In this study, we have performed a qHTS to identify compounds that inhibit the interaction between TRβ and SRC2-2. The titration-response screen of over 290,000 samples enabled us to determine the activity of each compound as well as the potency, efficacy, and other pharmacological measures of the actives. Using only the primary qHTS data, actives were prioritized, interfering compounds were identified, and nascent structure-activity relationships were derived. This analysis allowed the selection of a small number of compounds for retest and the inclusion of analogs and inactives for series of interest. Confirmation studies of select actives with sufficient potency and chemical tractability identified a MSNB containing series as a new class of thyroid hormone receptor-coactivator antagonists.
While the goal of this qHTS was to recover compounds that decreased FP via fluoroprobe displacement, the screen recovered 312 compounds that showed FP increases. The TF dataset indicated that 41% of these samples showed concentration dependent decreases in TF and an additional 28% showed increases (data not shown). These increases in FP and changes in TF may reflect compound effects on light scattering 22; 27
or the emission and polarization of the fluoroprobe. In the latter case, compounds may cause the fluoroprobe to aggregate resulting in both increased fluorescence and anisotropy. A less likely effect is compound autofluorescence because spectroscopic profiling of over 70,000 library samples shows that fewer than ten samples fluoresce in the far red excitation and emission spectra used in this assay 24
. There were a minority of compounds that showed increases in both FP and TF. Of the 312 compounds with increased FP and 466 compounds with increased TF, only 87 compounds showed increases in both.
Our assay used fluorescence polarization to detect the interaction of the TRβ LBD with a SRC2-2 fluoroprobe. However, other assay formats, like TR-FRET and AlphaScreen™, have been used to detect other NHR-coactivator interactions 11; 13; 31
. While all of these methodologies have been implemented to identify validated small molecules inhibitors, each has advantages and liabilities (reviewed in 30
). In general, FP formats are simpler, requiring the labeling of only one probe, which can result in lower assay costs and greater ease of implementation. However, FP assays typically require micromolar concentrations of target protein, resulting in lower sensitivity, and are susceptible to a variety of compound interferences 22
. Methods based upon TR FRET determine a ratio of two fluorescent emissions and are less susceptible to compound interferences, though artifacts do occur 23
. Antibody-based TR-FRET formats use nanomolar concentrations of target 11; 13
, permitting greater sensitivity in detecting potent inhibitors. However, these formats are more complex where probe reagents may be difficult to label or expensive to generate and may make large-scale screening cost prohibitive30
The TRβ-SRC2-2 qHTS was very selective in identifying actives. Only 299 FP inhibitors (0.1 % of screening collection) were recovered from the qHTS that were free of compound interference, as judged by total fluorescence activity. This percentage is similar to an earlier screen using this assay where 0.02% actives were identified by assaying compounds at a single concentration of 30 μM 2
. Our five-fold higher recovery of actives reported here is due likely to the titration-based screening method used 18
combined with a higher starting concentration of 92 μM for most of the screened compounds. The low yield of TRβ-SRC2-2 inhibitors may be attributed to several factors: the difficulty in disrupting protein-protein interactions with small molecules 9
, the relatively high concentration of target protein (0.6 μM), and the shallow pocket of TRβ that interacts with SRC2 8
. Indeed, many of the compounds that retested as positive in both FP assays, had IC50
values greater than 20 μM (Table S2
) indicating that most compounds weakly disrupted the interaction between TRβ and SRC2-2.
However, the qHTS identified one novel series that inhibited TRβ and SRC2-2 association with an IC50 value of 5 μM. This MSNB series represents a new chemotype inhibitor of this interaction. Eight MSNB analogs were tested in the qHTS, of which three were active. Because the qHTS assayed each compound at six concentrations, the determined potencies and efficacies allowed us to derive nascent structure-activity relationship for this series. All MSNB compounds containing either a hydrophobic imide (MLS000517530-01) or amide (MLS000389544-01 and MLS001003365-01) were active while those containing an aniline (MLS000517219-01 and MLS001017631-01), β-aminosulphone (MLS000336487-01), or oxadiazole (MLS001010708-01 and MLS001003284-01) were inactive (). These results suggest that a hydrophobic group is needed at this position for activity. Replacement of the MSNB group in MLS000517530-01 with other aromatic groups (MLS000517502-01, MLS000776485-01, and MLS000565662-01) also resulted in loss of the activity, implying that this group is essential. Further studies using a broader range of analogs will be needed to confirm these hypotheses.
The MSNB series is structurally distinct from the previously described β-aminoketones 2
and therefore represents a novel TR-SRC2 inhibitor scaffold. The β-aminoketone series of inhibitors has been successfully modified to improve potency, reduce cytotoxicity, and improve physiochemical determinants of bioavailability 3; 16
. However, this class has an inherent cardiotoxicity due to the interaction of the requisite aminoketone moiety with cardiac ion channels. While optimization has lessened the impact of this issue, the liability has not been eliminated. The new MSNB series does not possess chemical moieties expected to cause this liability and thus presents advantages over the earlier series. Structural studies indicate that β-aminoketones series members alkylate cysteine 298 in the AF-2 pocket of TRβ, which normally forms the interface for binding coactivators 8
. Initial characterization of the MSNB series indicates it binds irreversibly to the TRβ LBD and may inhibit in a similar manner 17
. Future studies will elucidate the mechanism of MSNB inhibition more fully.