Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be ‘undruggable.’ A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-279 is able to disrupt binding between EWS-FLI1 and RHA in an immunoprecipitation assay and blocks the transcriptional activity of EWS-FLI1, while (R)-YK-4-279 cannot. Enantiospecific effects are also established in cytotoxicity assays and caspase assays, where up to a log-fold difference is seen between (S)-YK-4-279 and the racemic YK-4-279. Our findings indicate that only one enantiomer of our small molecule is able to specifically target a protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be specific. Furthermore, proving YK-4-279 has only one functional enantiomer will be helpful in moving this compound towards clinical trials.
High‐affinity [3H]5‐hydroxytryptamine ([3H]5‐HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [3H]5‐HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [3H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high‐affinity binding appears to be to multiple sites, since displacement studies using 2 nM [3H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (nH): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [3H]5‐HT, since unlabeled 5‐HT did not discriminate between them. Thus, the high‐affinity [3H]5‐HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5‐HTIA, 5‐HTIB). In addition to the multiple high‐affinity spinal cord binding sites, a low‐affinity [3H]5‐HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [3H]5‐HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high‐and low‐affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [3H]5‐HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [3H]5‐HT in the spinal cord is associated with the 5‐HT uptake carrier, since fluoxetine, an inhibitor of 5‐HT uptake, does not alter binding characteristics. In addition, a 5‐HT binding site analogous to the site designated 5‐HT, was not apparent in the spinal cord. Ketanse‐rin and cyproheptadine, drugs that are highly selective for 5‐HT, sites, did not displace [3H]5‐HT from spinal tissue, and [3H]spiperone, a radioligand that binds with high affinity to 5‐HT2 sites, did not exhibit saturable binding in the tissue. Thus, the 5‐HT2 binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord.
A superfusion system employed to measure the K+-stimulated release of [3H]5-hydroxytryptamine [(3H]5-HT, [3H]serotonin) from a synaptosomal-rich spinal cord tissue preparation was carefully characterized, then used to examine the regulation of spinal 5-HT release. Spinal 5-HT release is apparently modulated by an autoreceptor. Exogenous 5-HT depressed, in a concentration-dependent manner, the K+-stimulated release of [3H]5-HT. Similarly, lysergic acid diethylamide (LSD) produced a concentration-dependent decrease in [3H]5-HT release. Methiothepin and quipazine blocked the inhibition of release induced by exogenous 5-HT. The 5-HT2 receptor antagonists spiperone and ketanserin failed to alter the action of 5-HT at the spinal 5-HT autoreceptor. Spiperone and ketanserin were shown, however, to alter the storage of [3H]5-HT. When used in concentrations greater than 10 nM, the drugs evoked increases in basal [3H]5-HT and [3H]5-hydroxyindoleacetic acid ( [3H]5-HIAA) effluxes which were independent of the presence of calcium ions. A good agreement existed between the potencies of drugs for modifying autoreceptor function and their abilities to compete for high-affinity [3H]5-HT binding in the spinal cord (designated 5-HT1). Furthermore quipazine, in concentrations that preferentially interact with the 5-HT1B subtype, antagonized the actions of exogenous 5-HT on K+-stimulated release. Spiperone, in a concentration that approximated the affinity constant of 5-HT1A sites for the drug, was ineffective in altering the ability of exogenous 5-HT to modulate K+-stimulated [3H]5-HT release. These results suggest that 5-HT1B sites are associated with serotonergic autoreceptor function in the spinal cord.
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