Aza-β3-amino acid containing peptidomimetics as cAMP-dependent protein kinase substrates

Article abstract of DOI:10.1016/j.bioorg.2010.05.004

Peptidomimetic analogs of the peptide RRASVA, known as the "minimal substrate" of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-β³ analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of structure modeling and also with the results of computational docking study of peptide and peptidomimetic substrates with the active center of PKA. It was concluded that the specificity determining factors which govern substrate recognition by the enzyme should be grouped along the phosphorylatable substrate, and such clustering might open new perspectives for pharmacophore design of peptides and peptide-like ligands.

Full text of DOI:10.1016/j.bioorg.2010.05.004

Bioorganic Chemistry 38 (2010) 229–233
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Aza-b3-amino acid containing peptidomimetics as cAMP-dependent protein
kinase substrates
Ksenija Kisseljova a,b, Aleksei Kuznetsov a, Michèle Baudy-Floc’h b, Jaak Järv a,
*
a Institute of Chemistry, University of Tartu, Ravila 14a, Tartu 50411, Estonia
b Groupe ‘Ciblage et Auto-Assemblages Fonctionnels’, UMR CNRS 6226, Institut de Chimie, Université de Rennes 1, 263 Av. du Général Leclerc, F-35042 Rennes Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 April 2010
Available online 1 June 2010
Peptidomimetic analogs of the peptide RRASVA, known as the ‘‘minimal substrate” of the catalytic sub-
unit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of nat-
ural amino acids by their aza-b3 analogs. The peptidomimetics were tested as PKA substrates and the
kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction
of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone
modification, while the maximal rate of the reaction was practically not affected by the structure of sub-
strates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of
structure modeling and also with the results of computational docking study of peptide and peptidom-
imetic substrates with the active center of PKA. It was concluded that the specificity determining factors
which govern substrate recognition by the enzyme should be grouped along the phosphorylatable sub-
strate, and such clustering might open new perspectives for pharmacophore design of peptides and pep-
tide-like ligands.
Keywords:
Aza-b3 amino acids
Peptidomimetics
Phosphorylation
cAMP-dependent protein kinase
Phosphorylation specificity
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
for the catalytic subunit of the cAMP-dependent protein kinase (EC
2.7.11.11) [8], further denoted as PKA, which is the simplest and a
Protein kinases, which constitute one of the largest gene families
encoded by human genome, control the majority of physiological
phenomena through regulatory phosphorylation of functionally
important proteins in eukaryotic cells as well as in plants and bac-
well-studied enzyme among protein kinases and therefore recog-
nized as a ‘‘model” of this kinase superfamily.
Molecular recognition of substrates by PKA can be presented on
three structural levels. Firstly, the peptide sequence around the
phosphorylatable serine is recognized and this fit seems to be
obligatory for the following phosphorylation step. For PKA, this se-
quence should contain two arginine residues in positions ꢀ3 and
ꢀ2 relatively to the phosphorylatable serine residue at position 0
[9,10], and the peptide RRASV, derived from the phosphorylation
teria [1–3]. In this phosphorylation reaction, the c-phosphate group
of ATP is transferred to the phosphorylatable residue of the protein
or peptide substrate and therefore, the accuracy of recognition of
the phosphorylation sites, implemented through high substrate
specificity of these enzymes, has a crucial impact on the effective-
ness of this signaling mechanism. On the other hand, however, it
has been found that all members of the protein kinase superfamily
have a structurally conserved catalytic core comprising the nucleo-
tide binding N-terminal lobe and the larger C-terminal lobe respon-
sible for binding of the phosphorylatable site of the protein/peptide
substrate [4–7]. These structural features have been first discovered
site of L-type pyruvate kinase and named a ‘‘minimum substrate”
for this enzyme [9], represents this recognition pattern. The idea
about the ‘‘primary substrate specificity” was confirmed by the re-
sults of the structural analysis of PKA complexes with peptides
[11,12], and also by the results of the kinetic study of phosphory-
lation of L-pyruvate kinase mutants with various peptide struc-
tures flanking the phosphorylatable serine [13]. Secondly,
substrate binding may occur apart from the primary binding site
of the catalytic center [14,15]. These interactions are probably
important in phosphorylation of proteins or long peptides
[16,17], and much less information is available about specificity
of these sites. Thirdly, some anchoring proteins may support the
fine targeting of the kinase to its protein substrates [18] in vivo.
As summarized, the most thorough understanding of sub-
strate specificity concerns the recognition of the peptide primary
Abbreviations: Boc, tert-butyloxycarbonyl; BSA, bovine serum albumin; DiPEA,
N,N0-diisopropylethylamine; DMF, N,N-dimethylformamide; Fmoc, 9H-fluoren-9-
ylmethoxycarbonyl; HOTB, 1-hydroxybenzotriazole; Pbf, 2,2,4,6,7-pentame-
thyldihydrobenzofuran-5-sulfonyl; PKA, cAMP dependent protein kinase catalytic
subunit, EC 2.7.11.11; SPPS, solid phase peptide synthesis; TBTU, O-(1H-benzotri-
azole-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic
acid; TRIS, 2-amino-2-hydroxymethyl-propane-1,3-diol.
* Corresponding author. Fax: +372 7375247.
E-mail address: jaak.jarv@ut.ee (J. Järv).
0045-2068/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.05.004

230
K. Kisseljova et al. / Bioorganic Chemistry 38 (2010) 229–233
structure around the phosphorylation site, since many peptides
have been involved in these studies. Moreover, an attempt has
been made to rationalize these data by quantitative structure–
activity relationships [19]. This analysis has revealed that the
primary specificity of PKA for short peptides cannot be expressed
by a single consensus sequence, while reactivity of these substrates
is determined by a sum of various structural effects of amino acid
residues depending on their position in the peptide. The same con-
clusion was reached through statistical analysis of the phosphory-
lation sites in natural substrates [14].
spectrum and HPLC. Peptidomimetic substrates RRASVazab3A,
RRASazab3VA, RRazab3ASVA, Razab3RASVA, and azab3RRASVA
were synthesized and characterized in this work as described be-
low. Chemicals for peptide synthesis were obtained from commer-
cial suppliers (Senn Chemicals, Iris Biotech GmbH, Fluka, and
Novabiochem) and were used without further purification. Analyt-
ical thin-layer chromatography was carried out on pre-coated sil-
ica gel plates (60 F254, Merck). Flash column chromatography
was carried out using silica gel 60 (230–400 mesh ASTM) from
Merck & Co., Germany.
All these investigations, including QSAR analysis, have been
concentrated on the study of
a
-peptides and do not concern the
2.2. Analytical methods
structure of the peptide backbone. Until recently, the most radical
alteration of the backbone structure was made by gradual replace-
NMR spectra were measured on a 300 MHz instrument (Bruker,
Germany) in DMSO-d6 or CDCl3 as solvent and internal reference. A
Waters HPLC system was used for purification and analysis of syn-
thetic products. Purification of aza-b3-peptides was performed on a
ment of
vealed that all these chimerical peptides were still
phosphorylated by PKA, except the substrate with -serine whose
L-amino acids with their D-isomers [20]. This analysis re-
D
OH-group was obviously wrongly orientated for the phosphoryl
transfer reaction.
C18 XTerraÒ RP18 (19 ꢁ 300 mm, 10
lm) column using water/ace-
tonitrile linear gradient (5–100%
B
in 40 min, 8 mL/min, rt,
In summary, all the results quoted above agree that the rules for
amino acid side group recognition in the peptide binding pocket of
PKA are rather ‘‘loose”. Therefore, it was interesting to analyze the
influence of alterations in the structure of the substrate backbone
upon their recognition by PKA. As no data on backbone-modified
peptidomimetic substrates phosphorylation have been reported
previously, we synthesized and tested a series of minimum sub-
strate RRASVA analogs, where each amino acid, except serine,
was sequentially replaced by an appropriate aza-b3-amino acid.
215 nm). The following buffers were used: (eluent A) water con-
taining 0.08% TFA by volume; (eluent B) acetonitrile containing
1% TFA by volume. Characterization of purified aza-b3-peptide hy-
brids were performed on a C18 XTerraÒ (4.6 ꢁ 250 mm, 5
lm) col-
umn using water/acetonitrile linear gradient (5–100% B in 30 min,
1 mL/min, 30 °C, 215 nm). High resolution mass spectra of the
products were measured on a LTQ Orbitrap (Thermo Electron)
spectrometer (positive ionization, static nanospray, boron silicate
emitters Proxeon, resolution 100,000 at m/z 400, external
calibration).
O
R
N
O
2.3. Synthesis of Fmoc protected aza-b3-amino acids
H2N
OH
H2N
OH
Fmoc-aza-b3-Ala-OH was prepared from methyl hydrazine, and
Fmoc-aza-b3-Val-OH and Fmoc-aza-b3-Arg(Boc)-OH were obtained
from hydrazine hydrate as previously described [25,26]. All prod-
ucts were purified chromatographically and their structure was
verified by NMR spectra. The details of these syntheses as well as
the results of product analysis are given in Supplement.
R
L-amino acid
aza-β3-amino acid
This replacement introduced additional degrees of conforma-
tional flexibility with the rotation around the C and Cb bonds that
a
has made the aza-b3-amino acid containing peptides as attractive
tools for design of peptide-like drugs of improved biological profile
and pharmacokinetic properties [21]. Secondly, introduction of
aza-b3-amino acids shifted the arginine side chains relatively to
each other. Therefore this replacement provided a good possibility
to analyze the role of mutual placement of these side groups in
protein kinase specificity and test tolerance of PKA against modifi-
cation of backbone of peptide-like ligands. As proteolytic stability
[22] and antigenic and immunogenic properties [23] of this type
of peptidomimetic compounds have been considered before, the
present study has opened further perspectives for use of these
compounds also for design of drugs acting on protein kinases,
which are generally recognized as the second most important
group of drug targets after G-protein-coupled receptors [24].
2.4. Synthesis of aza-b3-amino acid containing peptidomimetics
Peptide analogs were prepared on a 0.1 mmol scale by employ-
ing the Fmoc/tBu SPPS strategy on a Liberty Microwave-Enhanced
Peptide Synthesizer (CEM GmbH, Germany), using HOBt and TBTU
as activators, and DiPEA as base in DMF. The preloaded Fmoc-a-
Ala-OH Wang resin (0.40 or 0.81 mmol/g) was used. A coupling
time of 10 min was used for the introduction of Fmoc-aza-b3-ami-
no acids into peptides. For introduction of Fmoc-Arg(Pbf)-OH, dou-
ble couplings were performed. Cleavage was performed with TFA-
TIS-H2O 95:2.5:2.5 cocktail over 4 h. The yield of peptides with
aza-b3-amino acids was lower than the yield of the common pep-
tide, indicating that coupling of aza-derivatives was not so effec-
tive as natural amino acids, and was dependent upon the
particular amino acid derivative. The lowest yield was obtained
with aza-b3-arginine (Table 1). Crude peptides were purified chro-
matographically [27] and the purity of the products pooled was
above 95%, as found from the analytical HPLC runs and MS analysis.
The yields of these products were listed together with their exper-
imental HR molecular masses in Table 1.
2. Experimental
2.1. Chemicals
c
-[32P] ATP was obtained from Amersham (UK). The catalytic
subunit Calpha of mouse cAMP-dependent protein kinase (PKA),
recombinantly expressed in E. coli, 30 U/mg, 0.1 mg/mL, lot
040916, was obtained from Biaffin GmbH & Co. KG (Germany).
ATP, TRIS/HCl, BSA and H3PO4 were purchased from Sigma–Aldrich
(USA). Phosphocellulose paper P81 was acquired from Whatman
(UK). MgCl2 was purchased from Acros (Germany). Peptide sub-
strate RRASVA of purity above 95% was purchased from GL Bio-
chem Ltd. (Shanghai, China) and was characterized by MS
2.5. Kinetic measurements
The initial rate of peptide phosphorylation was measured at
30 °C as described previously [28,29]. Briefly, the reaction mixture
(final volume 100
c
l
L, 50 mM TRIS/HCl, pH 7.5) contained 100
lM
-[32P] ATP, 3–750
lM
peptide or peptidomimetic substrate,

K. Kisseljova et al. / Bioorganic Chemistry 38 (2010) 229–233
231
Table 1
Table 2
Characterization of aza-b3-amino acid containing peptidomimetics, derived from the
Phosphorylation of aza-b3-amino acid containing peptidomimetics by PKA at ATP
PKA ‘‘minimum substrate” RRASVA.
concentration 100
lM, 30 °C, 50 mM TRIS/HCl, pH 7.5.
aza
II
k
b3
Peptidomimetics
M observeda
Overall yield of synthesis (%)
Substrate
Km, lM
102 ꢃ V, (mg/
102 ꢃ kII, (mg/
RRASVA
II
ml) sꢀ1
ml) sꢀ1 Mꢀ1
l
k
RRASVazab3A
RRASazab3VA
RRazab3ASVA
Razab3RASVA
azab3RRASVA
674.4054
674.4051
674.4054
674.4052
674.4053
49
39
69
22
21
RRASVazab3A
43.7 8.8
26
5
4
1
2
5
3
0.6 0.2
0.18
0.09
0.16
0.52
0.03
1.0
RRASazab3VA 122.1 21.6 25
0.3 0.04
0.5 0.1
1.7 0.2
0.1 0.02
3.2 0.1
RRazab3ASVA
Razab3RASVA
59.8 9.4
19.1 2.9
27
32
azab3RRASVA 273.5 43.8 28
a
The calculated M is 674.4056.
RRASVA
11.1 3.5
36
10 mM MgCl2, and 0.3 lg/mL of the enzyme. The stock solution of
The sequence containing azab3-serine was not included into
this series of peptidomimetic substrates. As it was seen from
PKA was diluted 500-fold in 50 mM TRIS/HCl buffer (pH 7.5) con-
taining 1 mg/mL BSA, and 15 L of this solution was added into
the reaction mixture to initiate the phosphorylation reaction. At
known time intervals after starting the reaction, 10 L aliquots
l
RRASVA analogs, where the
L-amino acids were replaced with
D
-amino acids, the modification at the serine residue yielded a
l
non-phosphorylatable substrate [20]. Therefore, we decided not
to modify this part of the molecule, as this might similarly hinder
the transfer of the phosphate group from ATP to the substrate
molecule in the catalytic step.
It was found that all peptidomimetic compounds synthesized
were relatively good substrates of PKA and the results of kinetic
were taken from the reaction mixture and spotted onto pieces of
phosphocellulose paper, which were subsequently immersed into
ice-cold 75 mM phosphoric acid to stop the reaction. The pieces
were then washed four times with cold 75 mM H3PO4 (10 min each
time) to remove excess c
-[32P] ATP and were dried at 120 °C for
25 min. The radioactivity bound onto the paper was measured as
Cherenkov radiation using a Beckman LS 7500 scintillation coun-
ter. The values of the initial rate of the phosphorylation reaction
(v) were calculated from the slopes of the product concentration
vs time plots.
analysis, obtained at ATP concentration 100 lM, were listed in Ta-
ble 2. Additionally, the kinetic constants for RRASVA phosphoryla-
tion were determined. The initial velocities of phosphorylation of
all these substrates were well described by the common Michae-
lis–Menten equation:
2.6. Computational methods
kcat½Sꢂ½Eꢂ0
v
¼
;
ð1Þ
Km þ ½Sꢂ
Peptidomimetic structure modeling was performed using the
Spartan 4.0 software suite (Wavefunction, Inc., USA) and the min-
imum-energy conformations of the compounds were obtained.
Conformational searches were made by using molecular mechanics
with additional conditions of the aqueous medium for finding sta-
ble geometry. All peptides were represented as zwitterions for
these calculations. The peptidomimetic docking modeling with
PKA was carried out using the AutoDock Vina software (ver.
1.0.3) [30]. The initial structures of peptide analogs were calculated
as described above. The docking compatible structure formats of
the protein were prepared by AutoDockTools (ver. 1.5.4) [31].
The fitting box with 0.3 Å of grid spacing was defined once and
used for all docking calculations. The fitting area covers the peptide
binding site of PKA and the best docking pose with the lowest en-
ergy for each peptidomimetic within this area was obtained.
as illustrated in Fig. 1, and were used to calculate the V (=kcat[E]0)
and Km values listed in Table 2.
Additionally, we used the initial linear part of the Michaelis–
Menten plots, characterizing the phosphorylation reaction under
the second-order rate conditions, where [S] < Km, and calculated
the second-order rate constants kII:
v
¼ kII½Sꢂ½Eꢂ0:
ð2Þ
The kinetic parameters kII have the same meaning as the ratio
V
½Eꢂ0
kcat/Km, as kcat
¼
, but since their values ware determined from
different sets of experimental data, this approach can be used as
an internal test of applicability of the conventional reaction scheme.
It can be seen from Table 2 that the constants kII were, indeed, in
agreement with the kcat/Km values, confirming that there was no
substrate or product inhibition of the phosphorylation reaction.
As can be seen from Table 2, such variation in structure had
practically no effect on the maximal velocity of the phosphoryla-
tion reaction of peptidomimetics. Noteworthily, a similar trend
has been previously observed for an extensive series of short pep-
tide substrates, as summarized in [19]. On the other hand, how-
ever, and again similarly with normal peptide substrates,
variation in the structure of peptidomimetics had a significant
influence on the Km values which varied more than 20 times within
the studied series of substrates. The same specificity pattern was
also observed for the second-order rate constants (Table 2). There-
fore, the Km values should characterize the effectiveness of sub-
strate interaction with this enzyme. Then again, these data
should be comparable for peptidomimetic and peptide substrates,
as the catalytic step controls the positioning of the serine residue
of all these compounds in the enzyme active center.
2.7. Data processing
Calculations and statistical analysis of the data were carried out
using the GraphPad Prism (ver. 4.0, GraphPad Software Inc., USA)
software packages. The results were reported with standard errors.
3. Results and discussion
The methods of synthesis of Fmoc protected azab3-amino acids
and their application for preparation of peptidomimetics have sig-
nificantly developed in recent years [25,26,32,33] and were used in
this study to prepare a series of analogs of the peptide RRASVA
known as the ‘‘minimum substrate” for PKA. These synthetic pro-
cedures were based on the SPPS common strategy, with necessary
modifications of the coupling step of aza-b3-amino acids to in-
crease the overall yield of the synthesis (see Section 2). In this
way, a series of peptide RRASVA analogs with a successive replace-
ment of natural amino acids with their aza-b3-analogs was ob-
tained with reasonable yields (Table 1) which, however, still
remain below the results of the common peptide synthesis.
The results listed in Table 2 revealed that PKA was, indeed, sen-
sitive to the alteration of the backbone structure of substrates,
where –CHR– group of natural amino acids was replaced by
–NR–CH2–, but substituents R were the same. This sequence-
dependent effect of backbone modification is illustrated in Fig. 2,

232
K. Kisseljova et al. / Bioorganic Chemistry 38 (2010) 229–233
was made in position ꢀ2. This result might be confusing, as the
presence of two arginine residues in positions ꢀ3 and ꢀ2 has al-
ways been recognized as the most important substrate specificity
factor for PKA. Here, however, the observed results could be ex-
plained if we view the two arginine side groups as a single speci-
ficity cluster and consider that in Razab3RASVA these groups are
still close, while this localization is ruled out in azab3RRASVA
where the additional methylene group of the peptidomimetic
backbone separates these two residues.
The influence of the backbone shift on the peptide structure was
studied by computer modeling of Razab3RASVA, azab3RRASVA and
RRASVA, and the results were shown in Fig. 3. Indeed, the back-
bones of Razab3RASVA and RRASVA have rather similar structures
in their lowest energy state. At the same time, clear differences can
be observed in the case of the third compound, azab3RRASVA.
Moreover, this backbone alteration has also changed the spatial
orientation of the arginine side chains in azab3RRASVA, if com-
pared with Razab3RASVA and RRASVA, and most probably has im-
pact on the binding effectiveness of these substrates (Table 2).
Thus, an azab3-amino acid can efficiently replace a common amino
acid within a peptide sequence, providing that this replacement
does not hamper interaction of side chains with their recognition
site.
Fig. 1. Consolidated Michaels–Menten plot for phosphorylation of aza-b3-amino
acid containing peptidomimetic substrates RRASVazab3A (}), RRASazab3VA (s),
RRazab3ASVA (h), Razab3RASVA (
(30 °C, 50 mM TRIS/HCl, pH 7.5).
r
), azab3RRASVA (4), and RRASVA (d) by PKA
For modeling the substrate docking in the peptide binding cleft
of PKA, we proceeded from the structure of the enzyme complex
with AMPPNP-Mn [4], suggesting that this nucleotide inhibitor is
simulating the presence of ATP which is the second substrate of
the phosphorylation reaction. This docking analysis revealed that
peptidomimetic and peptide substrates had a somewhat different
orientation when bound in the enzyme active center (Fig. 4) and
were characterized by the following values of the docking energy:
ꢀ7.0 kcal/mol for Razab3RASVA, ꢀ6.3 kcal/mol for azab3RRASVA,
and ꢀ6.8 kcal/mol for RRASVA. At the same time, the distances be-
tween the oxygen atom of the substrate serine residue and the c-
Fig. 2. Sequence-dependent effect of replacement of L
-amino acids by their azab3-
phosphorus atom of the ATP derivative were all rather close for
these compounds: 3.6 Å, 4.0 Å and 3.2 Å, respectively. This result
was in agreement with the similar maximal rate values for sub-
strates (Table 2).
analogs in RRASVA upon the PKA-catalyzed phosphorylation of these substrates.
The effects on Km and kII were characterized by the following values:
ꢂ
ꢃ
ꢀ
ꢁ
azab3
KRmRASVA
Kamzab3
k
II
DpKm ¼ p
and
D
log kII ¼ log
.
RRASVA
k
II
The same explanation seems to be valid also for RRASazab3VA
where the azab3-group was located in position +1 and the mutual
positioning of two amino acids in the C-terminus of the substrate
was affected. This change produced more than a 10-fold decrease
in the binding effectiveness of the substrate as compared with
RRASVA (Fig. 2). At the same time, the backbone modifications
made in other places had smaller effects.
where the reactivity of the aza-analogs and RRASVA is compared
by using the relative
D
pKm and
D
log kII values, defined as
ꢂ
ꢃ
ꢀ
ꢁ
azab3
KRmRASVA
Kamzab3
k
II
DpKm ¼ p
and
D
log kII ¼ log
for different positions,
RRASVA
k
II
labeled as ꢀ3, ꢀ2, ꢀ1, 0, +1, +2 relatively to the serine residue, de-
noted as position zero.
It can be seen from Fig. 2 that the most significant effect was ob-
served if arginine was replaced by its azab3-analog in position ꢀ3,
while a relatively small effect was observed if this replacement
To sum up, the effect of backbone modification clearly de-
pended upon its location along the substrate molecule. As the loca-
tion of the most significant effects seems to be in correlation with
Fig. 3. Comparison of the calculated backbone structures of azab3RRASVA (left), RRASVA (center) and Raza b3RASVA (right). The backbones were shown as stick models, three
atoms of the serine residue, which were similarly fixed in all three molecules were shown as ball and stick models. The side groups of both arginine residues of the first
compound were directed ‘‘backwards”, while the same residues in the two following compounds were oriented ‘‘upwards”.

K. Kisseljova et al. / Bioorganic Chemistry 38 (2010) 229–233
233
Fig. 4. Docking of azab3RRASVA (left), RRASVA (center) and Razab3RASVA (right) with the substrate-binding cleft of the PKA complex with AMPPNP-Mn, simulating the
second substrate ATP of the phosphorylation reaction. The following color code was used for ligands: black – carbon, light gray – hydrogen, if shown, red – oxygen, blue –
nitrogen, orange – phosphorus, and yellow – Mn.
Pickeral, C. Shue, L.B. Vosshall, J. Zhang, Q. Zhao, X.H. Zheng, S. Lewis, Science
287 (2000) 2204–2215.
could be suggested that these phenomena are both governed by
[3] S.K. Hanks, T. Hunter, FASEB J. 9 (1995) 576–596.
the specificity pattern of the binding site for peptide side chains, it
interaction of the amino acid side chains with their recognition
sites on the protein molecule. These interactions can be changed
either by variation of the side chain structure, as has been shown
before, or by the backbone modification in the proper place. There-
fore, instead of recognition of a single side chain, the interaction of
the enzyme with clusters consisting of at least two amino acids
should be considered. Outside of these recognition clusters, the
backbone modifications are more tolerated and have smaller ef-
fects on ligand binding effectiveness. Therefore, the backbone
modification can be used to define the clusters and even to rank
them according to their stake into the overall specificity. On the
other hand, the regions between these clusters may have impor-
tance as the sites of substrate modification with minimal effect
on its activity. This might also be an important aspect to be consid-
ered in the design of peptidomimetic ligands.
[4] D. Bossemeyer, R.A. Engh, V. Kinzel, H. Ponstingl, R. Huber, EMBO J. 12 (1993)
849–859.
[5] M. Cherry, D.H. Williams, Curr. Med. Chem. 11 (2004) 663–673.
[6] T.G. Davies, M.L. Verdonk, B. Graham, S. Saalau-Bethell, C.C. Hamlett, T.
McHardy, I. Collins, M.D. Garrett, P. Workman, S.J. Woodhead, H. Jhoti, D.
Barford, J. Mol. Biol. 367 (2007) 882–894.
[7] S.K. Hanks, A.M. Quinn, T. Hunter, Science 241 (1988) 42–52.
[8] ExPASy, NiceZyme View of ENZYME: EC 2.7.11.11, , 2009, <www.expasy.org/
enzyme/2.7.11.11>.
[9] O. Zetterqvist, U. Ragnarsson, FEBS Lett. 139 (1982) 287–290.
[10] O. Zetterqvist, U. Ragnarsson, E. Humble, L. Berglund, L. Engstrom, Biochem.
Biophys. Res. Commun. 70 (1976) 696–703.
[11] S.S. Taylor, J. Zheng, E. Radzio-Andzelm, D.R. Knighton, L.F. Ten Eyck, J.M.
Sowadski, F.W. Herberg, W.M. Yonemoto, Philos. Trans. Roy. Soc. Lond. B Biol.
Sci. 340 (1993) 315–324.
[12] I. Tsigelny, B.D. Grant, S.S. Taylor, L.F. Ten Eyck, Biopolymers 39 (1996) 353–
365.
[13] M. Loog, N. Oskolkov, F. O’Farrell, P. Ek, J. Järv, Biochim. Biophys. Acta 1747
(2005) 261–266.
[14] A. Kreegipuu, N. Blom, S. Brunak, J. Järv, FEBS Lett. 430 (1998) 45–50.
[15] J.A. Ubersax, J.E. Ferrell Jr., Nat. Rev. Mol. Cell Biol. 8 (2007) 530–541.
[16] C. Kim, C.Y. Cheng, S.A. Saldanha, S.S. Taylor, Cell 130 (2007) 1032–1043.
[17] L.A. Pinna, M. Ruzzene, Biochim. Biophys. Acta 1314 (1996) 191–225.
[18] D. Mochly-Rosen, Science 268 (1995) 247–251.
[19] J. Järv, U. Ragnarsson, Bioorg. Chem. 19 (1991) 77–87.
[20] M. Eller, A. Sepp, R. Toomik, P. Ekman, J. Järv, U. Ragnarsson, L. Engstrom,
Biochem. Int. 25 (1991) 453–460.
[21] A. Grauer, B. König, Eur. J. Org. Chem. 2009 (2009) 5099–5111.
[22] J. Vagner, H. Qu, V.J. Hruby, Curr. Opin. Chem. Biol. 12 (2008) 292–296.
[23] H. Dali, O. Busnel, J. Hoebeke, L. Bi, P. Decker1, J.-P. Briand, M. Baudy-Floc’h, S.
Muller, Mol. Immunol. 44 (2007) 3024–3036.
Acknowledgments
This investigation was supported by the Estonian Ministry of
Education and Research Grant SF0180064s08 and by the Kristjan
Jaak travel scholarship for K.K.
Appendix A. Supplementary data
[24] P. Cohen, Nat. Rev. Drug Discov. 1 (2002) 309–315.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bioorg.2010.05.004.
[25] O. Busnel, L. Bi, M. Baudy-Floc’h, Tetrahedron Lett. 46 (2005) 7073–7075.
[26] O. Busnel, L. Bi, H. Dali, A. Cheguillaume, S. Chevance, A. Bondon, S. Muller, M.
Baudy-Floc’h, J. Org. Chem. 70 (2005) 10701–10708.
[27] A. Dif, F. Boulmedais, M. Pinot, V. Roullier, M. Baudy-Floc’h, F.M. Coquelle, S.
Clarke, P. Neveu, F. Vignaux, R.L. Borgne, M. Dahan, Z. Gueroui, V. Marchi-
Artzner, J. Am. Chem. Soc. 131 (2009) 14738–14746.
[28] A. Kuznetsov, A. Uri, G. Raidaru, J. Järv, Bioorg. Chem. 32 (2004) 527–535.
[29] R. Roskoski Jr., Methods Enzymol. 99 (1983) 3–6.
[30] O. Trott, A.J. Olson, J. Comput. Chem. 31 (2010) 455–461.
[31] M.F. Sanner, J. Mol. Graph. Modell. 17 (1999) 57–61.
[32] O. Busnel, M. Baudy-Floc’h, Tetrahedron Lett. 48 (2007) 5767–5770.
[33] H. Dali, O. Busnel, J. Hoebeke, L. Bi, P. Decker, J.-P. Briand, M. Baudy-Floc’h, S.
Muller, Mol. Immunol. 44 (2007) 3024–3036.
References
[1] T. Hunter, Cell 100 (2000) 113–127.
[2] G.M. Rubin, M.D. Yandell, J.R. Wortman, G.L. Gabor Miklos, C.R. Nelson, I.K.
Hariharan, M.E. Fortini, P.W. Li, R. Apweiler, W. Fleischmann, J.M. Cherry, S.
Henikoff, M.P. Skupski, S. Misra, M. Ashburner, E. Birney, M.S. Boguski, T.
Brody, P. Brokstein, S.E. Celniker, S.A. Chervitz, D. Coates, A. Cravchik, A.
Gabrielian, R.F. Galle, W.M. Gelbart, R.A. George, L.S. Goldstein, F. Gong, P.
Guan, N.L. Harris, B.A. Hay, R.A. Hoskins, J. Li, Z. Li, R.O. Hynes, S.J. Jones, P.M.
Kuehl, B. Lemaitre, J.T. Littleton, D.K. Morrison, C. Mungall, P.H. O’Farrell, O.K.