IL-25-inhibitory small molecule compounds and (ii) develop them towards lead compounds with suitable drug-like properties to be used as probes and starting point for future drug development.