Rational design of a Tn antigen mimic

Article abstract of DOI:10.1039/c1cc10192g

A novel Tn antigen mimic, in which the natural underlying amino acid has been replaced by the non-natural α-methylserine analogue, is reported. This derivative exhibits a similar affinity for a natural lectin as for the natural Tn and retains the bioactiv

Full text of DOI:10.1039/c1cc10192g

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Rational design of a Tn antigen mimicw
Francisco Corzana,*a Jesu
s H. Busto,a Filipa Marcelo,b Marisa Garcıa de Luis,a
´ ´
d
e
ef
Juan L. Asensio,c Sonsoles Martı
´
Jesus Jimenez-Barbero, Alberto Avenoza and Jesus M. Peregrina*
´ ´
´
n-Santamarıa, Yolanda Saenz, Carmen Torres,
´
´
a
b
a
Received 11th January 2011, Accepted 16th March 2011
DOI: 10.1039/c1cc10192g
A novel Tn antigen mimic, in which the natural underlying amino
acid has been replaced by the non-natural a-methylserine analogue,
is reported. This derivative exhibits a similar affinity for a
natural lectin as for the natural Tn and retains the bioactive
conformation observed in the Tn-containing glycopeptides with
anti-MUC1 antibodies.
The Tn-determinant (GalNAc-a-O-Ser/Thr, compound 1 in
Fig. 1a) is a specific human tumor-associated carbohydrate
antigen. It is expressed in many carcinogenic tumors, including
breast, prostate, lung and pancreatic cancers.1–3 The level
of expression often correlates with carcinoma differentiation
and aggressiveness.4,5 Consequently, this antigen serves as a
biomarker and it is being exploited for the development of
immunotherapies for cancer.6–9
It is well-known that cells of the immune system are
equipped with many lectin and lectin-like receptors.10 Thus,
carbohydrate moieties play important roles as major epitopes
for these receptors, and recent studies have shown that inter-
actions between tumor-associated carbohydrate antigens, such
as Tn, and lectins play a crucial function in the metastatic
cascade of some carcinoma cells.11–13 In this regard, the X-ray
structure of a monoclonal antibody that recognizes directly the
sugar moiety of a short glycopeptide carrying the Tn antigen
has been recently reported.14 Moreover, peptide fragments
containing the Tn antigen are recognized by different antibodies.
In particular, the Pro-Asp-Thr-Arg sequence comprises the
minimal epitope for MUC1 specific monoclonal antibodies,
Fig. 1 (a) Molecules studied in this work. (b) X-Ray structure
(pdb code 1sm3) of the TSAPDTRPAPGST peptide in complex with
SM3 antibody. The amino acid residues involved in the peptide
epitope are represented with bold letters. The helix-like conformation
of the Thr residue is highlighted.
such as SM3.15 The crystal structure of this antibody complexed
with a small peptide reveals that the region containing the Thr
residue adopts a helix-like conformation to properly fit in the
binding pocket.16 The glycosylation of the Thr residue with
GalNAc in the above-commented sequence does enhance the
interactions with the antibody.17
a
´
Departamento de Quımica, Universidad de La Rioja,
Centro de Investigacion en Sıntesis Quımica, UA-CSIC,
´
´
´
Madre de Dios, 51, 26006 Logron˜o, Spain.
E-mail: jesusmanuel.peregrina@unirioja.es,
francisco.corzana@unirioja.es; Fax: +34 941 299 621
b
c
d
´
Centro de Investigaciones Biologicas (CSIC), Madrid, Spain
Instituto de Quımica Organica General (CSIC), Madrid, Spain
Encouraged by these experimental observations and con-
sidering the role that these structural features could play for the
molecular recognition process, and consequently for the immune
response, we herein present a novel Tn mimic (compound 2,
Fig. 1a) in which the natural Thr amino acid has been replaced
by its non-natural a-methylserine analogue. In this regard,
different Tn-analogues have been previously prepared.18–21
However, from a rational design viewpoint, it is mandatory
that the novel derivatives retain the bioactive conforma-
´
´
´
Departamento de Quımica, Facultad de Farmacia,
Universidad San Pablo CEU, Madrid, Spain
e
f
´
´
´ ´
Area de Microbiologıa Molecular, Centro de Investigacion Biomedica
de La Rioja (CIBIR), 26006 Logron˜o, Spain
´
´
Area de Bioquımica y Biologıa Molecular, Universidad de La Rioja,
Area de Microbiologıa Molecular, Centro de Investigacion Biomedica
´
´
´
´
´
de La Rioja (CIBIR), 26006 Logron˜o, Spain
w Electronic supplementary information (ESI) available: Synthetic
procedures, NMR and MD data and STD experiments. See DOI:
10.1039/c1cc10192g
c
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Chem. Commun., 2011, 47, 5319–5321 5319

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Table 1 Dissociation (KD) and affinity constants (Ka) determined by
STD-NMR experiments and the corresponding thermodynamic bind-
ing parameters for EcorL at 298 K
Ligand
KD/mM
Ka/MÀ1
DG298/kcal molÀ1
GalNAc
1
2
0.75
2.50
1.64
1340a
400
À4.26
À3.55
À3.80
610
a
Ref. 32.
the a-methylserine analogue in this complex is fairly similar to
that observed in the crystal structure for the Thr-containing
peptide with the SM3 antibody (see Fig. 1b).
Fig. 2 Relative STD effects for derivatives 1 and 2 bound to EcorL
lectin. The results clearly indicate that the GalNAc unit is the major
epitope in both derivatives.
Although the essential epitope recognized by SM3 antibody
is the above-commented sequence Pro-Asp-Thr-Arg, following
the suggestion of a referee to demonstrate that derivative 2 is a
mimic of the Tn antigen, we carried out an enzyme-linked
immunoassay (ELISA Kit for Mouse Mucin 1) with glyco-
peptides 1 and 2. The analogues showed the same behaviour
with very weak binding (ca. 20 mM).
tion, being capable to serve as ligands for specific receptors.
Our experience indicates that this unnatural a-methylserine,
previously synthesized in our laboratory,22 favours a helix-like
conformation,23–27 thus maintaining the key structural features
required for the bioactivity.
Notably, although the synthesis of several biologically
active peptides containing Ca-methylated amino acids has
been described,28,29 only some of them have been incorporated
into glycopeptides.23,26,27,30 Moreover, to the best of our
knowledge, while the biological activity of some unnatural
glycopeptides has been reported,19,21 this particular type of
glycopeptides containing Ca-methylated amino acids has not
been tested yet in biological assays.
To go one step further, we also ran 25 ns MD simulations in
explicit water on the complex between SM3 antibody and the
glycopeptide Pro-Asp-MeSer*-Arg (* = aGalNAc), with our
non-natural Tn mimic, 2.
Notably, the conformation of this novel glycopeptide is
also very similar to that previously reported for the natural
derivative with Thr (Fig. 1b, 3c and 3d and ESIw).16 These
results indeed demonstrate that the non-natural Tn derivative 2
Once the compound was synthesized (see ESIw for details), we
decided to test it against Erythrina corallodendron lectin
(EcorL),31,32 which exhibits high affinity to GalNAc. Additionally,
the natural Tn antigen derivative 1 was also checked for com-
parative purposes.
The interaction of both Tn-determinants with the lectin was
monitored employing Saturation Transfer Difference (STD)
NMR experiments.33 Fig. 2 shows the epitope mapping of both
molecules. Major saturation transfer to the protons in the
pyranose ring was observed, in particular for the H2 proton.
The slight differences observed in the peptide backbone could
indicate the different conformation of both peptide moieties in the
bound state. As a next step, competitive STD experiments with
GalNAc were performed. It was shown that both Tn derivatives
competed with GalNAc for the contact site; this underscored their
bioactivity as lectin ligands (see ESIw for details).
STD competition binding experiments allowed us to obtain
an estimation of the affinity constant (Ka) values of molecules
1 and 2 to EcorL by comparison with a reference ligand of
known activity.34,35 In our case, the anomeric mixture of a and
b-GalNAc was used as a reference.32 The Ka values of the
glycopeptides are summarized in Table 1. Notably, both Tn
derivatives exhibited a similar affinity for EcorL lectin.
To rationalize the interaction at the molecular level and to
confirm our structural predictions about the conformational
behaviour of the unnatural Tn derivative 2, we also ran
unrestrained 25 ns MD simulations in explicit water on the
complex of compound 2 with EcorL lectin (Fig. 3a and ESIw).
All the interactions observed in the crystal structure for the
GalNAc–lectin complex36 were also conserved for derivative 2.
As shown in Fig. 3b, the conformation and molecular shape of
Fig. 3 (a) Superimposition of different frames obtained from 25 ns
MD simulations for the complex EcorL:2. (b) f/c distribution obtained
from the MD simulations for the peptide backbone of the a-methyl-
serine residue in the complex EcorL:2. (c) Superimposition of
different frames obtained from 25 ns MD simulations for the complex
SM3:Pro-Asp-MeSer*-Arg (* = aGalNAc). (d) f/c distribution
obtained from the MD simulations for the peptide backbone of the
a-methylserine residue in the complex with SM3.
c
5320 Chem. Commun., 2011, 47, 5319–5321
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can be regarded as an interesting mimic, not only because it
might show improved stability against chemical and enzymatic
hydrolysis,37 but also because it retains the bioactive con-
formation observed in the Tn-containing glycopeptides with
SM3 antibody. Finally, efforts towards the synthesis of larger
glycopeptides that include derivative 2 and further affinity
experiments with different biological targets are currently
underway in our group.
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