Development of selective LH receptor agonists by heterodimerization with a FSH receptor antagonist

Article abstract of DOI:10.1021/ml100229v

The structural resemblance of the luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) impedes selective agonistic targeting of one of those by low molecular weight (LMW) ligands. In the present study, we describe a series of dimeric ligands consisting of a LMW agonist with dual activity on the FSHR and the LHR linked to a selective FSHR antagonist. Biological evaluation shows these compounds to be potent and selective LHR agonists, since no agonistic activity on the FSHR was observed. Equimolar mixing of the monomeric counterparts did not yield the pharmacological profile observed for the heterodimeric ligands, and FSHR agonism of the monomeric LHR agonist was still observed. The here-described results show that ligands with unique pharmacological profiles can be obtained by dimerizing monomeric molecules with distinct apposite properties.

Full text of DOI:10.1021/ml100229v

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Development of Selective LH Receptor Agonists by
Heterodimerization with a FSH Receptor Antagonist
Kimberly M. Bonger,†, Sascha Hoogendoorn,†, Chris J. van Koppen,‡ C. Marco Timmers,§
Gijsbert A. van der Marel,*,† and Herman S. Overkleeft*,†
†Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands, ‡Department of Molecular
Pharmacology and MDPK, and §Department of Medicinal Chemistry, Merck Research Laboratories, MSD, P.O. Box 20, 5340 BH,
Oss, The Netherlands
ABSTRACT The structural resemblance of the luteinizing hormone receptor
(LHR) and follicle-stimulating hormone receptor (FSHR) impedes selective ago-
nistic targeting of one of those by low molecular weight (LMW) ligands. In the
present study, we describe a series of dimeric ligands consisting of a LMW agonist
with dual activity on the FSHR and the LHR linked to a selective FSHR antagonist.
Biological evaluation shows these compounds to be potent and selective LHR
agonists, since no agonistic activity on the FSHR was observed. Equimolar mixing
of the monomeric counterparts did not yield the pharmacological profile observed
for the heterodimeric ligands, and FSHR agonism of the monomeric LHR agonist
was still observed. The here-described results show that ligands with unique
pharmacological profiles can be obtained by dimerizing monomeric molecules
with distinct apposite properties.
KEYWORDS Luteinizing hormone receptor, follicle-stimulating hormone receptor,
G-protein-coupled receptor, dimeric ligands, bivalency
he luteinizing hormone receptor (LHR)1 and follicle-
stimulating hormone receptor (FSHR)2 are glycopro-
tein hormone receptors3,4 that belong to the large
A major research objective in pharmaceutical research on
human fertility is to identify low molecular weight (LMW)
agonists and antagonists that are selective for either LHR5-7
or FSHR.8-12 Relevant recent discoveries stemming from
such research and at the basis of the here-presented studies
are the LMW glycoprotein hormone receptor ligands, Org
41841 (1)5 and (R)-tetrahydroquinoline (2).8 Compound 1 is
a potent LHR agonist that appears to bind to LHR at a site
distinct from that employed by the natural glycoprotein
hormone LH, namely, within the trans-membrane region
of the receptor. Although at much higher concentrations, Org
41841 (1) also agonizes FSHR, and thus, the compound does
not emulate the glycoprotein hormone receptor selectivity
featured by LH itself. Interestingly, selective FSHR antago-
nists have been reported.8,12 For instance, tetrahydroquino-
line derivative (R)-2 is a potent FSHR antagonist for which no
activity on other glycoprotein hormone receptors has been
reported yet. Compound (R)-2 is a negative allosteric mod-
ulator and antagonizes the FSHR by binding to the trans-
membrane region and is thus noncompetitive with respect
to the glycoprotein ligand FSH.
T
family of G-protein-coupled receptors (GPCRs) and are
important mediators in human reproduction. Upon activa-
tion by luteinizing hormone (LH), the LHR initiates a signal
cascade resulting, among others, in androgen production. In
females, FSHR activation by FSH induces aromatase release
in the ovaries, upon which androgens are converted to
estrogens. Both LH and FSH are members of the so-called
glycoprotein hormone family and are heterodimers com-
posed of an identical R-subunit and a hormone-specific
β-subunit. The LHR can be agonized independently by yet
another glycoprotein hormone, namely, the human chorio-
nic gonadotropin (hCG), and for this reason, the LHR is also
referred to as the hCG receptor or CGR.1 The hCG glycopro-
tein combines the LHR/FSHR R-subunit with yet another
β-subunit. During reproductive cycles, both LHR and FSHR
are often activated at the same time. In exerting control over
human fertility, selective access to either the LHR- or the
FSHR-mediated signaling pathway is desirable. The unique
β-subunits of the glycoprotein hormones provide high selec-
tivity in their binding to the extracellular N terminus of their
respective receptors, and thus, selective therapeutic control
in either LHR- or FSHR-mediated signaling can be attained
by making use of recombinant LH/hCG or FSH, respectively.
Received Date: September 28, 2010
Accepted Date: November 1, 2010
Published on Web Date: November 08, 2010
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Figure 1. Monomeric and homo- and heterodimeric LHR and FSHR ligands subject to the here-described studies.
Ligand dimerization has recently emerged as an attractive
strategy in the search for receptor ligands with optimal
pharmaceutical properties. This holds true especially for
GPCR-based research, since several GPCRs, including LHR
and FSHR, were found to function at least in part as oligo-
meric assemblies.13-18 We investigated the merits of the
dimerization approach in both LHR agonistic19 and FSHR
antagonistic20 settings, using compounds 1 and 2, respec-
tively, as starting points (Figure 1). With respect to the first,
we previously established that acetylene derivative 3 ago-
nizes the LHR and FSHR with potencies comparable to that
of the parent compound, Org 41841 (1).19 We then prepared
homodimers 6 starting from acetylene derivative 3 and
employing a double copper-catalyzed Huisgen [3 þ 2] “click”
cycloaddition with the appropriate ethylene glycol-derived
bis-azides. We found that dimers 6 were less potent LHR
agonists than compounds 1 and 3 but that they outper-
formed their monomeric counterparts 5, composed of a
single copy of 3 linked to ethylene glycol bis-azide by means
of a single click event. In this study, we did not succeed in
separating LHR agonism from FSHR agonism, since dimeric
compounds 6 all possessed agonistic activity on the latter
receptor albeit that the maximal agonistic effect of 6 was
considerably lower for the FSHR. In a related study, we
investigated the panel of homodimeric tetrahydroquinoline
structures 8 on their FSHR antagonistic properties.20 Com-
pounds 8 were prepared from acetylene (R)-4 [itself an FSHR
antagonist with potency comparable to that of the parent
compound (R)-2] using the aforementioned “click” strategy
and employing the same set of ethylene glycol bis-azides.
Although much less potent than compounds (R)-2 and (R)-4,
dimers 8 were capable of antagonizing the FSHR in the
micromolar range. From the combined experiments and
from the finding that the dimers were noncompetitive with
the respective glycoprotein hormones, we concluded that
dimeric structures 6 and 8, although considerably more
bulky as compared to their monomeric counterparts, still
find access to the LHR/FSHR trans-membrane region. In all,
the results invite the design of a set of heterodimeric
structures that encompass both a LHR agonist entity and a
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Figure 2. Synthesis of compounds 9a-e. Conditions: (a) Bis-azide 10a-e (5 equiv), CuSO4 (0.2 equiv), sodium ascorbate (1 equiv), CH2Cl2/
H2O (33-56%). (b) Acetylene 3, CuSO4 (0.2 equiv), sodium ascorbate (1 equiv), CH2Cl2/H2O (75-97%).
FSHR antagonist moiety interconnected through ethylene
glycol spacers. With the aim to establish whether such
structures would be able to agonize the LHR without agoniz-
ing the FSHR, we set about to the synthesis of heterodimeric
structures 9 (Figure 2).
antagonists, whereas the compounds in which the two
pharmacophores are connected by longer spacers [n =
3-5, compounds (R)-9c-e] are low micromolar (IC50
values) FSHR antagonists. It should be noted, however, that
these compounds did not antagonize FSHR-mediated signal-
ing by 100% (Imax). A tentative conclusion at this stage
appears that the intrinsic agonistic activity of the LHR
selective pharmacophore based on the Org 41841 structure
(1) toward the FSHR is counterbalanced by the presence of a
dedicated FSHR antagonist within the dimeric structure. The
affinity of the latter for the FSHR is such that the apparent
binding affinity of the Org 41841 pharmacophore to this
receptor is reduced.
To exclude that steric bulk, rather than FSHR antagonistic
properties, is at the basis for exclusive LHR recognition, the
agonistic activity of compounds 5c and (S)-9a-e on both
receptors was measured (Figure 3, entries 10-14 in Table 1).
Compound 5c stems from our previous research on homo-
dimeric LHR agonists, and as we demonstrated before, this
compound has significant FSHR agonistic properties.19 Com-
pounds (S)-9a-e encompass the distomer of the FSHR antago-
nist 2 [the stereoisomer at the marked (*) chiral carbon atom]
and are otherwise the same as (R)-9a-e. Compounds (S)-9a-e
are in fact LHR agonists with potencies comparable to that of
(R)-9a-e but appear to exhibit a weak but significant FSHR
agonistic property, much more so than observed for com-
pounds (R)-9a-e. We previously20 demonstrated that the
(S)-enantiomer of parent compound 2 and its derivatives have
no activity on the FSHR, and this result thus underscores our
hypothesis that the lack of FSHR agonism in compounds
(R)-9a-e is caused by the presence of the FSHR-specific
(R)-tetrahydroquinoline antagonist in these structures.
Treatment of acetylene functionalized pharmacophore
4 with 5 equiv of 1,2-diazidoethane 10a, copper sulfate
(0.2 equiv), and sodium carbonate (0.1 equiv) in a 1:1
mixture of methylene chloride and water afforded mono-
meric compound 7a in 33% yield. The main side product in
this reaction is homodimer 8a, a side reaction that we could
not suppress further, but 8a and 9a are readily separable by
silica gel chromatography. In the next step, azide 7a and
acetylene derivative 3 are subjected to the same click con-
ditions to yield heterodimer 9a in 75%. Following the same
two-step procedure and starting from the appropriate ethy-
lene glycol bis-azides (10b-e), heterodimeric ligands 9b-e
were prepared in comparable overall yields.
We next set out to measure the LHR agonistic properties,
the FSHR agonistic properties, and the FSHR antagonistic
properties of the panel of heterodimeric compounds (R)-
9a-e. As control compounds, we included the monomeric
acetylene-modified compounds 3 and 4 in our assays.19,20
As shown in entries 3-7 in Table 1, all heterodimers are
potent, LHR agonists. Although not as potent as the parent
compound 3, all agonize the target receptor with an EC50
value in the nanomolar range. Importantly, whereas mono-
mer 3 agonizes the FSHR (EC50 = 126 nM), no agonistic
effect of any of the compounds (R)-9a-e on the FSHR at
concentrations up to 10 μM were seen. In an antagonistic
setup, the compounds composed of the shorter spacers [(R)-
9a and (R)-9b, with n = 0 or 1] appear moderate FSHR
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Table 1. Mean Agonistic Potency (EC50) on the FSHR/LHR and Mean Antagonistic Potency (IC50) and Maximal Inhibition (Imax) on the FSHR
of Heterodimeric Compounds (R)-9a-ea
d
e
compound
LHR agonistic assay EC50 (nM)b,c FSHR agonistic assay EC50 (nM)b (Emax
)
FSHR antagonistic assay IC50 (nM)b (Imax
)
1
2
3
4
5
6
7
8
9
3
4
1
126 (78%)
NA
NA
NA
10
27
43
61
55
NA
5
39
(R)-9a n = 0
(R)-9b n = 1
NA
65% at 10 μM
NA
75% at 10 μM
(R)-9c
n = 2
NA
920 (73%)
(R)-9d n = 3
(R)-9e n = 4
NA
1662 (65%)
NA
1023 (72%)
7c
5c
n = 2
n = 2
n = 0
n = 1
n = 2
n = 3
n = 4
NA
324
NA
NA
NA
NA
NA
NA
397 (81%)
43% at 10 μM
44% at 10 μM
18% at 10 μM
31% at 10 μM
39% at 10 μM
10 (S)-9a
11 (S)-9b
12 (S)-9c
13 (S)-9d
14 (S)-9e
24
80
75
57
39
a None of the compounds showed antagonistic activity on the LHR. For comparison, the potencies of the previously prepared compounds 3, 4, 7c,
and 5c and control compounds (S)-9a-e are listed. b EC50/IC50 values are determined from two independent experiments performed in duplicate. SD is
generally lower than 0.3. c All active compounds are full agonists for the LHR. d Maximal effect of the compounds on the FSHR. e Imax is the maximal
percentage of inhibition of FSH-mediated signaling observed for the compound at 10 μM. NA, not active.
Figure 3. Concentration-effect curves (agonistic setup) of selected compounds on CHO cells expressing either the LHR (left) or the FSHR
(right). Selected compounds are monomeric LH/FSHR agonist 5c and heterodimeric FSHR antagonist-LH/FSHR agonist (R)-9c and (S)-9c.
The mixture of FSHR antagonist 7c and LH/FSHR agonist 5c is also depicted. Concentration-effect curves were determined from two or
three independent experiments performed in duplicate.
In a next experiment, we set out to establish whether LHR
signaling, with concomitant FSHR silencing, could also be
attained by mixing the two individual components instead
of physically linking these. To this end, we mixed LH/FSHR
agonist 5c with monomeric FSHR antagonist 7c (Figures 2
and 3). While the potency of this mixture on the LHR
remains in the same order of magnitude as in the absence
of (R)-antagonist, the potency on the FSHR is reduced
significantly (P < 0.05). This is in accordance with inhibition
of FSHR binding of the agonist 5c by the antagonist 7c. Thus,
when the LH/FSHR agonist is interconnected to the active
(R)-FSHR antagonist, agonistic activity on the FSH receptor is
completely abolished up to 10 μM, whereas mixing the
individual pharmacophores still results in significant FSHR
agonistic activity. This demonstrates the additional effect of
the recognition units in the dimeric ligand (R)-9c to interact
with the FSHR.
FSHR with a FSHR antagonist may provide compounds that
are agonistic on the LHR and not on the FSHR. Although LHR
agonistic potency is somewhat compromised when com-
pared to the parent compounds, the gain is selective LHR
activation with no observed FSHR activation. We here reveal
that unique receptor selectivity profiles may be obtained by
linking two pharmacophores with two distinct activities but
undesired cross-reactivity. Such a receptor selectivity profile
may otherwise be difficult to achieve with monomeric LMW
compounds.
SUPPORTING INFORMATION AVAILABLE Experimental
procedures on the synthesis, analysis (NMR, HPLC, and MS), and
biological evaluation of all new compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *Tel: þ31-71-5274342. E-mail: marel_g@
chem.leidenuniv.nl (G.A.v.d.M.) or h.s.overkleeft@chem.leidenuniv.nl
(H.S.O.).
In conclusion, we have demonstrated that covalently
linking an agonist with dual activity on the LHR and the
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Author Contributions: These authors contributed equally to
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