Selective chemical rescue of a thyroid-hormone-receptor mutant, TRbeta(H435Y), identified in pituitary carcinoma and resistance to thyroid hormone.

The thyroid hormone receptors (TRs) are ligand-dependent transcriptional regulators that control critical genes in development and homeostasis in response to triiodothyronine (T3). As an important regulator of differentiation, TRb has been shown to be mutated in a high percentage of certain cancer types, including kidney, pituitary, liver, and thyroid cancer. These spontaneous TRb mutations cause the reduction or loss of TR function in a similar way to germline TRb mutants associated with inheritable genetic disease resistance to thyroid hormone (RTH). Paradoxically, RTH patients do not appear to be predisposed to these forms of cancer, although, in a few cases, identical TRb mutants have been identified in cancer and RTH. As part of our studies exploring applications of chemical rescue by small-molecule complementation, we previously examined how mutations to the TRb “His-Phe switch” motif, which mediates ligand-dependent transactivation response, can dramatically impair receptor function. Herein, we describe a new strategy to rescue a naturally occurring TRb mutant, His435! Tyr, by reorienting hydrogen-bonding interactions at the ligand–receptor interface. As TRb(H435Y) has been found in both RTH and pituitary carcinoma, our results serve perhaps as the first example of chemical rescue that targets a mutant protein involved in multiple disease states. Upon ligand binding, TR undergoes a conformational change that involves the repositioning of helix 12 to form a coactivator-binding interface (Figure 1a). For most nuclear receptors, the hormone does not make direct contact with helix 12, but rather interacts with residues on helix 11. These residues make contacts with helix 12 through a His-Trp or a His-Phe switch, which transduces ligand binding into a transcription response. Mutations to the His-Phe switch of TRb have been associated with dramatic (320–> 5000fold) reductions in ligand potency. 9] The high-resolution crystal structures of T3-bound TRb and TRa suggest that His435 forms a hydrogen bond with the 4’-OH group of T3 and participates simultaneously in aryl– aryl interactions with the Phe459 residue of helix 12 (Figure 2, left). We demonstrated previously that 4’-alkoxy derivatives of the thyroid-hormone-receptor agonist GC-1 have greater potency and efficacy with TRb(H435A) than the natural hormone T3; however, these analogues are ineffective in rescuing the activity of the His435 mutants TRb(H435Y) and TRb(H435L), which are known to be associated with RTH and cancer. Whereas TRb(His435L) is inactive at all T3 concentrations tested ( 5 mm), T3 is a full agonist (100% efficacy) for TRb(H435Y), although it is 390 times less potent with this mutant than with wild-type (wt) TRb. These results suggest that TRb(H435Y) retains its intrinsic ability to mediate ligand-dependent transcription response but requires extreme supraphysiological concentrations of T3 that would not be tolerated in vivo owing to the overstimulation of wildtype TRs. As in other studies in which the thyroid hormone receptor was targeted, the delicate balance of TR activity within the hypothalamic-pituitary-thyroid axis emphasizes the need for a ligand with subtype selectivity. Molecular modeling of TRb(H435Y) suggested that the Tyr435 side chain is still able to engage Phe459 through aryl– aryl interactions (Figure 2, right). Although the phenol hydroxy group of tyrosine is capable of forming a hydrogen bond, it is not appropriately positioned to interact with receptor-bound T3. We reasoned that appropriately designed hormone analogues may be able to rescue potency to TRb(H435Y) selectively by restoring hydrogen-bonding/ aryl–aryl interactions of the His-Phe switch through the creation of a novel Tyr-Phe switch. This strategy presented a unique challenge, as the side chain of tyrosine is considerably longer than that of histidine; therefore, it was necessary to introduce a hydrogen-bonding group while making the overall ligand structure smaller. As an initial approach, we reasoned that the outer phenyl ring of T3 could be replaced by a pyridyl ring (Figure 1b, right). For ease of synthesis and product stability, we chose to make analogues of the halogenfree thyromimetic GC-1 rather than analogues of T3 itself. We could then vary the alkyl substituent at the 3’-position with the aim of optimizing the hydrogen-bond geometry and hydrophobic contacts of the 3’ substituent (Scheme 1). As a control, we also synthesized a “phenyl” analogue of GC-1, QH9, in which the phenol hydroxy group has been replaced by a hydrogen atom. Pyridyl analogues were derived from the corresponding 2-substituted 4-cyanopyridines by the nucleophilic addition to 4-cyanopyridine of alkyl radicals generated by silver-promoted radical decarboxylation of the corresponding carboxylic acids. This method provided efficient access to the 2-alkyl pyridine series of ligands (see Scheme S1 in the Supporting Information). [*] A. Q. Hassan, J. T. Koh Department of Chemistry and Biochemistry, University of Delaware Newark, DE 19716 (USA) E-mail: johnkoh@udel.edu