Fig. 5: Profiling of rigid linker variation of ALP2 series and assessing IAP engagement, permeability and ternary complex formation by IPD hits.
From: Expanding the toolbox to develop IAP-based degraders of TEAD transcription factors
a Chemical structure of ALP2 series IPD A536 incorporating a spirocyclic linker and matched IAP- and TEAD- negative controls (A558 and A560, respectively). b Profiling of degradation of HiBiT-TEAD1 (NCI-H2052 cells) following 20 h dose–response treatment with ALP2 IPD (A536, green line) or matched IAP negative control (A558, black line) or TEAD1 negative control (A560, gray line) IPDs. HiBiT signal was normalized to CTG reading, and the HiBiT/CTG ratio was compared to a vehicle control to plot percentage of TEAD1 remaining. Plotted data represent individual data points from three independent biological experiments. Degradation DC50 and Dmax were fitted as described92. c Capillary-based western profiling of endogenous TEAD1 degradation (left panel) and cIAP1 auto-degradation (right panel) in NCI-H2052 cells (dose response, 20 h) by ALP2 IPD A536, its matched IAP negative control, A558 and TEAD negative control, A560 (color scheme as in b). Uncropped blot images are available in Supplementary Data 1. Dose response curves are represented with each concentration denoting mean \( \pm \) SD of n = 2 biologically independent experiments. d IAP cellular target engagement. A cellular IAP target engagement assay was developed based on the displacement of a fluorescent IAP tracer B678 from NanoLuc-tagged cIAP1184–618F616A or XIAP124–497V461E (HEK293T cells) and nanoBRET signal measured for IAP binders or IPDs treated in dose–response (left panel, refer Supplementary Fig. 5d and Supplementary Information Synthetic Chemistry methods for synthesis of B678). Percentage tracer occupancy (based on nanoBRET signal, normalized to DMSO vehicle) was measured for live cells (cIAP1 and XIAP) or cells permeabilized by pre-treatment with digitonin (cIAP1 only). For cIAP1, a cellular Availability Index (AI) was determined by first comparing the fitted IC50 values in live and permeabilized modes to obtain a Relative intracellular availability (RBA) value, then normalizing this to ASX series IAP ligand A255, selected as a cell-permeable control compound with high affinity to BIR3 of cIAP1 and XIAP47. cIAP1 was used for AI determination as most compounds tested have potent cIAP1 binding. Larger AI values represent lower intracellular availability relative to the permeable control A255. Right panel: tabulated IC50, RBA and AI values for ASX series IAP binder reference A255 and IPDs ALP1 A232, ALP2 A531, XB2 A538 and ALP2 A536. e Cellular ternary complex formation. Cellular IAP/IPD/TEAD1 ternary complex formation was measured by treating NCI-H2052 cells co-expressing NanoLuc-tagged cIAP1184–618F616A/Halo-TEAD1 or NanoLuc-tagged XIAP124–497V461E/Halo-TEAD1 with a dose response of IPDs (ALP1 IPD A531 and corresponding IAP or TEAD negative controls A557 and A423, or XB2 IPD A538, or ALP2 IPD A536), alongside NanoGlo Substrate and HaloTag 618 ligand. Measured nanoBRET signals relative to background were fitted to a Gaussian distribution model to calculate Emax and ECmax values for ternary complex formation with TEAD1 and either cIAP1 or XIAP. Data represent mean ± SD for n = 3 biologically independent experiments.
