A functionally orthogonal ligand-receptor pair created by targeting the allosteric mechanism of the thyroid hormone receptor

Article abstract of DOI:10.1021/ja060760v

Nuclear receptors are ligand-dependent transcription factors that are of interest as potential tools to artificially regulate gene expression. Ligand binding induces a conformational change involving helix-12 which forms part of the dimerization interface

Full text of DOI:10.1021/ja060760v

Published on Web 06/15/2006
A Functionally Orthogonal Ligand-Receptor Pair Created by
Targeting the Allosteric Mechanism of the Thyroid Hormone
Receptor
A. Quamrul Hassan and John T. Koh*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Delaware,
Newark, Delaware 19716
Received February 20, 2006; E-mail: johnkoh@udel.edu
Abstract: Nuclear receptors are ligand-dependent transcription factors that are of interest as potential tools
to artificially regulate gene expression. Ligand binding induces a conformational change involving helix-12
which forms part of the dimerization interface used to bind transcriptional coactivators. When triiodothyronine
(T3) binds the thyroid hormone receptor (TR) it indirectly contacts helix-12 through intermediary residues
His(435) and Phe(451) termed a His-Phe switch. The mutant TRâ(H435A) is nonresponsive to physiological
concentrations of T3 but can be activated by the synthetic hormone analogue QH2 which potently activates
His435fAla mutant at concentrations that do not activate the wild-type receptors TRR and TRâ. QH2
does not show antagonist behavior with the wild-type TRs. QH2’s functionally orthogonal behavior with
TRâ(H435A) is preserved on the three consensus thyroid hormone response elements.
Nuclear hormone receptors (NHRs) are ligand-dependent
transcription factors that regulate gene transcription in response
to small molecule hormones. NHRs directly bind DNA se-
quences in hormone responsive genes and activate transcription
in response to hormone. NHRs that have been engineered to
uniquely respond to synthetic ligands are of interest as artificial
gene regulatory systems. Several approaches have been used
to engineer NHRs including screening molecular libraries for
ligands that match nonfunctional receptor mutants or by
selecting libraries of receptor mutations that impart a response
to otherwise inactive ligand analogues.1-5 The highly flexible
nature of NHR ligand binding domains makes it difficult to
construct highly specific ligand-receptor pairs, as mutant
receptors often retain significant activity toward their natural
hormones and ligand analogues often have activities with
endogenous receptors. Here we describe a highly selective,
“functionally orthogonal,” ligand-thyroid hormone receptor
(TR) pair generated by targeting receptor residues directly
involved in the receptor’s ligand-dependent transactivation
allosterism. The site of receptor modification, identified by
examining naturally occurring mutations to TR associated with
genetic disease, should provide a general strategy for generating
highly selective ligand-receptor pairs from NHRs.
NHR ligand-binding domain undergoes a conformational change
that leads to the release of corepressor proteins and the
recruitment of transcriptional coactivators.6-8 Helix-12 of the
ligand-binding domain plays a central role in controlling the
ligand-induced receptor allosterism, as it has been observed to
undergo a substantial conformational change on ligand binding
and to form a part of the receptor/coactivator interface. The
proper positioning of helix-12 is required to create a proper
dimerization interface for “LXXLL” NR-box sequences of
transcriptional coactivators (Scheme 1A). The cocrystal struc-
tures of NHR ligand-binding domains with antagonists suggest
that many antagonists block transcriptional activation by
preventing helix-12 from adopting its active agonist conforma-
tion.9,10
Although the conformation of helix-12 is highly dependent
on the structure of the bound ligand, surprisingly, most of the
known NHR ligand-binding domain structures indicate that the
ligand does not directly contact helix-12 but rather the ligand
interacts with helix-12 through intermediary residues that bridge
between the ligand and helix-2. For example, in the T3-TRâ
cocrystal structure, the 4′-hydroxyl of T3 hydrogen bonds to
His435 that in turn forms favorable interactions (either pi-pi
or edge-face interactions) with Phe459, a critical residue on
helix-12 (Scheme 1B). This general motif was first recognized
in LXR involving His435 and Trp457 and was termed a His-
NHRs are composed of two fundamental functional do-
mains: a DNA binding domain that uniquely targets an NHR
to a specific subset of hormone-responsive genes and a ligand-
binding domain which selectively binds the hormone and
activates gene transcription. Upon binding the hormone, the
Trp switch or, in the case of the TR, a His-Phe switch.11
A
similar His-Phe switch is found in TRR and in VDR involving
His397 and Phe422 (Figure 1).12,13 Mutations to these residues
(1) Whelan, J.; Miller, N. J. Steroid Biochem. Mol. Biol. 1996, 58, 3-12.
(2) Shi, Y. H.; Koh, J. T. J. Am. Chem. Soc. 2002, 124, 6921-6928.
(3) Tedesco, R.; Thomas, J. A.; Katzenellenbogen, B. S.; Katzenellenbogen,
J. A. Chem. Biol. 2001, 8, 277-287.
(6) Hulbert, A. J. Biol. ReV. 2000, 75, 519-631.
(7) Katzenellenbogen, J. A.; Katzenellenbogen, B. S. Chem. Biol. 1996, 3, 87-
94.
(8) Yen, P. M. Physiol. ReV. 2001, 81, 1097-1142.
(9) Wurtz, J.-M.; Bourguet, W.; Renaud, J.-P.; Vivat, V.; Chambon, P.; Moras,
D.; Gronemeyer, H. Nat. Struct. Biol. 1996, 3, 87-94.
(10) Weatherman, R. V.; Fletterick, R. J.; Scanlan, T. S. Annu. ReV. Biochem.
1999, 68, 559-581.
(4) Schwimmer, L. J.; Rohatgi, P.; Azizi, B.; Seley, K. L.; Doyle, D. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 14707-14712.
(5) Chockalingam, K.; Chen, Z. L.; Katzenellenbogen, J. A.; Zhao, H. M. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 5691-5696.
9
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J. AM. CHEM. SOC. 2006, 128, 8868-8874
10.1021/ja060760v CCC: $33.50 © 2006 American Chemical Society

Functionally Orthogonal Ligand−Receptor Pair
A R T I C L E S
Scheme 1. Ligand-Induced Conformational Change of Nuclear Receptorsa
a (A) Hormone binding repositions helix-12 (H12, green) to create a dimerization interface for transcriptional coactivators containing a conserved “LXXLL”
NR-box (purple). (B) In the His-Phe switch of TRâ, T3 hydrogen bonds to His435, which interacts directly with Phe459 on helix-12. (C) The transducer role
of His435 may be replaced with appropriate hydrophobic groups.
Figure 1. The His-Trp and His-Phe switch serves as a transducer to relay ligand binding information to the AF-2 domain of nuclear receptors. (a) TRR,13
(b) VDR,12 and (c) LXR.17
that are directly involved in the mechanism of ligand-induced
allosterism can have a dramatic effect on hormone responsive-
ness to these receptors. For example, mutation to the His
member of His-Trp/His-Phe switches as in LXRâ(H435A),
LXRR(H421A), and VDR(H305Q) or mutation to the Trp/Phe
residues as in LXRR(W443F) and TRâ(F459C), TRâ(F459A)
cause inactivity or show severely reduced activity compared to
wild-type.11,14-16 The rickets-associated mutant VDR(H305Q)
is of particular interest because the Gln residue retains the
potential to hydrogen bond to the ligand. Although the natural
ligand, 1,25-dihydroxy vitamin D3, binds VDR(H305Q) with
near wild-type affinity in vitro, it is substantially less potent
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A R T I C L E S
Hassan and Koh
(80-fold less potent) in cellular transcription assays indicating
that the transduction mechanism is specifically impaired.16
diseases,21-24 we have developed thyroid hormone receptor
analogues that complement mutant forms of TRâ associated with
the genetic disease RTH (resistance to thyroid hormone). RTH
is characterized by a hyposensitivity to thyroid hormone
triiodothyronine (T3) in peripheral tissues. The vast majority
of RTH-associated mutations show dominant-negative inherit-
ance indicating that the presence of one mutant copy of the
receptor is able to compete with, and directly interfere with,
the function of wild-type copies of the receptor through the
formation of inactive receptor dimers.8,25 The dominant negative
activity of RTH-associated TR mutants implies that these
receptors have a relatively stable structure that can bind DNA
and form receptor dimers but are functionally impaired in their
ability to bind ligand and recruit coactivators.
Surprisingly, many TRâ mutants associated with RTH are
only mildly less responsive to T3 than wild-type TRâ (TRâ-
(wt)). For example, some of the most common RTH-associated
mutants, TRâ(R320C), TRâ(R320H), and TRâ(M310T), under
investigation in our laboratory show less than a 10-fold reduction
in responsiveness to T3 compared to wild-type, TRâ(wt).21,22,26
Evidently, even relatively modest changes in hormone respon-
siveness are sufficient to upset the normal balance of peripheral
tissue responsiveness and hormone synthesis needed to cause
RTH.
In general, nuclear receptors appear to accommodate a wide
range of mutations without gross impairment of receptor
function. Even when ligand potency is significantly reduced by
receptor mutations, ligands often show full efficacy though at
higher ligand concentrations.27 Similarly, NHRs can often
accommodate a range of ligand structures that would initially
appear to cause steric clashes with receptor side chains.28,29 In
contrast, some RTH-associated mutations are known to have
dramatic effects on receptor function. Mutations to His435,
His435fTyr, His435fLeu, and His435fGln have been shown
to have large effects on hormone responsiveness and have been
identified in RTH patients as well as in TSH secreting pituitary
adenoma (TSHoma) tumors.30,31 TRâ(H435Y) has a strongly
refractory response to T3 and is 230-times less responsive
toward T3 than TRâ(wt). TRâ(H435L) is completely devoid
of cellular activity within experimentally accessible concentra-
tions of T3.30 Examination of the T3-TRâ(wt) cocrystal
structure32 does not suggest any obvious structural perturbations
associated with these mutations. Rather, mutations to His435,
a part of TR’s His-Phe switch, may disrupt the normal molecular
transduction of the ligand-binding signal to helix-12. Similar
Several groups have developed analogue-selective forms of
hormone receptors as ligand-inducible regulators of gene
expression by screening ligand libraries to match mutant
receptors or by applying selection/directed evolution methods
to identify receptors that respond to otherwise inactive ligand
analogues.2,4,5,18,19 One of the challenges associated with
generating orthogonal ligand-receptor pairs from NHRs is
finding mutations that efficiently prevent activation by the
endogenous hormone without otherwise compromising the
receptor’s structure or intrinsic ability to activate gene transcrip-
tion. A few groups have used genetic selections in yeast to
identify mutant receptors that selectively respond to synthetic
ligands; however, the ligand’s selectivity for the mutant versus
the wild-type receptor can change substantially when these
ligand-receptor pairs are transferred to mammalian cell lines.1,4,5
In some cases, receptors selected for their ability to respond to
synthetic ligands may also retain significant activity for their
natural ligands.4
For consideration as artificial transcriptional regulators,
engineered receptors should not be responsive to physiological
concentrations of the natural hormone but respond to concentra-
tions of a synthetic ligand analogue that do not activate wild-
type (or other endogenous) receptors. We have termed ligand-
receptor pairs that match this practical definition for functioning
independent of the endogenous hormone-receptor pair as
“functionally orthogonal.”2,20 Though of potential academic
interest, the change in the receptor’s selectivity for the synthetic
versus natural ligand (i.e., the receptor’s “ligand selectivity”)
will have little practical importance if the concentrations of
synthetic ligand needed to activate the engineered receptors also
activate endogenous receptors. Provided that a modified receptor
is no longer responsive to endogenous concentrations of the
natural ligand, the synthetic ligand’s receptor selectivity, defined
as EC50(mutant)/EC50(wt) for a given ligand, represents a
practical measurement of selectivity of a system. By this
criterion ligand-receptor pairs developed by directed evolution
strategies do not show better receptor selectivity than the best
systems developed by rational design.2 This suggests that
judicious choice of the site of receptor modification is necessary
for any ligand-receptor engineering strategy.
As part of our program to generate small molecules that
rescue function to mutant NHRs associated with human genetic
(21) Ye, H. F.; O’Reilly, K. E.; Koh, J. T. J. Am. Chem. Soc. 2001, 123, 1521-
1522.
(11) Williams, S.; Bledsoe, R. K.; Collins, J. L.; Boggs, S.; Lambert, M. H.;
Miller, A. B.; Moore, J.; McKee, D. D.; Moore, L.; Nichols, J.; Parks, D.;
Watson, M.; Wisely, B.; Willson, T. M. J. Biol. Chem. 2003, 278, 27138-
27143.
(22) Shi, Y.; Ye, H.; Link, K. H.; Putnam, M. C.; Hubner, I.; Dowdel, S.; Koh,
J. T. Biochem. J. 2005, 44, 4612-4626.
(23) Swann, S. L.; Bergh, J.; Farach-Carson, M. C.; Ocasio, C. A.; Koh, J. T.
J. Am. Chem. Soc. 2002, 124, 13795-13805.
(12) Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.; Moras, D. Mol. Cell
2000, 5, 173-179.
(24) Swann, S. L.; Bergh, J. J.; Farach-Carson, M. C.; Koh, J. T. Org. Lett.
2002, 4, 3863-3866.
(13) Wagner, R.; Apriletti, J. W.; McGarth, M. E.; West, B. L.; Baxter, J. D.;
Fletterick, R. J. Nature 1995, 378, 690-697.
(25) Yen, P. M. Trends Endocrinol. Metab. 2003, 14, 327-333.
(26) Furlanetto, T. W.; Kopp, P.; Peccin, S.; Gu, W. X.; Jameson, J. L. Mol.
Genet. Metab. 2000, 71, 520-526.
(14) Cheng, S. Y.; Ransom, S. C.; Mcphie, P.; Bhat, M. K.; Mixson, A. J.;
Weintraub, B. D. Biochemistry 1994, 33, 4319-4326.
(15) Tone, Y.; Collingwood, T. N.; Adams, M.; Chatterjee, V. K. J. Biol. Chem.
1994, 269, 31157-31161.
(27) Shi, X. B.; Ma, A. H.; Xia, L.; Kung, H. J.; White, R. W. D. Cancer Res.
2002, 62, 1496-1502.
(16) Gardezi, S. A.; Nguyen, C.; Malloy, P. J.; Posner, G. H.; Feldman, D.;
Peleg, S. J. Biol. Chem. 2001, 276, 29148-29156.
(28) Katzenellenbogen, J. A.; Muthyala, R.; Katzenellenbogen, B. S. Pure Appl.
Chem. 2003, 75, 2397-2403.
(17) Wilkinson, J. R.; Towle, H. C. J. Biol. Chem. 1997, 272, 23824-23832.
(18) Koh, J. T.; Putnam, M.; Tomic-Canic, M.; McDaniel, C. M. J. Am. Chem.
Soc. 1999, 121, 1984-1985.
(29) Koh, J. T.; Biggins, J. B. Curr. Top. Med. Chem. 2005, 5, 413-420.
(30) Nomura, Y.; Nagaya, T.; Tsukaguchi, H.; Takamatsu, J.; Seo, H. Endo-
crinology 1996, 137, 4082-4086.
(19) Doyle, D. F.; Braasch, D. A.; Jackson, L. K.; Weiss, H. E.; Boehm, M. F.;
Mangelsdorf, D. J.; Corey, D. R. J. Am. Chem. Soc. 2001, 123, 11367-
11371.
(31) Ando, S.; Sarlis, N. J.; Oldfield, E. H.; Yen, P. M. J. Clin. Endocrinol.
Metab. 2001, 86, 5572-5576.
(32) Wade, C. B.; Robinson, S.; Shapiro, R. A.; Dorsa, D. M. Endocrinology
2001, 142, 2336-2342.
(20) Koh, J. T. Chem. Biol. 2002, 9, 17-23.
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Figure 2. Cellular dose response behavior of (green square) TRR(wt), (blue diamond) TRâ(wt), and (red down-triangle) TRâ(H435A) toward (A) T3 and
(B) GC-1.
Scheme 2. Synthesis of Alkoxy Analoguesa
Scheme 3. Synthesis of Alkyl Substituted Analoguesa
a (a) n-BuLi, THF, -78 °C; (b) H2, 10%Pd/C, 9% AcOH/EtOH; (c)
Cs2CO3, BrCH2CO2R3, DMF, then NaOH, MeOH.
magnitude effects were observed with mutations to Phe459
which is the interacting partner of His435 located on helix-
12.14,15 The low responsiveness of the mutations at His435
prompted us to exploit this critical transducer residue to create
highly selective ligand-receptor pairs from TRâ.
We have generated the His435fAla mutation in TRâ
(pSG5hTRâ) using oligonucleotide directed site-specific mu-
tagenesis and evaluated its activity in transiently transfected
HEK293 cells that were cotransfected with pSG5hTRâ(H435A),
the hormone responsive promoter DR4-Luc+, and pRLbasic, a
renilla luciferase control. The alanine substitution was selected
as a cavity forming mutation that would maintain a high helical
propensity at position 435, which is centrally located in helix-
11. Reporter gene assays conducted with this mutation dem-
onstrate that TRâ(H435A) is 769-fold less responsive to T3
(EC50 ) 392 ( 84 nM) than TRâ(wt) (EC50 ) 0.51 ( 0.05
nM). The synthetic halogen-free thyromimic GC-1 is more than
2380-fold less potent with TRâ(H435A) (EC50 >5000 nM) than
with TRâ(wt) (EC50 ) 2.1 nM) (Figure 2). Although the TRâ-
(H435A) is still responsive to extremely high concentrations
of T3, the mutant is no longer responsive to physiological
concentrations of T3, which are generally well below 2 nM (total
T3 ) 2 nM; free T3 ) 3-8 pM),6 suggesting that TRâ(H435A)
is nonresponsive to physiological concentrations of T3.
We envisioned that the His-Phe switch found in TRâ(wt)
a (a) n-BuLi, THF, -78 °C; (b) H2, 10%Pd/C, 9% AcOH/EtOH; (c)
Cs2CO3, BrCH2CO2R3, DMF, then NaOH, MeOH; (d) K2CO3, DMF,
p-NO2PhOTf; (e) PdCl2(dppf), R4MgCl; (f) TBAF, THF.
which uses both hydrogen bonding and edge-pi aromatic
interactions could be replaced by an appropriate hydrophobic
cluster (Scheme 1C). Based on a site-model generated from an
appropriately modified TRâ/T3 cocrystal structure, we designed
two series of potential, mutant-selective analogues of the
halogen-free thyromimetic GC1. The general synthetic strategy
is based on the synthesis of Scanlan et al.33,34 Three 4′-O-alkyl
substituted ethers of GC-1, QH1, QH2, and QH3 were synthe-
sized from the corresponding 2-isopropylphenol ethers (Scheme
2). Similarly, 4′-alkyl substituted analogues of GC-1 were
synthesized via Kumada coupling of the intermediate aryl triflate
7 (Scheme 3). While the synthesis of QH4, QH5, and QH7 by
(33) Chiellini, G.; Nguyen, N. H.; Yoshihara, H. A. I.; Scanlan, T. S. Bioorg.
Med. Chem. Lett. 2000, 10, 2607-2611.
(34) Chiellini, G.; Apriletti, J. W.; al Yoshihara, H.; Baxter, J. D.; Ribeiro, R.
C.; Scanlan, T. S. Chem. Biol. 1998, 5, 299-306.
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Our ability to rescue the His435fAla mutant of TRâ
prompted us to examine if these same ligands may be able to
rescue related RTH mutants, TRâ(H435L) and TRâ(H435Y).
Cell based reporter gene assays indicate that QH1 thru QH7
were devoid of activity below 5 µM (data not shown). The lack
of activity observed with any of our ligands with these related
His-to-hydrophobic mutations further suggest that successful
rescue at this position requires a precise match in size, shape,
and functionality.
The mutant TRâ(H435A) is not responsive to physiological
concentrations of T3. As a potential tool for independently
regulating gene expression, it is important to further demonstrate
that the concentrations of QH2 used to fully activate the mutant
TRâ(H435A) do not activate either of the two known TR
subtypes, TRâ(wt) or TRR(wt). Cell based reporter gene assays
Figure 3. Cellular response of synthetic ligands toward TRâ(H435A).
this route was straightforward, Kumada coupling with the more
stericly demanding isopropyl grignard afforded 8c which
contained the proteo-analogue 8e as an inseparable impurity.
The “benzene” analogue QH9 was synthesized independently
by another route and was found to be inactive in cell based
assays with TRâ(H3435A) (Scheme 3). Therefore, QH6 was
synthesized and evaluated as an 85% mixture of QH6 and QH9.
The analogues QH1-QH7 were evaluated in cell based
reporter gene assays. All of the analogues show some activity
below one micromolar with the mutant TRâ(H435A); however,
only QH2, the O-ethyl ether of GC1, has low nanomolar activity
(EC50 ) 6.4 ( 0.57) with the Ala mutation (Figure 3). QH2 is
61-fold more potent than T3 and 778-fold more potent than the
parent analogue GC1 with the mutant TRâ(H435A). The
structure appears to be quite sensitive to the exact size of the
alkoxy subtituent at the 4′ position as the methoxy substituted
analogue QH1 (EC50 ) 55 nM) is 9-times less potent with half
the efficacy (45%). The propoxy substituted analogue QH3
(EC50 ) 130 nM) is also significantly less active than QH2.
Interestingly, the isosteric 4′-propyl analogue QH5 is not very
potent suggesting that the ether oxygen may still be important
for high affinity binding to the mutant. The propyl and isopropyl
substituted analogues, QH5 and QH6, are very poor agonists
for TRâ(H435A); however, the isobutyl analogue, QH7 (EC50
) 123 nM), is slightly more active.
show that QH2 is a weak agonist for both TRR(wt) (EC50
)
737 nM) and TRâ(wt) (EC50 ) 341 nM). QH2 is therefore 53-
fold selective for the mutant TRâ(H435A) over the TRâ(wt)
and 115 times more potent than TRR(wt). Concentrations of
QH2 that almost fully activate with TRâ(H435A) do not
significantly activate the endogenous receptor TRâ(wt) or TRR-
(wt) (Figure 4a, Table 1).
The thyroid hormone receptor can act on hormone responsive
genes bearing several consensus types of thyroid hormone
response elements in their promoters.8 The DR4 promoter is
the most common thyroid hormone response element (TRE);
however, it is also important to evaluate the potency, efficacy,
and selectivity of our compounds on the other common TREs,
PAL and F2. Reporter gene assays conducted using F2-luc and
PAL-luc promoters show that QH2 retains mutant selectivity,
potency, and efficacy on genes controlled by these common
TREs. The actions of QH2 on the TRâ(H435A) mutant were
also found to be functionally orthogonal on two other common
thyroid hormone response elements, F2 and PAL (Figure 4B
and 4C). Furthermore, QH2 does not compete with T3 in TRâ-
(wt) demonstrating that QH2 is not an antagonist and only serves
as a potent and selective agonist for the engineered mutant
(Figure 5, Table 2).
Figure 4. Comparative dose response behavior of QH2 with TRR(wt), TRâ(wt), and TRâ(H435A) on three consensus TRE reporters. (3) TRR + T3, (0)
TRR + QH2, (1) TRâ + T3, (9) TRâ + QH2.
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Table 1. Potencies and Efficacies of T3 and Synthetic Ligands for TRâ(H435A)a
a Cellular reporter gene assays in HEK293 cells transiently transfected with pSG5TRâ(H435A), DR4-Luc+. Potencies are measured as (SEM except as
indicated. b Potencies of weak agonists are reported as (min/max from two separate experiments performed in triplicate.
Table 2. Summary of Activities on Three Consensus Promotorsa
T3, nM
QH2, nM
GC-1, nM
TRE
receptor
(% efficacy)
(% efficacy)
(% efficacy)
DR4
TRR
0.12 ( 0.03
(100)
737 ( 59.0
(90)
6.2 ( 1.6
(88)
TRâ
0.51 ( 0.05
(100)
341 ( 25
(98)
2.1 ( 0.9
(90)
TRâ(H435A)
TRR
392 ( 84
(97)
0.25 ( 0.08
(100)
6.43 ( 0.57
(93)
1596 ( 84
(76)
>5000
(40)
n.d.
F2
TRâ
0.86 ( 0.11
(100)
774 ( 53
(82)
0.12 ( 0.04
(100)
0.44 ( 0.05
(100)
1240 ( 122
(58)
27.9 ( 7.1
(93)
1439 ( 142
(75)
594 ( 37
(87)
n.d.
n.d.
n.d.
n.d.
n.d.
TRâ(H435A)
TRR
Pal
TRâ
TRâ(H435A)
747 ( 119
(90)
22.9 ( 5.9
(93)
Figure 5. Cellular response of T3 activated TR toward QH2.
a % efficacy: maximum inducible activity compared to TR(wt) with T3.
n.d.: not determined.
Compared to other known mutations in the TRâ ligand
binding pocket, mutations to His435 which disrupt the receptors
“His-Phe switch” represent a hypersensitive region of the ligand
binding pocket that can be effectively manipulated to generate
highly selective ligand-receptor pairs. In this example, receptor
function is greatly impaired (769-fold) by His435fAla muta-
tion, which is almost completely rescued by the synthetic ligand
QH2 which is more than 778-fold more potent than the parent
analogue GC-1. Significantly, though a similar motif is used to
relay ligand binding to helix-12 in several other nuclear
receptors, the use of hydrogen bonding and edge-pi aromatic
interactions to act as transducers of ligand binding to the AF-2
domain is not essential and can be replaced with a hydrophobic
cluster. Subtle structural modifications of the ligand at this
position lead to large changes in ligand potencies and efficacies.
The His435-Phe459 switch of TRâ is only part of a complex
network of molecular interactions involved in ligand-dependent
switching of TRâ to its active conformation. Therefore, even
though T3 is 769-fold less potent in TRâ(H435A) than TRâ-
(wt), it is still a full agonist in the mutant. It is important to
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recognize that ligand binding and activity are energetically
coupled as has been shown that the presence of coactivators
can affect ligand off-rates and observed cellular potencies
(EC50’s).36 Here we target residues involved in transactivation
but are in principle effecting binding affinity as well. The low
affinity of T3 for TRâ(H435A) made determination of QH2’s
binding affinity by standard radio-ligand displacement assays
intractable. His435 plays a role in both ligand binding and
transactivation function, and QH2 is a very weak agonist toward
TRâ(wt) and does not show antagonist activity with TRâ(wt).
The selectivites observed with QH2 are not as good as the
best analogues identified through multiple rounds of selection
which have reported selectivites of 10-300-fold or the best
systems developed by design (up to 400-fold). For example,
Schwimmer et al. identified an RXR mutant that was 300-times
more responsive to the synthetic ligand LG335 than wild-type
RXR; however the selected receptor retains substantial activity
with the natural ligand 9-cis-retinoic acid.37 Zhao et al. reported
that, in HEC-1 cells, dihydroxybenzil (DHB) had a receptor
selectivity of 178-fold with a modified estrogen receptor (ER)
mutant identified through multiple rounds of selection. This
mutant identified through selection also exploited residues at
the interface between helix-11 and helix-12. Interestingly, this
same ligand exhibited only 8-fold receptor selectivity between
this same mutant and wild-type in the original yeast strain from
which it was identified. The 53-fold receptor selectivity of QH2
for TRâ(H435A) is above the median selectivity reported for
other single point mutation rescues reported in the literature.
Mutant-selective ligands (or substrates) for engineered pro-
teins are an important chemical biological tool for the study of
cellular function. The design of functionally orthogonal ligand-
receptor pairs from flexible proteins such as NHRs presents a
unique challenge in molecular design. Steric-based strategies
or “bump-and-hole” engineered ligand-receptor pairs often fail
to provide receptors that no longer respond to endogenous
ligands or ligands that do not also activate endogenous recep-
tors.20
A variety of strategies have been used to develop mutant
selective ligands including structure-based ligand design as well
as selection/directed evolution strategies. Regardless of the
design strategy, it is critical to judiciously select the site of
receptor modification so as to effectively abolish response to
the natural ligand without disrupting the intrinsic function of
the receptor.
Here we describe a new ligand-receptor design strategy
based on targeting critical residues involved in the ligand-
dependent transactivation mechanism. Of note, His435 mutants
of TRâ have been reported in human pituitary cancer as well
as resistance to thyroid hormone, suggesting that similar ligand
design strategies might someday be used to rescue receptor
function associated with these important disease states.
35
Acknowledgment. We thank the National Institutes of Health
for financial support of this work (NIH RO1DK54257). Q.H.
was supported in part by a University of Delaware Competitive
Graduate Fellowship.
Supporting Information Available: Experimental procedures
and spectroscopic characterization of compounds available. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(35) Gee, A. C.; Carlson, K. E.; Martini, P. G. V.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. Mol. Endocrinol. 1999, 13, 1912-1923.
(36) Szapary, D.; Huang, Y.; Simons, S. S., Jr. Mol. Endocrinol. 1999, 13, 2108-
2121.
(37) Schneekloth, J. S.; Fonseca, F. N.; Koldobskiy, M.; Mandal, A.; Deshaies,
R.; Sakamoto, K.; Crews, C. M. J. Am. Chem. Soc. 2004, 126, 3748-
3754.
JA060760V
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