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. Author manuscript; available in PMC: 2022 Jul 19.
Published in final edited form as: J Am Chem Soc. 2022 Jan 7;144(2):701–708. doi: 10.1021/jacs.1c03980

Figure 2. Characterization of EN106 binding with FEM1B.

Figure 2.

(a) Synthesis of NJH-2-030. (b) Dose-response of NJH-2-030 inhibition of FEM1B and TAMRA-conjugated FNIP1 interaction assessed by fluorescence polarization. (c, d) NJH-2-030 engagement of FEM1B in cells. HEK293T cells were treated with DMSO or NJH-2-030 (10 μM) for 4 h. Cell lysates were subjected to CuAAC with biotin picolyl azide, probe-modified proteins were avidin-enriched, eluted, and analyzed by SDS/PAGE and Western blotting for FEM1B or loading control GAPDH (c) or analyzed by TMT-based quantitative proteomic profiling (d). Data are from n=3/group. Proteins annotated and highlighted in red showed >2-fold enrichment with probe over DMSO with adjusted p-values=0.001 or p-values<0.001. (e, f) Flow cytometry analysis of GFP-FNIP1 degron levels compared to mCherry levels with EN106 treatment in HEK293T cells for 12 h with either basal levels of FEM1B or with transient FEM1B overexpression. Representative flow cytometry traces of DMSO and EN106 (20 μM) treatment groups shown in (e) and quantified data shown in (f). (g) EN106 inhibits oxygen consumption in HEK293T cells. HEK293T cells were treated with DMSO vehicle or EN106 (10 μM) for 16 h after which mitochondrial oxygen consumption was read out with MitoXpress Xtra reagent. Shown in (b, f, g) are individual biological replicate values and/or average ± sem for n=2-4/group. Significance in (f, g) is expressed as *p<0.05 compared to vehicle-treated controls in each group in (f) or compared to each paired control in (g).