Trigonal scaffolds for multivalent targeting of melanocortin receptors

Article abstract of DOI:10.1039/c4ob02094d

Melanocortin receptors can be used as biomarkers to detect and possibly treat melanoma. To these ends, molecules bearing one, two, or three copies of the weakly binding ligand MSH(4) were attached to scaffolds based on phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane by means of the copper-assisted azide-alkyne cyclization. This synthetic design allows rapid assembly of multivalent molecules. The bioactivities of these compounds were evaluated using a competitive binding assay that employed human embryonic kidney cells engineered to overexpress the melanocortin 4 receptor. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (～2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed.

Full text of DOI:10.1039/c4ob02094d

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DOI: 10.1039/C4OB02094D
Organic and Biomolecular Chemistry
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ARTICLE
Trigonal scaffolds for multivalent targeting of
melanocortin receptors
Cite this: DOI: 10.1039/x0xx00000x
N. G. R. Dayan Elshan,a Thanuja Jayasundera,b Bobbi L. Anglin,a Craig S.
Weber,b Ronald M. Lynch,b, c and Eugene A. Mash*a
Received 00th January 2015,
Accepted 00th January 2015
Melanocortin receptors can be used as biomarkers to detect and possibly treat melanoma. To
these ends, molecules bearing one, two, or three copies of the weakly binding ligand MSH(4)
were attached to scaffolds based on phloroglucinol, tripropargylamine, and 1,4,7ꢀ
triazacyclononane by means of the copperꢀassisted azideꢀalkyne cyclization. This synthetic
design allows rapid assembly of multivalent molecules. The bioactivities of these compounds
were evaluated using a competitive binding assay that employed human embryonic kidney
cells engineered to overexpress the melanocortin 4 receptor. The divalent molecules exhibited
10ꢀ to 30ꢀfold higher levels of inhibition when compared to the corresponding monovalent
molecules, consistent with divalent binding, whereas the trivalent molecules were only
statistically (~2ꢀfold) better than the divalent molecules, still consistent with divalent binding
but inconsistent with trivalent binding. Possible reasons for these behaviors and planned
refinements of the multivalent constructs targeting melanocortin receptors based on these
scaffolds are discussed.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
a homology model based on the crystal structure of the GPCR
rhodopsin suggested distances ranging from 20ꢀ50 Å between
the binding sites of abutted receptors.20 In previous articles, we
described multivalent constructs based on linear21ꢀ24 and
spherical25 scaffolds, typically with ligand spacing in this range.
Using a competitive binding assay and a human embryonic
kidney cell line, HEK293, genetically engineered to
overexpress the human melanocortin 4 receptor (hMC4R),9
these constructs were shown to exhibit enhanced binding
consistent with statistical effects, but inconsistent with
simultaneous binding of two or more ligands to receptors on the
cell surface.
Melanoma is the most aggressive form of skin cancer in the
world and accounts for over 75% of skin cancerꢀrelated deaths.1
As with many serious cancers, melanoma has a good prognosis
when detected in its early stages.2 Malignant cells often
overexpress characteristic receptors on their cell surfaces when
compared to normal cells,3ꢀ5 affording a potential means of
detection. For example, the melanocortin 1 receptor, a Gꢀ
protein coupled receptor (GPCR), is overexpressed in most
forms of melanoma.6,7 Among other research groups,8ꢀ10 we are
pursuing detection strategies that employ multivalent
interactions by two or more weakly binding ligands attached to
a scaffold. Such multivalent molecules can selectively bind
with high avidity to cells overexpressing the targeted ligand
receptors.11ꢀ12
Recently, multivalent binding of molecules that incorporate
MSH(4) ligands was demonstrated using monoꢀ, diꢀ, and
trivalent compounds 4-6 18
.
The divalent and trivalent
exhibited 16ꢀfold and 350ꢀfold
enhancements, respectively, in the measured IC50 values
compared to the monovalent construct in a competitive
binding assay against an NDPꢀ ꢀMSHꢀbased fluorescent probe.
The interꢀligand distances in and were estimated to be 24 ±
Factors that can influence the interactions of multivalent
molecules with cell surface receptors include the potency of the
ligands utilized, the interꢀligand distances, and the possible
geometries of ligand display permitted by the scaffold. Peptide
ligands that are known to bind to melanocortin receptors
constructs
5
and
6
4
α
5
6
include AcꢀSerꢀTyrꢀSerꢀNleꢀGluꢀHisꢀ
ProꢀValꢀNH2 ( , NDPꢀ
ꢀMSH, high affinity),13 SerꢀNleꢀGluꢀ
PheꢀArgꢀTrpꢀNH2
, MSH(7), medium affinity),14 and
PheꢀArgꢀTrpꢀNH2 (
, MSH(4), low affinity).14 Because
DPheꢀArgꢀTrpꢀGlyꢀLysꢀ
5 Å by molecular modeling. Structurally related dendrimeric
constructs bearing six or nine MSH(4) ligands exhibited
1
α
Hisꢀ
Hisꢀ
D
D
(2
3
somewhat reduced inhibitory potencies.19
These studies
strongly suggest that narrow limits on ligand spacing exist for
observation of true multivalent binding to hMC4 receptors, at
least for the engineered HEK293 cell line used in the
competitive binding assays.
cooperative binding leading to enhanced avidity is more evident
when weakly binding ligands are utilized,11,12 we (and others15ꢀ
19) have used MSH(4) in our multivalent constructs.
Control of ligand number, spacing, and geometry depends
on the scaffold used to display the MSH(4) ligand. In the
absence of crystallographic data on the melanocortin receptors,
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DOI: 10.1039/C4OB02094D
Results
HN R2
O
Chemistry. Commercially available phloroglucinol (
alkylated according to
7) was
a
reported method26 to produce
O
O
trisalkyne 10 (Scheme 1). Using microwaveꢀassisted copperꢀ
catalyzed azideꢀalkyne cycloaddition (CuAAC) reactions, the
known serinamide azide 1124 was attached to the
H
N
N
NH2
R1
N
H
H
phloroglucinolꢀderived hub, leading to 12a-c
reactions of 12b 12c, and 10 with the known MSH(4) azide
1324 afforded monoꢀ, bisꢀ, and trisꢀMSH(4) constructs 14a-c
.
CuAAC
R3
N
,
H
,
4 R1 = W, R2 = R3 = Ac
respectively. Although the reaction vials and solvents were
purged with argon to exclude oxygen from the system, if air is
introduced during handling oxidation of the reactive Cu(I)
species can occur. We observed that inclusion of sodium
ascorbate in the reaction mixtures enhanced the repeatability of
the product yields.
5 R1 = Ac, R2 = R3 = W
6 R1 = R2 = R3 = W
O
O
MSH(4)
N
N
W =
N
Serinamideꢀcontaining constructs 15a-c were prepared from
commercially available tripropargylamine (
similar manner (Scheme 2). However, CuAAC reactions of
15b 15c, and with MSH(4) azide 13 under the conditions
used to prepare 14a-c were unsatisfactory. For the synthesis of
16a and 16b, acceptable yields were obtained when tris[(1ꢀ
8) and azide 11 in a
,
8
In light of these observations, we wished to more fully
evaluate the effects of ligand spacing and orientation on avidity
while simplifying the scaffold to enhance the economy and
versatility of the synthetic approach to useful multivalent
molecules. In this article, we present syntheses and bioassay
results from MSH(4)ꢀbearing multivalent molecules based on
benzylꢀ1
H
ꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl]amine
(TBTA)
was
omitted from the reaction mixture. Normally TBTA stabilizes
Cu(I),27 but in these reactions the product triazoles might serve
this function. Along with added TBTA, the products might
phloroglucinol
(
7
), tripropargylamine
(8), and 1,4,7ꢀ
complex Cu(I) ions, reducing the reaction rate. Reaction of
with azide 13 to produce 16c was more challenging.
Microwaveꢀassisted reactions with different reagent
compositions, solvents, and reaction times failed. However,
reaction of with azide 13 in the presence of
8
triazacyclononane (
9
) scaffold cores. As a part of this study,
we also describe new protocols that improve the signalꢀtoꢀnoise
ratio for in vitro binding assays when using the engineered
HEK293 cells.
8
tetrakis(acetonitrile)copper(I) hexafluorophosphate (TACP)
catalyst and 2,6ꢀlutidine in a mixture of water and acetonitrile
(MeCN) for 4 days at room temperature provided 16c in 62%
yield.
OH
H
H
N
N
N
N
H
HO
OH
7
8
9
Scheme 1. Synthesis of phloroglucinolꢀbased compounds 5a and 14a-c.
O
O
O
N3
R3
R1
OH
O
Ser-NH2
O
Propargyl bromide,
11
HO
R2
O
Cu(CH3CN)4PF6, TBTA,
Sodium ascorbate, DMF,
Microwave (100 oC, 4 h)
K2CO3,
DMF, rt, 4 d
48%
OH
O
12a R1 = R2 = R3 = X (83%)
12b R1 = R2 = X; R3 = Y (31%)
12c R1 = X; R2 = R3 = Y (48%)
7
10
O
O
H
R3
O
R3
N3
N
O
O
MSH(4)
N
N
NH2
N
13
X =
O
O
O
OH
R2
R2
Cu(CH3CN)4PF6, TBTA,
Sodium ascorbate, DMF,
Microwave (100 oC, 4 h)
O
Y =
Z =
R1
R1
12b R1 = R2 = X; R3 = Y
12c R1 = X; R2 = R3 = Y
10 R1 = R2 = R3 = Y
14a R1 = R2 = X; R3 = Z (16%)
14b R1 = X; R2 = R3 = Z (23%)
14c R1 = R2 = R3 = Z (51%)
MSH(4)
N
N
N
O
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C4OB02094D
Scheme 2. Synthesis of tripropargylamineꢀbased compounds 15a and 16a-c.a
R2
O
H
11
N
R3
N
N
N
N
NH2
R1
N
Cu(CH3CN)4PF6, TBTA
X =
O
Sodium ascorbate, DMF,
Microwave (100 oC, 4 h)
OH
15a R1 = R2 = R3 = X (100%)
15b R1 = R2 = X; R3 = Y (30%)
8
15c R1 = X; R2 = R3 = Y (71%)
Y =
R2
R2
MSH(4)
O
13
N
N
N
N
R3
N
Z =
R3
R1
R1
Conditions (A) or (B)
15b R1 = R2 = X; R3 = Y
15c R1 = X; R2 = R3 = Y
16a R1 = R2 = X; R3 = Z (33%)
16b R1 = X; R2 = R3 = Z (40%)
16c R1 = R2 = R3 = Z (62%)
8
R1 = R2 = R3 = Y
aConditions A: TACP, sodium ascorbate, DMF, microwave irradiation to maintain 100 °C, 4 h; used in the synthesis of 16a and 16b.
Conditions B: TACP, 2,6ꢀlutidine, H2O/MeCN (1:1), rt, 4d; used in the synthesis of 16c.
Scheme 3. Synthesis of 1,4,7ꢀtriazacyclononaneꢀbased compound 18.
Z
Z
Propargyl bromide,
KOH
MSH(4)
H
H
N
N
Z
N
N
13
N
N
N
N
N
Z =
O
N
Cu(CH3CN)4PF6,
N
N
H
EtOH/Toluene (1:1),
24 h, 69%
Sodium ascorbate, DMF,
Microwave (100 oC, 4 h), 42%
9
17
18
Reaction of 1,4,7ꢀtriazacyclononane (9) with propargyl assays were performed using the known timeꢀresolved
bromide using a reported method28 gave 17 along with fluorescence (TRF) probe, EuꢀDTPAꢀPEGOꢀMSH(7)ꢀNH2
14,32
quaternary ammonium salts due to overꢀalkylation (Scheme 3).
(
19
)
and HEK293 cells engineered to overexpress hMC4R
By reducing the number of equivalents of propargyl bromide (approximately 640,000 copies per cell) and human
and controlling the reaction temperature during its addition, 17 cholecystokinin 2 receptor (hCCK2R, approximately 1,100,000
was produced in 69% yield. The CuAAC reaction between 17 copies per cell).9 Saturation binding curves are depicted in
and azide 13 under the conditions used for preparation of 14a-c Figure 1. The Kd for 19 calculated from these assays was 21 ±
but without TBTA afforded compound 18 in 42% yield. 3 nM, which is consistent with the value of 27 ± 4 nM reported
Removal of copper ions from the crude product 18 was from saturation binding assays using an older protocol.14
especially difficult, possibly due to metal ion chelation by the
O
side arms and the 1,4,7ꢀtriazacyclononane core. It was
necessary to remove the copper ions by extraction,29,30 because
O
H
N
preparative HPLC does not remove them.
prolonged contact with dithizone in chloroform gave complete
removal of the copper ions.31
Fortunately,
N
O
N
O
Eu
N
O
OH
O
O
O
O
O
Compounds 14a-c, 16a-c, and 18 used in biological assays
O
were ≥95% pure as determined by HPLC analysis (see
Electronic Supplementary Information).†
H
Ser-Nle-Glu-His-DPhe-Arg-Trp-NH2
N
O
O
O
Biological Assays. Using a new protocol (see the Discussion
19
and the Experimental Section for details), saturation binding
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