ABSTRACT
Molecular conjugates of hormone receptor-ligands with molecular probes or functional domains are finding diverse applications in chemical biology. Whereas many examples of hormone conjugates that target steroid hormone receptors have been reported, practical ligand conjugates that target the nuclear thyroid hormone receptor (TRbeta) are lacking. TR-targeting conjugate scaffolds based on the ligands GC-1 and NH-2 and the natural ligand triiodothyronine (T3) were synthesized and evaluated in vitro and in cellular assays. Whereas the T3 or GC-1 based conjugates did not bind TRbeta with high affinity, the NH-2 inspired fluorescein-conjugate JZ01 showed low nanomolar affinity for TRbeta and could be used as a nonradiometric probe for ligand binding. A related analogue JZ07 was a potent TR antagonist that is 13-fold selective for TRbeta over TRalpha. JZ01 localizes in the nuclei of TRbeta expressing cells and may serve as a prototype for other TR-targeting conjugates.
Subject(s)
Amides/chemical synthesis , Amides/metabolism , Benzyl Compounds/chemistry , Fluoresceins/chemistry , Receptors, Thyroid Hormone/metabolism , 3T3 Cells , Amides/chemistry , Animals , Benzyl Compounds/chemical synthesis , Fluoresceins/chemical synthesis , Fluorescence Polarization , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Ligands , Mice , Protein Binding , Receptors, Thyroid Hormone/agonists , Receptors, Thyroid Hormone/antagonists & inhibitors , Thyroid Hormone Receptors alpha/agonists , Thyroid Hormone Receptors alpha/antagonists & inhibitors , Thyroid Hormone Receptors alpha/metabolism , Thyroid Hormone Receptors beta/agonists , Thyroid Hormone Receptors beta/antagonists & inhibitors , Thyroid Hormone Receptors beta/metabolismABSTRACT
The thyroid hormone receptors (TRs) are ligand-dependent transcription factors that control the expression of multiple genes involved in development and homeostasis in response to thyroid hormone (triiodothyronine, T3). Mutations to TRbeta that reduce or abolish ligand-dependent transactivation function are associated with resistance to thyroid hormone (RTH), an autosomal dominant human genetic disease. A series of neutral alcohol-based compounds, based on the halogen-free thyromimetic GC-1, have been designed, synthesized, and evaluated in cell-based assays for their ability to selectively rescue three of the most common RTH-associated mutations (i.e., Arg320 --> Cys, Arg320 --> His, and Arg316 --> His) that affect the basic carboxylate-binding arginine cluster of TRbeta. Several analogues show improved potency and activity in the mutant receptors relative to the parent compound GC-1. Most significantly, two of these mutant-complementing thyromimics show high potency and activity with a strong preference for the mutant receptors over wild-type TRalpha(wt), that is associated with the cardiotoxic actions of T3. The compounds were evaluated in reporter gene assays using the four common thyroid hormone response elements, DR4, PAL, F2 (LAP), and TSH, and show activities and selectivites consistent with their unique potential as agents to selectively rescue thyroid function to these RTH-associated mutants.
