Multiple N-methylation by a designed approach enhances receptor selectivity

Article abstract of DOI:10.1021/jm701044r

An unselective cyclic peptide integrin ligand was sequentially N-methylated by a designed approach, where only the externally oriented (solvent exposed) amide bonds were N-methylated. The N-methylation resulted in tremendous enhancement in selectivity amo

Full text of DOI:10.1021/jm701044r

5
878
J. Med. Chem. 2007, 50, 5878-5881
Multiple N-Methylation by a Designed Approach
Enhances Receptor Selectivity
sequence is the fibronectin receptor R5â1, which is expressed
on activated endothelial cells, immune cells, and fibroblasts.9
It plays a key role during development of the vascular system
and pathological angiogenesis, such as that in solid tumors.11
We show here that a systematic multiple N-methyl scan can be
employed to achieve surprisingly enhanced receptor selectivity
between these receptor subtypes.
,10
†
⊥
‡
Jayanta Chatterjee, Oded Ovadia, Grit Zahn,
#
⊥
£
Luciana Marinelli, Amnon Hoffman, Chaim Gilon, and
Horst Kessler*,†
Center for Integrated Protein Science at the Department Chemie,
Lehrstuhl II f u¨ r Organische Chemie, Technische UniVersit a¨ t
M u¨ nchen, Lichtenbergstrasse 4, Garching D85747, Germany,
Pharmaceutics and Organic Chemistry Departments, The Hebrew
UniVersity of Jerusalem, Jerusalem 91904, Israel, Jerini A.G.,
InValidenstrasse 130, 10115 Berlin, Germany, and Farmaceutica e
Tossicologica, UniVersit a` di Napoli ‘Federico II’, Via D.
Montesano, 49-80131 Napoli, Italy
12
N-Methylation is a precious tool to modify lipophilicity,
1
3
3
proteolytic stability, and bioavailability and to induce con-
1
4
formational rigidity to the peptide backbone. Mono-N-
methylation of peptide ligands has been employed over the years
1
5
16
to enhance potency, new receptor subtype selectivity, and
17
tuning of an agonist to antagonist. However, multiple N-
methylation has been seldom employed, probably owing to
18
ReceiVed August 22, 2007
the availability of N-methylated amino acids, subsequent
difficult coupling, and unpredictable conformational change.
19
14,20
In this work, we envisioned a “design approach” instead of
the commonly used “library approach”. For this purpose, the
prerequisite is the knowledge of the bioactive conformation of
the stem peptide (lead structure). Due to numerous studies of
substituted cyclic penta- and hexapeptides containing the
tripeptide sequence RGD, those peptides were chosen for
this approach. We used the cyclic hexapeptide cyclo
Abstract: An unselective cyclic peptide integrin ligand was sequen-
tially N-methylated by a designed approach, where only the externally
oriented (solvent exposed) amide bonds were N-methylated. The
N-methylation resulted in tremendous enhancement in selectivity among
the different integrin receptor subtypes (R5â1, Rvâ3, and RIIbâ3).
Conformational and docking studies were performed, which suggested
that the receptor selectivity is principally caused by reduced backbone
flexibility due to N-methylation.
1
2
3
4 5 6
(
-G R G D f L -), for which 63 different N-methylated ana-
Integrins are heterodimeric proteins that are important for
cell-cell and cell-extracellular matrix interactions and are
composed of R and â subunits.1 Integrins serve as transmem-
brane linkers between their extracellular ligands and the
cytoskeleton and modulate various signaling pathways; thus,
they have the capacity to influence cell migration, differentiation,
and survival during embryogenesis, angiogenesis, wound heal-
ing, immune and nonimmune defense mechanisms, hemostasis,
21
logues are possible (Figure 1A). However, we synthesized a
small library of seven derivatives in which only the externally
oriented (solvent exposed) amide protons were N-methylated.
This should lead to structures in which the overall conformation
is only slightly modified, retaining at least some activity, but
in addition, it may positively influence permeability when orally
administered. So far, there is no orally available integrin
antagonist in use; however, there are some compounds for Rvâ3
reported by the Merck group which are orally available, but
,2
1
and oncogenic transformation. The combination of different R
and â subunits determines the ligand specificity and therefore
the biological function of the integrins. Many integrins are linked
with pathological conditions; therefore, the specific inhibition
of a certain integrin is a very promising tool for effective
therapeutic intervention with limited side effects.
22
not yet as drugs on the market.
The cyclic peptides merely act as a scaffold to hold the side
chains in proper spatial orientation. Detailed study of the impact
14
of N-methylation on cyclic pentapeptides and hexapeptides
(unpublished results) suggested to us that N-methylation of the
The RIIbâ3 is the most abundant integrin on the surface of
platelets, which mediates aggregation of platelets to form
thrombi. The activated RIIbâ3 in the final step of blood clot
formation binds to blood glycoprotein fibrinogen to cross-link
platelets in a growing thrombus. Thus, compounds that compete
with fibrinogen in binding to RIIbâ3 can act as potent
antithrombotic agents.3 The peptidomimetic “Tirofiban” is
externally oriented solvent-exposed amide protons may not
drastically change the backbone peptide conformation (e.g.,
trans-cis peptide bond interconversion); however, it helps in
rigidifying the backbone conformation by (i) restricting the
peptide bond flip (180° rotation of the peptide bond about the
R
20,23
adjacent C ’s)
due to steric hindrance of the N-methyl group
and (ii) ruling out the possibility of conformational equilibrium
4,5
already on the market for this purpose. The vitronectin
receptor Rvâ3, which also recognizes other extracellular matrix
proteins like fibronectin and osteopontin, is expressed on
endothelial cells, osteoclasts, and tumor cells and is involved
between interchangeable turn structures, for example, âII′ to γ
24
and vice versa. The reason for choosing the cyclic hexapeptide
rather than the pentapeptide is the “rigidity”, as cyclic hexapep-
tides unlike cyclic pentapeptides usually exhibit a conformation
with two internally oriented peptide bridges (often two â turns).
Cyclic hexapeptides correspond to cyclohexanes (see discussion
6
-8
in processes of angiogenesis and bone remodeling.
A third
important integrin which recognizes the RGDa tripeptide
2
5
26
in Heller et al.) and prefer to adopt a chairlike conformation.
*
To whom correspondence should be addressed. E-mail: Kessler@
1
2
3
4 5 6
The cyclic peptide cyclo(-G R G D f L -) (1), which was
ch.tum.de. Tel: 498928913300. Fax: 498928913210.
†
27
Technische Universit a¨ t M u¨ nchen.
Pharmaceutics Department, The Hebrew University of Jerusalem.
Organic Chemistry Department, The Hebrew University of Jerusalem.
Jerini A.G..
Universit a` di Napoli ‘Federico II’.
Abbreviations: RGD, arginine glycine aspartic acid; HATU, N-[(dim-
reported by Pfaff et al. to be selective toward RIIbâ3 compared
⊥
to Rvâ3, reveals a âII′ turn about D-Phe-Leu and a âII turn
£
21
‡
about Arg-Gly, which is the recognition motif, with two
4
1
1
#
internal hydrogen bonds between Asp CO-HNGly and Gly -
a
4
CO-HNAsp. This stem peptide cyclo(-GRGDfL-) has some
ethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methyl-
methanaminium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; Caco-
flexibility (due to the two Gly residues) but allows investigation
of the effect of N-methylation on the backbone to obtain highly
2
, colon carcinoma cell monolayer; PAMPA, parallel artificial membrane
28
29
permeation assay; Papp, apparent permeability.
active and selective RIIbâ3 integrin antagonist. We describe
1
0.1021/jm701044r CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/01/2007

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 5879
Figure 1. (A) Lead structure (1) with two â turns and the solvent-
exposed amide protons (in red). (B) Stereoview of cyclo(-GRGDfL-)
(
1). Note the stabilizing γ turns about aspartic acid and glycine 1,
Figure 2. Stereoview of cyclo(-GRGDfL-) (8). The stabilizing γ turns
about aspartic acid and glycine 1 are absent, resulting in a flat structure.
Note the âI′ turn about Arg and Gly.3
presenting the peptide in a “folded conformation”.
Table 1. The Seven N-Methylated Cyclic Analogues with the Stem
1
2
3
4 5 6
Peptide cyclo(-G R G D f L -) and Their Binding Affinity (IC50 in nM)
toward Three Different Integrins; the N-Methylated Residues Are
Highlighted in Bold
the peptidic backbone in an extended orientation. It is to be
noted that the side chain of arginine is very flexible and could
fit into Rvâ3 and RIIbâ3; thus, the main selectivity was brought
about by the rigidity in the scaffold by N-methylation.
In the stem peptide 1 or when leucine (2) and/or phenylalanine
no.
analogue
R5â1
Rvâ3
RIIbâ3
Rvâ3/RIIbâ3
1
2
3
4
5
6
7
8
c(-GRGDfL-)
c(-GRGDfL-)
c(-GRGDfL-)
c(-GRGDfL-)
c(-GRGDfL-)
c(-GRGDfL-)
c(-GRGDfL-)
c(-GRGDfL-)
740
3900
4300
1200
>20000
∼20000
>20000
>20000
100
103
490
195
560
2000
12
620
15
0.5
0.2
0.2
64
2
86
(3) were N-methylated, there was considerable flexibility in the
770
âII turn, resulting in unspecific binding. In all of the peptides
lacking the N-methylated arginine residue, the NMR and the
MD showed the possibility of forming a γ turn about the glycine
in position 3, which ultimately brought the side chains of the
aspartic acid and arginine close to each other, resulting in a
comparatively better fit into the Rvâ3 binding pocket. In addition
to the γ turn, we observed a “kink” in the backbone conforma-
1200
1300
2730
12200
165
30
16
406
here a biased small library in which all externally oriented amide
bonds except Gly , which is involved in the receptor binding,
were N-methylated (Table 1).
3
30
36
tions of 1 and 2, giving rise to a “folded” structure, resulting
1
in two further γ turns about Asp and Gly (Figure 1B).
The linear peptides were obtained by standard solid-phase
This kinked conformation is probably favored for Rvâ3 as
both 1 and 2 bind better to Rvâ3 than to RIIbâ3. This kink was
lost by N-methylation of D-Phe and/or Arg, which blocked
the γ turns and presented the peptide in a flattened conforma-
tion. In the case of 8, there was no indication of any γ
31
techniques, with N-methylation either in solution or on a solid
32
support, and finally, the cyclization was carried out in solution
using HATU/HOBt.33
It is worth noting that, despite our previous report, we found
no receptor selectivity for 1, and there was an inclination toward
Rvâ3 selectivity with N-methylated leucine (2) or D-phenyla-
lanine (3). The different IC50 values and, consequently, the
different receptor selectivity can be explained by other experi-
mental conditions used in this work compared to the work by
Pfaff and colleagues.27 The main difference is the composition
of used integrins and thus the applied protein concentration,
which causes other IC50 values (details in Supporting Informa-
tion).
3
turn about Gly , and the peptide was in a flat conformation
(Figure 2), which eventually resulted in holding the aspartic
acid and arginine side chains apart, fitting well into the RIIbâ3
pocket.
The conformation of peptide 8 is very similar to that of 4;
the only difference is the slight clockwise rotation of the phenyl
ring and counterclockwise rotation of the isopropyl group of
the leucine side chain (as suggested by the ROE’s). In addition,
we observed a close resemblance to a âI′ turn about Arg and
3
Significant selectivity was first obtained in 4 with the
N-methylated arginine residue, which corroborates with previous
Gly in 8 owing to the reduced flexibility in this region (due to
the N-Me-D-Phe), and thus, there is no indication of the
3
4
3
results. Extending further the N-methylation of 4 to leucine,
there was almost no loss in the activity in 6 but a further gain
in selectivity. Exchanging the site of N-methylation from leucine
to phenylalanine in 7, there was a sudden loss in the activity.
However, interestingly, the activity was gained back with an
additional N-methylation of 7, giving rise to 8, with a
tremendous enhancement in the selectivity and still high activity
for RIIbâ3. It is really surprising that a single N-methyl group
when present at phenylalanine is responsible for the loss of
activity in 7 and the gaining back the activity and enormous
selectivity when present at leucine in 8.
These results prompted us to study the solution conformation
of these analogues. It is well-known that the selectivity between
Rvâ3 and RIIbâ3 can be achieved by fine-tuning the distance
between the carboxyl group of aspartic acid and the guanidine
group of arginine in the ligands. In our case, the selectivity
first arose by N-methylation of the arginine residue (4), which
was primarily owing to the reduction in the flexibility about
arginine and glycine 3 (which resembles a âI′ turn), presenting
formation of γ turn about Gly , whereas the âI′ turn in 4 was
flexible, and there was a close resemblance to a γ turn about
3
Gly (Figure 3). This is probably one of the reasons for low
binding of 8 to Rvâ3, in contrast to that for 4.
To have an insight into the binding modes of 4 and 8 into
3
7
RIIbâ3, docking studies were performed using the software
Autodock. In both peptides, the carboxylate group of Asp was
found to coordinate the metal ion at the MIDAS region, whereas
the Arg side chain extended into the deep â-propeller pocket,
forming a hydrogen bond to the RIIb-Asp224 carboxylate group.
The main difference between the 4 and 8 binding modes comes
5
from the upper part of the peptides in the region from D-Phe -
6
Leu (Figures 2 and 3). Due to multiple N-methylation
introduced in 8, and especially due to the N-methylation of
D-Phe residue, 8, when compared to 4, seems to lower its π-π
interaction with â3-Tyr122 (shown by the yellow arrow in
Figure 4) and did not properly orient the Leu carbonyl group
to hydrogen bond with the R214 side chain (shown by the white
arrow in Figure 4).
35

5
880 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Letters
understanding of the bioactive conformation. Thus, multiple
N-methylation of peptides could be a straightforward and
simplistic approach to obtain highly potent and selective ligands.
Acknowledgment. We gratefully acknowledge the Hum-
boldt Foundation for the support via the Max-Planck-For-
schungspreis. This work was also, in part supported, by the
German-Israel Foundation. We thank Thorsten Lanz for
performing ELISA assays.
Supporting Information Available: NMR spectrum, HRMS,
and detailed experimental and computational procedures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
3
Figure 3. Stereopicture of cyclo(-GRGDfL-) (4). About Gly , the
molecule has a tendency to adopt a γ turn (shown by the curved line),
forming a hydrogen bond between AspNH and ArgCO. Also interesting
to note is the kink in the peptide backbone about Asp. This kink is
stabilized by formation of a closed γ turn involving the PheNH and
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