A subtype-selective thyromimetic designed to bind a mutant thyroid hormone receptor implicated in resistance to thyroid hormone [12]

Full text of DOI:10.1021/ja003442j

J. Am. Chem. Soc. 2001, 123, 1521-1522
A Subtype-Selective Thyromimetic Designed to Bind
1521
a Mutant Thyroid Hormone Receptor Implicated in
Resistance to Thyroid Hormone
Hai Fen Ye, Kathryn E. O’Reilly, and John T. Koh*
Department of Chemistry and Biochemistry
UniVersity of Delaware, Newark, Delaware 19716
ReceiVed September 20, 2000
Two recent reports have demonstrated that small organic
compounds (MW < 800) can act to restore function to forms of
p53 and the human growth hormone receptor complex that are
functionally impaired by specific genetic mutations.1-2 These
examples of “pharmacological rescue” of genetically impaired
proteins suggest that it may be possible to design new drugs to
recover activity from many proteins known to be mutated in a
number of genetically based diseases. However, thus far, com-
pounds used to restore function to proteins bearing natural
mutations associated with human disease are of too low potency
(micromolar activity) to act as practical therapeutics.1 Mutations
to the family of nuclear and steroid hormone receptors are
implicated in a diverse set of genetic diseases.3-4 In many cases
these mutations have been shown to reside within or around the
hormone-binding pocket of the receptor and disrupt normal
transactivation function.5-7 In this work we demonstrate that by
using a known receptor agonists as a structural scaffold, potent
(nanomolar active) hormone analogues can be rationally designed
to complement a mutant form of the human thyroid hormone
receptor beta (hTRâ) implicated in the genetic disease resistance
to thyroid hormone (RTH) (Figure 1).
The thyroid hormone receptor (TR) functions as a ligand-
dependent transcriptional regulator that controls the expression
of a specific set of genes involved in development and homeo-
stasis in response to triiodothyronine (T3).8-9 There are two
known TR subtypes: TRR which has been found in high
concentration in skeletal muscle and brain and is closely linked
to cardiac function, and TRâ which is undetectable in kidney and
heart tissues.10 Many RTH-associated mutations to TRâ are known
to impair or abolish ligand-dependent transactivation function
which can lead to a range of clinical presentations such as goiter,
learning disabilities, impaired bone maturation, and mental
retardation.9,11 Although many mutant receptors show only
reduced activity toward T3, clinical treatment of RTH with
supraphysiological concentrations of T3 to recover TRâ activity
can lead to over-stimulation of TRR which is implicated with
undesirable side effects such as tachycardia and heart arhythmia.12-14
Figure 1. (a) Normally, T3 binds to the thyroid hormone receptor (TR)
and regulates expression of specific genes. (b) Mutations to TR* prevent
normal T3 binding; however, appropriate analogues can recover normal
TR function.
Figure 2. Thyroid hormone (T3) and analogues.
The RTH-associated mutation, TRâ(R320C) exhibits a reduced
affinity for T3.15-16 Ligand-dependent transactivation assays
performed with HEK293 cells transiently transfected with mutant
or “wild-type” (Wt) receptor, show that T3 is 7-fold less active
with the mutant hTRâ(R320C) (EC50 ) 4.3 ( 0.1 nM) than the
“wild-type” hTRâ(Wt) (EC50 ) 0.66 ( 0.02 nM).17 Furthermore,
concentrations of T3 required to significantly activate the mutant
TRâ(R320C), impart an undesirable saturating response to TRR-
mediated transactivation (EC50 ) 0.14 ( 0.24 nM) (Figure 3).
Clinically, treatment of RTH patients harboring the hTRâ(R320C)
mutation with supraphysiological levels of T3 was indeed
observed to affect cardiac function in some patients.16 Therefore,
compounds having high affinity and selectivity for mutant forms
of TRâ over the R-subtype are sought for RTH therapy.
In some instances thyroid hormone analogues (Figure 2) have
been used in RTH therapy; however, their use is largely empirical,
and in some instances treatment is also associated with cardio-
toxicity.18-20 The potent nonhalogenated thyromimetic GC1 is
therefore of particular interest because it preferentially binds TRâ
(Kd ) 67 pM) over TRR (Kd ) 440 pM).12 However, GC1 shows
a significantly reduced activity toward the mutant receptor TRâ-
(R320C) (EC50 ) 37.7 ( 10.8 nM) than to the TRâ(Wt) (EC50
3.67 ( 1.1 nM) in cultured cells and is therefore no longer selec-
tive for the mutant â-subtype over TRR(Wt) (EC50 ) 6.6 ( 1.0
nM)(Figures 3 and 5).
(1) Foster, B. A.; Coffey, H. A.; Morin, M. J.; Rastinejad, F. Science 1999,
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(17) HEK293 cells were transiently transfected with pSG5 encoding hTRâ,
hTRâ(R320C) or hTRR, enhanced luciferase reporter DR4-tk-luc+, and control
plasmid pRL-CMV (See Supporting Information).
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10.1021/ja003442j CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/30/2001

1522 J. Am. Chem. Soc., Vol. 123, No. 7, 2001
Communications to the Editor
Figure 3. Luciferase reporter gene activity reported as relative light units
(RLU) induced by various ligand-receptor pairs.
Figure 5. (a) GC1 shows selectivity for TRâ over TRR but is not
selective for the RTH mutant TRâ‚R320C). (b) HY1 has improved activity
and selectivity for the mutant TRâ(R320C). TRR, (O); TRâ, (1); TRâ-
(R320C), (9).
greater levels of subtype-selectivity may be desirable, these data
suggest that HY1 may have unique potential as a therapeutic
capable of recovering activity from the mutant form of TRâ while
potentially avoiding the undesirable side effects associated with
TRR over stimulation.
This work demonstrates that by making compensatory modi-
fications to known hormone agonists, new, highly potent ligands
can be made which are selective for mutant receptors implicated
in human disease. This paper focuses on one particular mutation
associated with the relatively rare genetic disease RTH. However,
many genetic diseases including Cushings disease (GR), rickets
(VDR), androgen insensitivity syndrome (AR), adrenal hypoplasia
congenita (DAX1),4 as well as certain forms of diabetes (PPAR),7
leukemia (RAR),24 and prostate cancer (AR),25 are also associated
with mutations to hormone receptor ligand-binding domains.26
Although in principle this general strategy may require a unique
drug to be designed for each mutation associated with a particular
disease, as demonstrated by this work on hTRâ and the related
work of Soprano et al. on RAR,22 similar design strategies may
be used to complement structurally similar mutations in related
receptors.
Figure 4. Model of HY1 bound to hTRâ(R320C) generated in Flo98/
QXP based on the reported T3-TRâ cocrystal structure.
The TR(R320C) mutant is similar to mutations to charged
(basic) residues of the retinoic acid receptor that we and others
have shown to alter ligand-binding specificity to favor binding
neutral ligands.21-22 On the basis of site-models generated from
the coordinates of the T3/TRâ crystal structure,5 we designed the
neutral alcohol HY1 as a potential subtype-selective ligand for
the mutant receptor hTRâ(R320C) (Figures 2 and 4).23 This ligand
receptor design is chemically related to a study by Soprano et
al., who showed that an artificial mutation in the retinoic acid
receptor, hRARâ(R269A), binds the neutral ligand retinol in
preference to its natural ligand retinoic acid,22
Assays of transactivation function show that HY1 (EC50 )
7.01 ( 3.0 nM) is 5-times more potent an agonist toward TRâ-
(R320C) than the parent compound GC1, indicating that our
designed ligand was indeed more potent than GC1 (Figure 5).
Importantly, HY1 is also capable of eliciting substantial trans-
activation response from the mutant TRâ at concentrations that
show only partial activation of TRR (EC50 ) 37.69 ( 10.4 nM)
and TRâ (EC50 ) 32.05 ( 8.7 nM).(Figure 3). Although even
Acknowledgment. K.E.O. was supported a grant from HHMI through
the Undergraduate Medical Science Education Program. We gratefully
acknowledge financial support from Johnson & Johnson and the National
Institutes of Health Grant No. RO1 DK54257-01.
Supporting Information Available: Synthesis and characterization
of HY1, experimental details of cell-culture experiments (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org.
JA003442J
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Chem. Soc. 1999, 121, 1984-1985.
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C.; Claassen, E.; van Rooij, H. C.; Trapman, J.; Brinkmann, A. O.; Mulder,
E. Biochem. Biophys. Res. Commun. 1990, 173, 534-40.
(26) GR, glucocorticoid receptor; VDR, vitamin D3 receptor; AR, androgen
receptor; Dax1, an orphan receptor; PPAR, peroxisome proliferator-activated
receptor; RAR, retinoic acid receptor.
(22) Tairis, N.; Gabriel, J. L.; Soprano, K. J.; Soprano, D. R. J. Biol. Chem.
1995, 270, 18380-18387.
(23) McMartin, C.; Bohacek, R. S. J. Comput.-Aided Mol. Des. 1997, 11,
333-344.