Bright and stable luminescent probes for target engagement profiling in live cells

Resolving the deceptive isoform and complex selectivity of HDAC1/2 inhibitors

“…We next profiled a HDACi reference set (Figure 1A), which, in addition to Cpd-60, included CI-994 and the hydroxamates SAHA (vorinostat) and LBH-589 (panobinostat). Dose-response titration using optimized assay conditions for recombinant HDAC isoforms and lysate-based CoREST complex yielded K D values for LBH-589, SAHA, and CI-994 that are in good agreement with literature data and our previously reported TR-FRET platform for isoform-specific HDAC1 profiling (Figure 1D and Table 1) (Bantscheff et al, 2011;Becher et al, 2014;Fuller et al, 2019;Payne et al, 2021). However, we were surprised to find that, even after long (6 h) incubation to ensure equilibrium binding, the affinity of Cpd-60 (K D = 143 mM; 95% confidence interval [CI] = 70-505 mM) was more similar to published values for other HDAC1/2 corepressor complexes (e.g., SIN3 and NuRD) than reported for immunoprecipitated CoREST complex (half-maximal inhibitory concentration [IC 50 ] = 0.16 mM) (Fuller et al, 2019).…”

“…To address this problem and enable selective profiling of HDA-Cis for specific HDACs in the presence of other isoforms, we recently reported a new class of TR-FRET probes, termed Cora-Fluors, and applied them in the development of isoform-specific, cell-free, and live-cell ligand displacement assays for HDAC1 (Payne et al, 2021). We speculated that the pairwise combination of a TR-FRET-donor-labeled antibody, specific for the complex of interest, with an acceptor-labeled HDAC tracer ligand (fluorescein isothiocyanate labeled suberoylanilide hydroxamic acid [SAHA-FITC], 1; Figure 1A) would provide a reliable assay platform capable of directly probing endogenous HDAC-containing corepressor complexes in cell extracts (Figure 1B).…”

“…To better understand the molecular basis of the contextdependent activity of HDACis, we here establish an extension of our recently reported CoraFluor time-resolved Fo ¨rster resonance energy transfer (TR-FRET) assay methodology to enable the complex-specific profiling of small molecule HDACis (Payne et al, 2021). Our data provide strong evidence that Cpd-60 is devoid of HDAC1/2 selectivity over HDAC3.…”

“…Commercial IgG antibodies (Anti-6xHis, Abcam 18184; Anti-GST, Abcam 19256; anti-LSD1, Abcam 224270; anti-FLAG M2, Sigma F1804) and nano-secondary alpaca anti-rabbit IgG (ChromoTek, shurbGNHS-1) were labeled with CoraFluor-1-Pfp and AF488-Tfp ester (ThermoFisher A37570) as previously described (Payne et al, 2021). The following extinction coefficients were used to calculate protein concentration and degree-of-labeling (DOL): IgG ε 280 = 210,000 M -1 cm -1 , ChromoTek shurbGNHS-1 ε 280 = 24,075 M -1 cm -1 , CoraFluor-1-Pfp ε 340 = 22,000 M -1 cm -1 , AF488-Tfp ε 495 = 71,000 M -1 cm -1 .…”

Luminescent Lifetime Regulation of Lanthanide-Doped Nanoparticles for Biosensing

“…An efficient energy transfer pathway could be established in fluorescence dye-loaded rare-earth nanocrystals, which enables luminescence lifetime tuning [ 53 ]. In contrast to conventional molecular donor–acceptor pairs, the energy transfer efficiency is related to the distances between lanthanide-doped nanoparticles, and thus significantly depends on the nanoparticle diameter.…”