Fig. 4. Structure-activity relationship of Abd LMO2-binding compounds.

Hits identified from the HTS library screen were classified and a family of related compounds identified as the basis of SAR analysis. The chemical structures of the hit matter from the BRET screen were examined and a family of compounds was identified. (A) Chemical structures of the 8 re-synthesized hits (Abd-L5 to Abd-L12) with their respective molecular weight (MW). (B) Dose response inhibition of LMO2-iDAb LMO2_dm3 interaction by compounds Abd-L5 to Abd-L12 (concentration range: 1, 10, and 20 μM). (C) SAR study of Abd-L9 compound as template. The compound was divided into four substituents (named A to D) that were substituted by various other chemical groups to give new compounds. The colored boxes (red, pink, blue, and green) represent the different moieties substituted in the SAR study in positions A, B, C, and D, respectively. Structures are shown of representative compounds from Abd-L15 to Abd-L25. The new compounds were tested by BRET assays with LMO2-iDAb LMO2_dm3 interaction. The different SAR-derived compounds are shown with their MW and the percentage of BRET inhibition of the interaction LMO2-iDAb LMO2_dm3 at 20 μM including Abd-L24 and Abd-L25 modified on their positions B and C, respectively, which do not affect the BRET interaction LMO2-iDAb LMO2_dm3. (D) Dose response effect of the Abd-L15 to Abd-L25 compounds on LMO2-iDAb LMO2_dm3 interaction (Abd concentration used: 5, 10, and 20 μM). Experiments in (B) and (D) were performed twice. Error bars presented in (B) and (D) correspond to mean values ± SD of biological repeats.