Heteroditopic binding of magnetic resonance contrast agents for increased relaxivity

Article abstract of DOI:10.1002/anie.201007689

Kept within bounds: For peptide-targeted contrast agents, internal motion limits the relaxivity gain. A focused library approach identified an N-terminal thymine peptide nucleic acid (PNA) that reaches an additional binding pocket on the protein fibrin. T

Full text of DOI:10.1002/anie.201007689

DOI: 10.1002/anie.201007689
Imaging Agents
Heteroditopic Binding of Magnetic Resonance Contrast Agents for
Increased Relaxivity**
Zhaoda Zhang, Andrew F. Kolodziej, Matthew T. Greenfield, and Peter Caravan*
Contrast agents for magnetic resonance imaging (MRI)
provide anatomical and functional detail and increasingly
can convey information at the molecular level.[1] The field of
molecular MRI has advanced to the point that clinical studies
with molecularly targeted agents are now appearing.[2]
Despite the tremendous strengths of molecular MRI (molec-
ular specificity superimposed on a high spatial resolution
anatomical image, deep tissue penetration, three-dimensional
imaging, and lack of ionizing radiation), the field remains
limited by the relatively low sensitivity for contrast agent
detection.[1b,3] Sensitivity of contrast agents is typically
described by the extent to which they can induce relaxation
of tissue water, and this is termed relaxivity (r1). Molecular
relaxivity can be increased either by increasing the number of
paramagnetic ions in the molecule, or optimizing the molec-
ular factors that influence relaxation, or some combination of
both.
For targets present at high concentrations, such as fibrin or
extracellular matrix components, it is possible to develop
effective peptide-targeted agents with one or more gadoli-
nium chelates for positive signal enhancement.[4] Unlike
nanoparticles, these relatively small molecules can rapidly
reach targets in extravascular spaces and can be readily
excreted through the kidneys to reduce or even avoid long-
term gadolinium retention and toxicity.
Clinical MRI is performed at relatively low fields (0.2–3 T,
with a majority of scanners at 1.5 T) compared to NMR
spectroscopy. One of the most effective ways to increase
relaxivity at these field strengths is to slow the rotational
dynamics of the contrast agent.[1b,3] For targeted agents,
binding to the protein target slows rotation and can increase
relaxivity several-fold over the unbound agent. While protein
binding generally increases relaxivity, the gains are often
limited because of internal motion. This is especially true for
peptide-based agents where there may be many single bonds
between the rigidly bound peptide pharmacophore and the
gadolinium chelate, resulting in increased flexibility at the
gadolinium ion. For agents that employ multiple chelates, it is
a challenge to conjugate these chelates in a way that
minimizes internal motion and yet does not deleteriously
impact targeting.
One relaxation enhancing strategy is to introduce two
binding moieties to further rigidify the molecule upon protein
binding.[5] This was successfully applied in a serum albumin-
targeted gadolinium tetramer where it was demonstrated that
a tetramer with two binding groups resulted in ca. 50% higher
relaxivity than the analogous tetramer with one binding group
when relaxivity was measured in albumin solution.[6] We
recently reported two GdDTPA tetramers (DTPA = diethyl-
ene triamine pentaacetic acid) targeted to fibrin containing
either one or two fibrin-specific peptides.[7] While the agent
with two peptides had higher affinity to fibrin, the relaxivity
of both compounds bound to fibrin was approximately the
same. This suggested that both peptide moieties were not
simultaneously bound to fibrin.
Recently, we showed that the relaxivity of a fibrin-bound
peptide conjugated to four GdDTPA moieties (Gd2-Pep-
Gd2) was limited by internal motion.[8] Although the relax-
ivity of Gd2-Pep-Gd2 increased when it was bound to fibrin,
this increase was much lower than theoretically possible due
to the flexibility inherent in the molecule. To increase the
sensitivity of this agent one could increase the number of
gadolinium chelates per molecule. However this would also
increase the relaxivity of the unbound agent (background
signal). Further, it is challenging to add more chelates in a way
that does not decrease fibrin affinity nor increase internal
motion and lower per gadolinium relaxivity. Another
approach would be to use a second peptide and vary the
linker length between the peptides to identify a molecule
where both peptides are bound and the molecule is further
rigidified. Both approaches are uncertain and significantly
increase the complexity of the molecule.
In this report we employ a much simpler approach that
was inspired by the success in the fragment-based drug
discovery field.[9] We reasoned that it is possible to identify a
second small pharmacophore at the peptide N-terminus by
screening a small library of peptides with N-terminal varia-
tion. The binding of this second pharmacophore would serve
to rigidify the N-terminal part of the molecule and boost
relaxivity while at the same time increasing overall affinity for
the target. This is shown conceptually in Figure 1. A peptide
nucleic acid (PNA) group is a DNA-mimicking molecule and
these have been widely used in molecular biology procedures,
diagnostic assays and antisense therapies. PNA moieties can
render the molecule more resistant to endo- and exonuclease-
mediated degradation, as well as to protease digestion, and
can also introduce hydrogen bonding and p–p interactions
[*] Dr. Z. Zhang, Prof. P. Caravan
A. A. Martinos Center for Biomedical Imaging
Massachusetts General Hospital, Harvard Medical School
149 13th St, Suite 2301, Charlestown, MA 02129 (USA)
Fax: (+1)617-726-7422
E-mail: caravan@nmr.mgh.harvard.edu
Dr. A. F. Kolodziej, M. T. Greenfield
Epix Pharmaceuticals, Lexington, MA 02124 (USA)
[**] This work was supported in part by the National Institute of
Biomedical Imaging and Bioengineering, R01EB009062.
Supporting information for this article (details of compound
syntheses, protein binding, and relaxivity assays) is available on the
WWW under http://dx.doi.org/10.1002/anie.201007689.
Angew. Chem. Int. Ed. 2011, 50, 2621 –2624
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2621

Communications
Table 1: Inhibition constants (Ki; standard deviation in parentheses)
showing binding of peptides to soluble fibrin fragment DD(E).
Compound NH2-Pep A-Pep
C-Pep
G-Pep
T-Pep
Ki [mm] 1.8 (0.2) 1.3 (0.1) 1.0 (0.1) 1.0 (0.1) 0.7 (0.1)
observed. The thymine derivative, T-Pep, showed the greatest
increase in affinity (160%) relative to the parent peptide.
Based on the peptide screen, we incorporated the thymine
PNA into the contrast agent Gd2-T-Pep-Gd2 (Scheme 1)
using the same synthetic approach that was used to prepare
Gd2-Pep-Gd2.[8] As a control Gd2-Gly2-Pep-Gd2 was also
synthesized. This latter compound has the same number of
bonds between the peptide and the bis(GdDTPA) moiety as
the PNA derivative, that is, the same degree of rotational
flexibility in the absence of protein binding. These com-
pounds were compared to data for Gd2-Pep-Gd2 reported
previously.[8] Conjugation of four GdDTPA moieties to the
peptide results in somewhat lower fibrin affinity compared to
the peptide itself, as may be expected (Table 2). However,
addition of the PNA group results in greater affinity as
compared to the two other contrast agents suggesting a
positive binding interaction between the PNA and the
protein.
Figure 1. Mechanism of increased relaxivity by heteroditopic protein
binding. A, B) In absence of protein (fibrin), molecule is flexible,
undergoes fast tumbling, relaxivity is low. C) Fibrin binding reduces
rotational motion and relaxivity is increased, but local motion limits
relaxivity. D) Addition of second binding group PNA limits internal
motion upon fibrin binding and boosts relaxivity.
with protein targets to improve affinity. We synthesized four
new peptides (T-Pep, G-Pep, C-Pep, and A-Pep, Scheme 1)
and compared their fibrin affinity to the parent peptide, NH2-
Pep. Peptides were screened for binding to the soluble fibrin
fragment DD(E), and the results are given in Table 1. For all
four PNA derivatives a modest increase in affinity was
Table 2: Inhibition constants for fibrin fragment DD(E) binding, Ki, and
per gadolinium relaxivities, r1 [mmÀ1 sÀ1], in Tris-buffered saline (TBS),
human plasma, or bound to human fibrin.[a]
Compound
Ki [mm] r1 (TBS) r1 (Plasma) r1 (Fibrin) % Inc
Gd2-Pep-Gd2
Gd2-Gly2-Pep-Gd2 4.0
Gd2-T-Pep-Gd2 3.5
4.7
13.0
12.4
13.3
13.1
13.0
13.1
18.0
17.8
28.0
37
37
114
[a] Determined at 1.5 T, 378C. “% Inc” refers to the percentage increase
in r1 going from plasma to fibrin. Uncertainties estimated at Æ10%.
The relaxivity of the new contrast agents was measured in
pH 7.4 Tris-buffered saline (TBS), human plasma, or a 30 mm
fibrin gel in TBS. Per gadolinium relaxivities at 1.5 T, 378C
are listed in Table 2. The relaxivities of the three compounds
in buffer or plasma were very similar and quite high compared
to GdDTPA itself (3.3 and 4.1 mmÀ1 sÀ1, respectively).[10] This
increase can be traced to the much larger size of the peptide–
gadolinium multimers which results in a longer rotational
correlation time.[8] As expected, the relaxivities increase when
the peptide–chelate conjugates are bound to fibrin, due to a
further increase in the correlation time. However Gd2-T-Pep-
Gd2 showed greater than 50% higher fibrin bound relaxivity
than the other two compounds. Since Gd2-T-Pep-Gd2 and
Gd2-Gly2-Pep-Gd2 have an equivalent number of single
bonds between the peptide pharmacophore and the bis-
(GdDTPA) moiety, an equivalent relaxivity would be
expected if the PNA group did not interact with the protein.
The much higher relaxivity for Gd2-T-Pep-Gd2 bound to
fibrin is most likely due to a positive binding interaction
between the PNA and the protein which serves to reduce
rotational flexibility at the N-terminus and increase relaxivity.
Scheme 1. Compounds synthesized and discussed in this study. Gd2-
Pep-Gd2 was described previously.[8]
2622 www.angewandte.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 2621 –2624

The relaxivities in Table 2 are per Gd ion, but on a
molecular basis, the relaxivity is four times higher. Thus the
relaxivity of Gd2-T-Pep-Gd2 bound to fibrin is 112 mmÀ1 sÀ1 at
1.5 T, which should enable sensitive thrombus detection. We
note that the fibrin-bound relaxivity of Gd2-T-Pep-Gd2 is
over 50% higher than that of EP-2104R, another peptide-
based agent that has shown thrombus imaging efficacy in
human trials.[2,4d]
To better understand the mechanism of increased relax-
ivity, nuclear magnetic relaxation dispersion (NMRD) meas-
urements were performed. Here relaxivity is measured as a
function of applied field and this technique is very sensitive to
the rotational dynamics at the GdIII ion. Figure 2A shows
The NMRD profiles were modeled using Solomon–
Bloembergen–Morgan theory (see Supporting Information
for more details). We used the simplest model that could
reproduce the NMRD data and results in averaged correla-
tion times for the four GdDTPA moieties. In the absence of
protein, the data could be well fit by an isotropic model with a
rotational correlation time of about 400 ps. Fibrin binding
results in restricted motion and here a simple isotropic model
was not sufficient to reproduce the data; instead the Lipari–
Szabo formalism was used.[11] Here two correlation times
describe rotational diffusion: a slow, global correlation time
(tg) for the protein-bound compound and a shorter, local
correlation time (tl) for internal motion. These are weighted
by an order parameter, 1 ꢀ F2 ꢀ 0, where F2 = 1 represents
isotropic global motion and F2 = 0 represents local motion
decoupled from the slow global motion. Both F2 and tl
increased in the order Gd2-T-Pep-Gd2 > Gd2-Pep-Gd2 >
Gd2-Gly2-Pep-Gd2 indicating that the increased relaxivity
of Gd2-T-Pep-Gd2 was due to restricted internal motion
likely caused by binding of the PNA residue to the protein.
In summary, we have shown that the small structural
perturbation of incorporating a PNA group into a fibrin-
targeted contrast agent has a profound impact on relaxivity.
The PNA moiety increases molecular weight by 3% but
increases relaxivity by 50% compared to Gd2-Gly2-Pep-Gd2.
The effect of the PNA group on relaxivity is the equivalent of
synthesizing an agent with six GdDTPA moieties to achieve
equivalent relaxivity. The PNA group has a modest positive
impact on fibrin binding and serves to rigidify the N-terminal
portion of the molecule upon fibrin binding. Importantly, the
PNA group does not increase non-specific protein binding. As
a result, relaxivity of Gd2-T-Pep-Gd2 bound to fibrin is more
than 50% increased compared to Gd2-Pep-Gd2 while the
relaxivity of the two compounds in plasma is comparable. This
should result in much greater clot:blood contrast for Gd2-T-
Pep-Gd2.
Figure 2. NMRD showing per Gd relaxivity at 358C of Gd2-Pep-Gd2
~
*
(&), Gd2-Gly2-Pep-Gd2 ( ) and Gd2-T-Pep-Gd2 ( ) in A) pH 7.4 Tris
buffer, B) human plasma, and C) bound to human fibrin. Solid lines
are fits to the data as described in the text.
NMRD profiles for the three compounds in TBS at 358C. In
the absence of protein, the NMRD profiles of the three
compounds are very similar as may be expected for their
similar structures. These profiles are flat and indicative of fast
rotation. Figure 2B shows relaxivities in human plasma. The
relaxivities are slightly increased in plasma suggesting some
weak plasma protein binding. Again, all three compounds
show very similar NMRD profiles in plasma, suggesting that
all compounds show the same degree of weak binding.
Figure 2C shows the NMRD profiles of the compounds
bound to fibrin. Fibrin binding causes an increase in relaxivity
for all three compounds. For Gd2-Gly2-Pep-Gd2 and Gd2-
Pep-Gd2 the increase in relaxivity upon fibrin binding ranged
from 30–70% depending on field (Table 3). For the PNA
derivative the relaxivity increase was much more remarkable:
90–140% higher than in the absence of fibrin. Comparing the
relaxivity of each compound in fibrin we note that the
introduction of the PNA group increases relaxivity by 30–
60% when compared to the other two agents.
This strategy of rigidifying peptide-based MR contrast
agents upon binding should be broadly applicable. Focused
libraries combining N- or C-terminal fragments can be rapidly
prepared and screened to identify elongated peptides with
enhanced affinity. Derivatization of the N- or C-terminus with
metal chelates would then show restricted internal motion
upon binding and enhanced relaxivity.
Received: December 7, 2010
Revised: December 30, 2010
Published online: February 8, 2011
Keywords: fibrin · gadolinium · magnetic resonance imaging ·
.
peptides · rotational dynamics
Table 3: Average NMRD parameters for the three contrast agents bound
to fibrin at 358C.[a]
Compound
tv [ps]
D2
F2
tl [ps]
[ꢀ10À18 s2]
[1] a) R. Uppal, P. Caravan, Future Med. Chem. 2010, 2, 451; b) E.
Terreno, D. D. Castelli, A. Viale, S. Aime, Chem. Rev. 2010, 110,
3019.
[2] J. Vymazal, E. Spuentrup, G. Cardenas-Molina, A. J. Wiethoff,
M. G. Hartmann, P. Caravan, E. C. Parsons, Jr., Invest Radiol.
2009, 44, 697.
Gd2-Pep-Gd2
18.2Æ1.5
9.6Æ0.3 0.06Æ0.01
818Æ18
720Æ20
Gd2-Gly2-Pep-Gd2 16.0Æ1.6 10.6Æ0.4 0.04Æ0.01
Gd2-T-Pep-Gd2
10.4Æ0.6
8.3Æ0.4 0.10Æ0.02 1507Æ31
[a] The global correlation time, tg, was greater than 20 ns for all
compounds. The water residency time, tm, was assumed to be 140 ns as
determined previously for Gd2-Pep-Gd2.[8]
[3] P. Caravan, Chem. Soc. Rev. 2006, 35, 512.
Angew. Chem. Int. Ed. 2011, 50, 2621 –2624
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2623

Communications
[4] a) C. Burtea, S. Laurent, M. Port, E. Lancelot, S. Ballet, O.
[6] Z. Zhang, M. T. Greenfield, M. Spiller, T. J. McMurry, R. B.
Lauffer, P. Caravan, Angew. Chem. 2005, 117, 6924; Angew.
Chem. Int. Ed. 2005, 44, 6766.
[7] S. Nair, A. F. Kolodziej, G. Bhole, M. T. Greenfield, T. J.
McMurry, P. Caravan, Angew. Chem. 2008, 120, 4996; Angew.
Chem. Int. Ed. 2008, 47, 4918.
[8] Z. Zhang, A. F. Kolodziej, J. Qi, S. A. Nair, X. Wang, A. W. Case,
M. T. Greenfield, P. B. Graham, T. J. McMurry, P. Caravan, New
J. Chem. 2010, 34, 611.
[9] a) M. Congreve, G. Chessari, D. Tisi, A. J. Woodhead, J. Med.
Chem. 2008, 51, 3661; b) P. J. Hajduk, J. Greer, Nat. Rev. Drug
Discovery 2007, 6, 211.
Rousseaux, G. Toubeau, L. Vander Elst, C. Corot, R. N. Muller,
J. Med. Chem. 2009, 52, 4725; b) V. Amirbekian, J. G. Aguinaldo,
S. Amirbekian, F. Hyafil, E. Vucic, M. Sirol, D. B. Weinreb, S.
Le Greneur, E. Lancelot, C. Corot, E. A. Fisher, Z. S. Galis,
Z. A. Fayad, Radiology 2009, 251, 429; c) F. Ye, E. K. Jeong, Z.
Jia, T. Yang, D. Parker, Z. R. Lu, Bioconjugate Chem. 2008, 19,
2300; d) K. Overoye-Chan, S. Koerner, R. J. Looby, A. F.
Kolodziej, S. G. Zech, Q. Deng, J. M. Chasse, T. J. McMurry, P.
Caravan, J. Am. Chem. Soc. 2008, 130, 6025; e) P. Caravan, B.
Das, S. Dumas, F. H. Epstein, P. A. Helm, V. Jacques, S. Koerner,
A. Kolodziej, L. Shen, W. C. Sun, Z. Zhang, Angew. Chem. 2007,
119, 8319; Angew. Chem. Int. Ed. 2007, 46, 8171.
[10] M. Rohrer, H. Bauer, J. Mintorovitch, M. Requardt, H. J.
Weinmann, Invest. Radiol. 2005, 40, 715.
[11] G. Lipari, A. Szabo, J. Am. Chem. Soc. 1982, 104, 4546.
[5] F. Kielar, L. Tei, E. Terreno, M. Botta, J. Am. Chem. Soc. 2010,
132, 7836.
2624 www.angewandte.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 2621 –2624