From proteomics to discovery of first-in-class ST2 inhibitors active in vivo
Abdulraouf M. Ramadan, Etienne Daguindau, Jason C. Rech, Krishnapriya Chinnaswamy, Jilu Zhang, Greg L. Hura, Brad Griesenauer, Zachary Bolten, Aaron Robida, Martha Larsen, Jeanne A. Stuckey, Chao-Yie Yang, Sophie Paczesny
Abdulraouf M. Ramadan, Etienne Daguindau, Jason C. Rech, Krishnapriya Chinnaswamy, Jilu Zhang, Greg L. Hura, Brad Griesenauer, Zachary Bolten, Aaron Robida, Martha Larsen, Jeanne A. Stuckey, Chao-Yie Yang, Sophie Paczesny
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Research Article Therapeutics Transplantation

From proteomics to discovery of first-in-class ST2 inhibitors active in vivo

Abstract

Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. Here, we report the discovery of small-molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, 3 compounds were selected for evaluation in 2 experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2.

Authors

Abdulraouf M. Ramadan, Etienne Daguindau, Jason C. Rech, Krishnapriya Chinnaswamy, Jilu Zhang, Greg L. Hura, Brad Griesenauer, Zachary Bolten, Aaron Robida, Martha Larsen, Jeanne A. Stuckey, Chao-Yie Yang, Sophie Paczesny

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Figure 3

IC50 values of iST2-1 and its analogs, SAXS studies of apo-ST2, ST2/iST2-1, and potential iST2-1 binding sites identified from computational simulations.

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IC50 values of iST2-1 and its analogs, SAXS studies of apo-ST2, ST2/iST2...
(A) Chemical structures of racemic iST2-1, (R)-iST2-1, (S)-iST2-1, analogs of iST2-1, and structural alignment of (R)-iST2-1 (purple) with (S)-iST2-1 (green). IC50 values of the inhibitors are shown in parentheses. (B) Inhibition curves and IC50 values (in parentheses) of 4 representative compounds. Each data point is an average of triplicate measurements ± SD. (C) SAXS profiles, the residual plot between the SAXS profiles of apo-ST2 and ST2/iST2-1, comparison of the Kratky plot based on the SAXS profiles of apo-ST2 and ST2/iST2-1, the pair-wise distance distribution [P(r)] of apo-ST2 and ST2/iST2-1 calculated from the SAXS profiles. Dmax values are shown in parentheses. (D) Ab initio shape reconstruction of apo-ST2 (gray) and ST2/iST2-1 (purple). (E) Mapping of the (R)-iST2-1 and (S)-iST2-1 binding sites (surface envelop) in ST2 based on 32-ns MD simulations. Maps detected from 13- and 15-Å octahedron boxes are colored in purple and green, respectively. Consensus binding sites (S1r, S2r and S1s) are circled. The D1 and D2 domains of ST2 are labeled. (F) Binding sites of (R)-iST2-1 (green mesh) and (S)-iST2-1 (red mesh) that block interaction between ST2 and IL-33 (orange) are highlighted. (G) Binding sites of (R)-, (S)-, (S,S)-iST2-1 in the glycosylated ST2 from the MD simulations. (S,S)-iST2-1 is (S)-iST2-1 in which the proton on the pyrrolidine group is in the S form and its mapped site is shown in cyan mesh. SAXS, small-angle X-ray scattering.