Tetrazole as a Replacement of the Electrophilic Group in Characteristic Prolyl Oligopeptidase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00394

4-Phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines were studied as prolyl oligopeptidase inhibitors. The compounds were more potent than expected from the assumption that the tetrazole would also here be a bioisostere of the carboxylic acid group and the corresponding carboxylic acids are at their best only weak inhibitors. The aminoacyl groups l-prolyl and l-alanyl gave potent inhibitors with IC₅₀ values of 12 and 129 nM, respectively. This was in line with typical prolyl oligopeptidase inhibitors; however, we did observe a difference with N-methyl-l-alanyl, which gave potent inhibitors in typical prolyl oligopeptidase inhibitors but not in our novel compound series. Furthermore, all studied 4-phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines decreased α-synuclein dimerization at the concentration of 10 μM, also when they were only weak inhibitors of the proteolytic activity of the enzyme with an IC₅₀ value of 205 μM. Molecular docking studies revealed that the compounds are likely to bind differently to the enzyme compared to typical prolyl oligopeptidase inhibitors represented in this study by 4-phenylbutanoyl-aminoacyl-2(S)-cyanopyrrolidines.

Full text of DOI:10.1021/acsmedchemlett.9b00394

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Letter
Tetrazole as a Replacement of the Electrophilic Group
in Characteristic Prolyl Oligopeptidase Inhibitors
Tommi P Kilpeläinen, Jonna K Tyni, Maija Lahtela-Kakkonen, Tony
S Eteläinen, Timo T Myöhänen, and Erik Aarne Anders Wallén
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00394 • Publication Date (Web): 11 Nov 2019
Downloaded from pubs.acs.org on November 11, 2019
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Tetrazole as a Replacement of the Electrophilic Group in
Characteristic Prolyl Oligopeptidase Inhibitors
Tommi P. Kilpeläinen†, Jonna K. Tyni‡, Maija K. Lahtela-Kakkonen‡, Tony S. Eteläinen†, Timo T.
Myöhänen† and Erik A. A. Wallén⊥,*
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†Drug Research Program, Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, P.O. Box 56,
00014, Helsinki, Finland.
⊥Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box
56, 00014, Helsinki, Finland.
‡School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, 70211, Kuopio, Finland.
KEYWORDS: Prolyl oligopeptidase, inhibitor, synthesis, tetrazole, alpha-synuclein aggregation, molecular docking
ABSTRACT: 4-Phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines were studied as prolyl oligopeptidase inhibitors. The
compounds were more potent than expected from the assumption that the tetrazole would also here be a bioisostere of the carboxylic
acid group and the corresponding carboxylic acids are at their best only weak inhibitors. The aminoacyl groups L-prolyl and L-alanyl
gave potent inhibitors with IC50 values of 12 and 129 nM, respectively. This was in line with typical prolyl oligopeptidase inhibitors,
however, we did observe a difference with N-methyl-L-alanyl, which gave potent inhibitors in typical prolyl oligopeptidase inhibitors
but not in our novel compound series. Furthermore, all studied 4-phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines decreased α-
synuclein dimerization at the concentration of 10 μM, also when they were only weak inhibitors of the proteolytic activity of the
enzyme with an IC50 value of 205 µM. Molecular docking studies revealed that the compounds are likely to bind differently to the
enzyme compared to typical prolyl oligopeptidase inhibitors represented in this study by 4-phenylbutanoyl-aminoacyl-2(S)-
cyanopyrrolidines.
Prolyl oligopeptidase (PREP) is a serine protease with
endopeptidase activity on short proline containing peptides
cleaving the peptide on the carboxyl side of the proline residue,
and many proline containing peptides, such as oxytocin and
several neuropeptides, have been identified as its substrates in
vitro (see for review Garcia-Horsman et al. 2007).1 PREP is
mainly found in the brain, more specifically in caudate nucleus
and putamen, hippocampus, and cortex.2 However, significant
PREP activities and protein levels have been measured also in
peripheral tissues such as skeletal muscle,3 renal cortex, liver,
ovaries, testis, and in ovarian and colorectal tumors.4-6
Alterations in the enzyme activity of PREP have been observed
in several diseases, including Parkinson’s disease, Alzheimer’s
disease, Lewy body dementia,7 and in several types of tumors.8,9
Apart from its proteolytic activity, PREP has also other
biologically important functions mediated by protein-protein
interactions.10,11 PREP is able to enhance dimerization of α-
synuclein (αSyn), the key player in cellular toxicity in
Parkinson’s disease, via a direct protein-protein interaction.12
PREP has highly flexible regions that are in equilibrium
between many conformations and inhibitor binding stabilizes
these flexible regions into one conformation.13 Inhibited PREP
has been shown to increase autophagy which is known to
enhance clearance of aggregated forms of proteins and decrease
dimerization of αSyn in vitro and in vivo.14,15
and it is typically a pyrrolidine with or without an electrophilic
group in its 2-position. However, also five-membered
heteroaryls such as 2-thienyl and 2-furanyl in the P1-position
have resulted in potent inhibitors.17 The P2 moiety can be a wide
variety of aminoacyl groups or aminoacyl mimetics with Pro
being the most preferred natural aminoacyl group, and the P3
moiety is typically a lipophilic acyl group. There are many
examples of variations among the published inhibitors16,
however, the two carbonyl groups of the connecting amide
bonds have been difficult to replace and only few inhibitors lack
one or both of them in their structures.18-21
The design for the new compounds came from one compound
synthesized by the last author of this paper in his earlier research
in the search for different replacements for the electrophilic
group in PREP inhibitors, where the tetrazolyl group gave a
surprisingly potent compound (unpublished data). In the present
study, our primary aim was to investigate if 4-phenylbutanoyl-
aminoacyl-2(S)-tetrazolylpyrrolidines as PREP inhibitors had
the same structure-activity relationship as typical PREP
inhibitor compound series such as 4-phenylbutanoyl-
aminoacyl-pyrrolidines and 4-phenylbutanoyl-aminoacyl-2(S)-
cyanopyrrolidines. A further aim was to study what effect the
novel compound series had on protein-protein-mediated
functions of PREP, such as catalysis of αSyn dimerization. We
were intrigued by the fact that the tetrazole ring is quite different
from typical 2-substituents on the P1 pyrrolidine ring,
especially its polar character is exceptional.
Almost all known highly potent PREP inhibitors are
structurally related to or recognizable mimetics of the peptide
substrates with distinct P1, P2 and P3 sites.16 The P1 moiety is
mimicking the important L-proline (Pro) residue in the substrate
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.
1
H-Xaa-OH
2
3
4
5
O
g
1a
: Xaa=Pro
: Xaa=Gly
: Xaa=Sar
: Xaa=Ala
f
Boc Xaa OSu
Xaa
N
Boc Xaa
6c 6f
N
1d
1e
1h
5b
5c
: Xaa=Ala
: Xaa=MeAla
: Xaa=D-Ala
,
7c 7e 7f 7g 7h
,
,
,
,
e
5f
6
a
h
7
8
9
O
O
i,d
g
d
O
Xaa
N
Xaa OH
Boc Ala
N
Ala
N
CO2H
CO2Me
10
11
12
13
14
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10a 10b
8b
4a 4d 4e 4g 4h
,
,
,
,
,
9b
CO2Me
j
d
10b
k (only
)
O
c
Aib OMe
O
g
l
Xaa
N
3g
Boc MeAla OH
MeAla
N
Boc
11c
CONH2
CONH2
13a-e
12c
m
H-Aib-OMe
2g
O
O
Xaa
N
b
n
Xaa
N
N
N
H-Aib-OH
CN
HN
15a-e
14a-e
N
1g
Scheme 1. Synthesis of the compounds, where a specific aminoacyl group is always with the same lower case letter in compound
numbering. Reagents and conditions (a) 1. 10% aqueous Na2CO3, 4-phenylbutanoyl chloride / Et2O; (b) SOCl2 / MeOH, reflux; (c)
4-phenylbutanoyl chloride, DIPEA / DCM; (d) LiOH / water, MeOH; (e) 1. pivaloyl chloride (or in its place ethyl chloroformate for
4g), Et3N / DCM, 0 °C, 2. pyrrolidine, Et3N / DCM; (f) pyrrolidine / DCM; (g) 1. TFA / DCM, 0 °C, 2. 4-phenylbutanoyl chloride,
Et3N / DCM (or alternatively for 6f, 2. 1 M NaOH, 4-phenylbutanoyl chloride, / Et2O); (h) L-proline methyl ester, DIPEA / DCM;
(i) 1. ethyl chloroformate, Et3N / DCM, 0 °C, 2. L-proline methyl ester, Et3N / DCM; (j) 1. pivaloyl chloride, Et3N / DCM, 0 °C, 2.
L-prolinamide, Et3N, / DCM; (k) 1. ethyl chloroformate, Et3N / THF, -10 °C, 2. NH3 (7 M in MeOH); (l) 1. ethyl chloroformate, Et3N
/ DCM, 0 °C, 2. L-prolinamide, Et3N / DCM; (m) TFAA, Et3N / THF; (n) NaN3, NH4Cl / DMF, 100 °C.
There are PREP inhibitors with five-membered
heteroaromatics, which look slightly similar to our
tetrazoles.22,23 However, all of these have an electrophilic
carbonyl group in exactly the same position as the typical
electrophile. In addition, the tetrazole ring is negatively charged
at physiological pH unlike the five-membered heteroaromatics
in the published PREP inhibitors.
intermediates.
The
4-phenylbutanoyl-aminoacyl-L-
prolinamides were converted to the corresponding nitriles by
dehydration with trifluoroacetic anhydride, and then further to
the corresponding tetrazoles with sodium azide.
The inhibition of the proteolytic activity of PREP (IC50) for
the compounds is presented in Table 1. The IC50 values were in
the beginning of the project determined using mouse brain
homogenate, but we changed later to purified recombinant
porcine PREP. Most unsbstituted pyrrolidines and prolinamides
were tested with brain homogenate and all nitriles and tetrazoles
were tested with purified porcine PREP. The IC50 values for a
few compounds were determined with both homogenate and
pure enzyme to verify that the values were comparable.
The synthetic routes for accessing the compounds in this
study are presented in Scheme 1. Compounds 7a (SUAM-
1221), 7b and 14a (KYP-2047) were obtained from our old
compound library at the University of Eastern Finland (14a was
also synthesized here only as a synthetic intermediate on the
route to 15a but not used in the testing). The connecting amide
bonds were synthesized using activation of carboxylic acid
groups to corresponding acid chlorides or mixed anhydrides.
These activation methods could not be used for non-N-alkylated
chiral amino acids due to their susceptibility for racemization,
and a milder activation to hydroxysuccinimide esters was used
for them. Furthermore, in the case of 2-aminoisobutyric acid
(Aib) the low solubility of the amino acid in the solvent of the
first step of the synthetic route had to be circumvented, and in
the case of N-methyl-L-alanine (MeAla) the slightly modified
synthetic route gave improved yields and more easily separable
In the beginning of the project earlier uncharted aminoacyl
groups, Aib, sarcosine (Sar), MeAla and β-alanine (βAla) were
evaluated at the P2 site in 4-phenylbutanoyl-aminoacyl-
pyrrolidines, which is the most typical PREP inhibitor structure,
and compared to the two natural aminoacyl groups Pro and L-
alanine (Ala) which have been reported to give the most potent
compounds.24 In this study we did not carry out comparisons to
L-phenylalanine, L-methionine, L-leucine, L-valine and
glycine (Gly) as they have been previously shown to strongly
decrease the inhibitory activity.24
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Table 1. Inhibitory activities of the synthetized compounds.
1
O
2
Xaa
N
3
R
4
5
6
7
IC50 (nM)
enzyme
IC50 (nM)
homogenate
Compound
Xaa
R
IC50 95% CI
IC50 95% CI
7a (SUAM-1221)
Pro
H
12
10-15
-
-
-
8
9
7b
Ala
H
147
115-191
-
7c
MeAla
Sar
H
-
-
298
142-571
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7e
H
-
-
5141
3950-6604
12600-105300
8752-22570
7912-17790
-
7f
D-Ala
Aib
H
-
-
35180
7g
H
-
-
-
13510
7h
H
-
12080
Ala
Pro
13a
CONH2
CONH2
CONH2
CONH2
CONH2
CN
4371
-
3841-4965
-
13b
Ala
-
-
-
-
6213
3325-11340
9792-97350
95 170-249 800
329 600-637 700
13c
MeAla
Gly
-
28 800
13d
-
153 800
13e
Sar
-
457 000
a
a
14a (KYP-2047)
14b
Pro
0.86
4.06
5.4
-
0.3
-
Ala
CN
2.0-7.7
4.5-6.4
3.28
2.06-5.22
14c
MeAla
Gly
CN
-
-
14d
CN
220
269
12
117-454
264
138-493
b
14e
Sar
CN
-
-
-
-
15a
Pro
tetrazolyl
tetrazolyl
tetrazolyl
tetrazolyl
tetrazolyl
CO2H
CO2H
9.9-14.7
71-225
-
15b
Ala
129
27 180
205 400
10 640
3626
17 540
91
-
55-146
15c
MeAla
Gly
22 150-33 410
-
-
-
-
-
c
15d
-
-
15e
Sar
8110-14 390
2821-4965
-
10a
Pro
-
10b
Ala
14 100-22 100
-
a Confidence interval could not be determined since IC50 value is lower than lowest used concentration.
b Confidence interval could not be determined since Hillslope is greater than -2.
c Confidence interval could not be determined since IC50 value is too high.
All explored aminoacyl groups were in fact one
methyl/methylene group changes from Ala and of these only
MeAla gave a potent compound 7c. The other aminoacyl groups
Sar, D-Ala, Aib and βAla gave compounds 7e, 7f, 7g and 7h,
respectively, with significantly reduced inhibitory activities.
129 nM, respectively. Surprisingly, MeAla gave only a weak
inhibitor 15c, which was over 200-fold lower in inhibitory
activity than 15b. It is obvious that the N-methyl group on Ala
is not favorable in combination with the tetrazole group. Gly
and Sar gave the tetrazoles 15d and 15e, which had weak
inhibitory activities of 205 and 10.6 µM, respectively. The
carboxylic acid analogues 10a and 10b of the most potent
tetrazoles 15a and 15b, respectively, had only weak inhibitory
activities in the micromolar range, indicating no bioisosterism
between the tetrazole and the carboxylic acid group in this
series of compounds.
Pro, Ala, MeAla, Gly and Sar were selected as the aminoacyl
groups for our novel tetrazoles. They were synthesized via the
corresponding prolinamide and nitrile intermediates, which
were also tested for their inhibitory activities. As expected, the
prolinamide intermediates 13a-13e had only weak inhibitory
activities and the nitrile intermediates 14a-14e were highly
potent inhibitors. The electrophilic nitrile group is known to
have favorable interaction with the catalytic Ser554 residue at
the active site of the enzyme, and for 14a (which is the well-
known reference compound KYP-2047)25 this has been
described as a reversible covalent interaction.26,27 For the
nitriles, also Pro, Ala and MeAla gave the most potent inhibitors
14a, 14b and 14c, with IC50 values of 0.86, 4.06 and 5.4 nM,
respectively. These IC50 values should be used with caution as
at least 14a is a covalently binding compound with slow, tight
binding enzyme kinetics.
For evaluating the ability of the tetrazoles 15a-15e to reduce
αSyn dimerization we used a slightly modified PCA (protein
fragment complementation assay) method described in
Savolainen et al. 2015.12 We used mouse N2A cells in the assay
which has high endogenous PREP activity (Suppl. Fig 1.).
Results of the assay are shown in Figure 1. Interestingly, all
tetrazoles 15a-e decreased αSyn dimerization. The αSyn
dimerization assay was performed at the concentration of 10
µM and even tetrazole 15d with an IC50 value of 205 µM
reduced αSyn dimerization with the same magnitude as
tetrazoles 15a and 15b with IC50 values 12 and 129 nM,
respectively.
Among our target tetrazoles 15a-15e, Pro and Ala gave the
most potent inhibitors 15a and 15b, with IC50 values of 12 and
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100
50
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***
*
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Figure 1. Results of the αSyn-αSyn2 dimerization in PCA (protein fragment complementation assay) with 4 h treatment and 10 µM
concentration. Tetrazoles 15a-e reduced the amount of αSyn dimers 18-25% compared to control. 14a (KYP-2047), 14d and 14e were the
only compounds from nitrile group which were able to reduce αSyn dimerization. Carboxyl acids 10a and 10b were not able to reduce
formation of αSyn dimers. Lactacystin serves as positive control for αSyn dimerization. Data are presented as means+SEM (n≥3, ***
p<0.001, **p< 0.01, *p<0.05)
We decided also to examine the nitrile intermediates as a
comparison. The nitrile 14a (KYP-2047) had been verified to
reduced αSyn dimerization in several earlier studies, but the
other nitriles had not been studied.12,14 To our surprise, the
nitriles 14b and 14c with aminoacyl groups Ala and MeAla did
not have an effect on αSyn dimerization although they are 4-5
nM inhibitors of PREP, and on the other hand, the nitriles 14d
and 14e with aminoacyl groups Gly and Sar had an effect
although they are only 220-260 nM inhibitors of PREP. It is
important to highlight here that only compounds 14a (89%,
p<0.05), 14d (81%, p<0.05), 15b (75%, p<0.01), 15c (75%,
p<0.05) and 15d (77%, p<0.05) had statistically significant
decrease in aSyn dimerization (Student’s t-test compared to
DMSO control, n≥3).
differently than the nitrile group. This can be seen most clearly
between compounds 14a and 15a, and compounds 14d and 15d.
The two most potent tetrazoles 15a and 15b were inclined to
form an interaction between their negatively charged tetrazole
group to the positively charged Arg643 instead of Ser554
(Suppl. Fig. 7 and 8). For the other tetrazoles the tetrazole ring
at S1 was not forming any interactions with amino acid residues
in the binding pocket (Suppl. Fig. 9-11). Moreover, the pose of
15e was tilted when compared to other tetrazoles or nitriles, and
its phenyl group positioned outside the pocket (Suppl. Fig. 11.).
The docking results proposed a putative binding pose in
which all the tetrazoles could fit to the binding pocket. In this
hypothetical pose the phenyl group was at S1 instead of S3
(Suppl. Fig. 12-16). Tetrazoles might prefer to position the
lipophilic benzene ring rather than the hydrophilic tetrazole ring
into the hydrophobic S1 pocket. The tetrazole ring does not
have a similar ability as the nitrile group to form a covalent
bond to Ser554, which is an important interaction for at least
some nitriles in anchoring them at S1. As a less hydrophilic
moiety, the nitrile group might also be more easily placed into
the S1 pocket than the tetrazole ring. Overall, these results
suggest that the tetrazoles might have two putative binding
poses: the phenyl group at S3 (Figure 2E) or at S1 (Figure 2F).
The αSyn dimerization assay results for both nitriles and
tetrazoles clearly indicate that the structure-activity relationship
for affecting this function of PREP is slightly different from
inhibiting the proteolytic activity. To study the binding of the
nitriles 14a-e and tetrazoles 15a-e to PREP, molecular docking
studies were performed (Figure 2A-F). The binding pocket
included the commonly known S1, S2, and S3 subsites (Figure
2C). Among the nitriles, 14a is known to bind covalently to the
catalytically active serine residue (Ser554) at S1.28 Other
nitriles could be assumed to orient similarly to 14a directing the
nitrile group towards S1 and Ser554 (Figure 2D). Indeed, all
nitriles could place the nitrile group at S1 and the phenyl group
at S3. The most potent nitriles 14a, 14b, and 14c directed the
nitrile group towards Ser554. However, in docking studies 14d
and 14e could not orient the nitrile group towards Ser554. This
could maybe explain why they are less potent inhibitors than
the other nitriles. In the docking protocol the covalent
interaction between the nitrile group and Ser554 was not
assessed. Thus, the possible covalent bond formation could
actually force the nitrile groups to S1.
In conclusion, although the tetrazole group is a known
common bioisostere for the carboxylic acid group, the tetrazole
is clearly not a bioisostere of a carboxylic acid group in PREP
inhibitors. The tetrazoles were more potent inhibitors of the
proteolytic activity and αSyn dimerization catalyzing effect of
PREP than the corresponding carboxylic acids. The present
study further hypothesized that the tetrazoles might have
another binding mode in addition to the known binding mode
of PREP inhibitors. In this putative binding mode, the tetrazole
ring is placed at S3 and the phenyl group at S1. Remarkably, all
tetrazoles reduced αSyn dimerization despite the fact that some
of them only were weak inhibitors of the proteolytic activity.
These findings taken together possibly also indicate that there
could be another alternative binding site in enzyme, which
affects more the αSyn dimerization catalyzing effect than the
proteolytic activity of the enzyme.
Interestingly, none of the tetrazoles formed an interaction to
Ser554 even though they could place the tetrazole ring at S1.
The poses with the tetrazole ring at S1 were compared to the
poses of the corresponding nitriles with the nitrile group at S1
(Suppl. Fig. 2-6). The comparison revealed that the tetrazole
ring might be positioned in the binding pocket slightly
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Figure 2. Putative binding site of the tetrazoles with PREP. (A) The crystal structure of PREP. The catalytically active serine residue (Ser554)
is marked with black, and the inhibitor-binding site is marked with green mesh. (B) The crystal structure of PREP from site of Figures C-F.
(C) The ligand-binding pocket with the S1, S2, and S3 subsites. Green indicates lipophilic, yellow aromatic, red electronegative, and blue
electropositive areas. (D) Compound 14a at the inhibitor-binding site. The nitrile points towards Ser554 and forms hydrogen bond to it (not
shown in the figure). (E) Compound 15a at the inhibitor-binding site in the commonly known binding mode. (F) A suggested hypothetical
binding mode for the tetrazoles with compound 15a as a representative compound.
EXPERIMENTAL SECTION
AUTHOR INFORMATION
Synthesis and characterization of novel compounds.
Synthesis and characterization of all compounds are
reported in detail in supporting information.
Corresponding Author
*Erik A. A. Wallén, orcid.org/0000-0002-2269-5179, E-mail:
erik.wallen@helsinki.fi
Author Contributions
Expression and purification of recombinant porcine PREP.
Porcine PREP enzyme was expressed and purified
according protocol described by Venäläinen et al. 2002.29
The manuscript was written through contributions of all authors.
Chemistry was contributed by TPK and EAAW, pharmacology by
TPK, TSE and TTM, and molecular docking studies by JKT and
MKLK. All authors have given approval to the final version of the
manuscript.
Determination of IC50 value. Preparation of mice brain
homogenates and detailed procedure for in vitro assay for
inhibitory activity are found in supporting information.
Funding Sources
This study was supported by grants from Academy of Finland
(318327, 305710), HiLIFE (proof-of-concept), Jane and Aatos
Erkko Foundation, Business Finland TUTLI funding and Sigrid
Juselius Foundation to TTM, and by salary support from Drug
Research Doctoral Program at University of Helsinki to TPK and
Drug Research Doctoral Program at University of Eastern Finland
to JKT. The computational capacity and licenses support provided
by CSC-IT Center for Science, Finland, is acknowledged.
PCA for αSyn dimerization. Detailed cell culture, procedure
and used DNA constructs are found in supporting information.
Molecular docking studies. Protocol is described in supporting
information.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
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ACS Medicinal Chemistry Letters
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models of Parkinson's disease. Br. J. Pharmacol. 2012, 166, 1097-
1113.
15. Savolainen, M. H.; Richie, C. T.; Harvey, B. K.; Männistö, P. T.;
Maguire-Zeiss, K. A.; Myöhänen, T. T. The beneficial effect of a prolyl
oligopeptidase inhibitor, KYP-2047, on alpha-synuclein clearance and
autophagy in A30P transgenic mouse. Neurobiol. Dis. 2014, 68, 1-15.
16. Lawandi, J.; Gerber-Lemaire, S.; Juillerat-Jeanneret, L.;
Moitessier, N. Inhibitors of prolyl oligopeptidases for the therapy of
human diseases: Defining diseases and inhibitors. J. Med. Chem. 2010,
53, 3423-3438.
ACKNOWLEDGMENT
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8
Dr Reinis Svarcbahs is acknowledged for assistance with PREP-
purification and Ms Nina Sipari for mass spectrometry. Reference
compound 7a (SUAM-1221), 7b and 14a (KYP-2047) were
obtained from our old compound library at the University of
Eastern Finland (earlier University of Kuopio).
ABBREVIATIONS
PREP, prolyl oligopeptidase; αSyn, alpha-synuclein; Aib, 2-
aminoisobutyric acid; Sar, sarcosine; MeAla, N-methyl-L-alanine;
βAla, beta-alanine; PCA, protein fragment complementation assay.
17. Jarho, E. M.; Venäläinen, J. I.; Poutiainen, S.; Leskinen, H.;
Vepsäläinen, J.; Christiaans, J. A. M.; Forsberg, M. M.; Männistö, P.
T.; Wallén, E. A. A. 2(S)-(Cycloalk-1-enecarbonyl)-1-(4-phenyl-
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butanoyl)pyrrolidines
and
2(S)-(aroyl)-1-(4-
phenylbutanoyl)pyrrolidines as prolyl oligopeptidase inhibitors.
Bioorg. Med. Chem. 2007, 15, 2024-2031.
18. Nakajima, T.; Ono, Y.; Kato, A.; Maeda, J.; Ohe, T. Y-29794 a
non-peptide prolyl endopeptidase inhibitor that can penetrate into the
brain. Neurosci. Lett. 1992, 141, 156-160.
19. Tarrago, T.; Kichik, N.; Segu, J.; Giralt, E. The natural product
berberine is a human prolyl oligopeptidase inhibitor. ChemMedChem.
2007, 2, 354-359.
REFERENCES
1. García-Horsman, J. A.; Männistö, P. T.; Venäläinen, J. I. On the
role of prolyl oligopeptidase in health and disease. Neuropeptides.
2007, 41, 1-24.
2. Myöhänen, T. T.; Venäläinen, J. I.; Tupala, E.; Garcia-Horsman,
J. A.; Miettinen, R.; Männistö, P. T. Distribution of immunoreactive
prolyl oligopeptidase in human and rat brain. Neurochem. Res. 2007,
32, 1365-1374.
3. Daly, D. J.; Maskrey, P.; Pennington, R. J. T. Characterization of
proline endopeptidase from skeletal muscle. Int. J. Biochem. 1985, 17,
521-524.
4. Goossens, F. H.; De Meester, I.; Vanhoof, G.; Scharpé, S.
Distribution of prolyl oligopeptidase in human peripheral tissues and
body fluids. Eur. J. Clin. Chem. Clin. Biochem. 1996, 34, 17-22.
5. Myöhänen, T. T.; Venäläinen, J. I.; García-Horsman, J. A.;
Piltonen, M.; Männistö, P. T. Distribution of prolyl oligopeptidase in
the mouse whole-body sections and peripheral tissues. Histochem. Cell
Biol. 2008, 130, 993-1003.
6. Myöhänen, T. T.; Pyykkö, E.; Männistö, P. T.; Carpen, O.
Distribution of Prolyl Oligopeptidase in Human Peripheral Tissues and
in Ovarian and Colorectal Tumors. J. Histochem. Cytochem. 2012, 60,
706-715.
7. Mantle, D.; Falkous, G.; Ishiura, S.; Blanchard, P. J.; Perry, E. K.
Comparison of proline endopeptidase activity in brain tissue from
normal cases and cases with Alzheimer's disease, Lewy body dementia,
Parkinson's disease and Huntington's disease. Clin. Chim. Acta. 1996,
249, 129-139.
8. Liu, J.; Kusinski, M.; Ilic, V.; Bignon, J.; Hajem, N.;
Komorowski, J.; Kuzdak, K.; Stepien, H.; Wdzieczak-Bakala, J.
Overexpression of the angiogenic tetrapeptide AcSDKP in human
malignant tumors. Anticancer Res. 2008, 28, 2813-2817.
9. Larrinaga, G.; Perez, I.; Blanco, L.; López, J. I.; Andrés, L.;
Etxezarraga, C.; Santaolalla, F.; Zabala, A.; Varona, A.; Irazusta, J.
Increased prolyl endopeptidase activity in human neoplasia. Regul.
Pept. 2010, 163, 102-106.
10. Brandt, I.; Gérard, M.; Sergeant, K.; Devreese, B.; Baekelandt,
V.; Augustyns, K.; Scharpé, S.; Engelborghs, Y.; Lambeir, A. Prolyl
oligopeptidase stimulates the aggregation of a-synuclein. Peptides.
2008, 29, 1472-1478.
11. Di Daniel, E.; Glover, C. P.; Grot, E.; Chan, M. K.; Sanderson,
T. H.; White, J. H.; Ellis, C. L.; Gallagher, K. T.; Uney, J.; Thomas, J.;
Maycox, P. R.; Mudge, A. W. Prolyl oligopeptidase binds to GAP-43
and functions without its peptidase activity. Mol. Cell. Neurosci. 2009,
41, 373-382.
12. Savolainen, M. H.; Yan, X.; Myöhänen, T. T.; Huttunen, H. J.
Prolyl oligopeptidase enhances a-Synuclein dimerization via direct
protein-protein interaction. J. Biol. Chem. 2015, 290, 5117-5126.
13. Tsirigotaki, A.; Van Elzen, R.; Van Der Veken, P.; Lambeir, A.
; Economou, A. Dynamics and ligand-induced conformational changes
in human prolyl oligopeptidase analyzed by hydrogen/deuterium
exchange mass spectrometry. Sci. Rep. 2017, 7.
14. Myöhänen, T. T.; Hannula, M. J.; Van Elzen, R.; Gerard, M.;
Van Der Veken, P.; García-Horsman, J. A.; Baekelandt, V.; Männistö,
P. T.; Lambeir, A. M. A prolyl oligopeptidase inhibitor, KYP-2047,
reduces a-synuclein protein levels and aggregates in cellular and animal
20. Kumar, R.; Bavi, R.; Jo, M. G.; Arulalapperumal, V.; Baek, A.;
Rampogu, S.; Kim, M. O.; Lee, K. W. New compounds identified
through in silico approaches reduce the a-synuclein expression by
inhibiting prolyl oligopeptidase in vitro. Sci. Rep. 2017, 7.
21. Tarragó, T.; Kichik, N.; Claasen, B.; Prades, R.; Teixidó, M.;
Giralt, E. Baicalin, a prodrug able to reach the CNS, is a prolyl
oligopeptidase inhibitor. Bioorg. Med. Chem. 2008, 16, 7516-7524.
22. Tsutsumi S.; Okonogi T.; Shibahara S.; Ohuchi S.; Hatsushiba
E.; Patchett A. A.; Christensen B. G. Synthesis and structure-activity
relationships of peptidyl alpha-keto heterocycles as novel inhibitors of
prolyl endopeptidase. J. Med. Chem. 1994, 37, 3492-3502
23. Bal G.; Van der Veken P.; Antonov D.; Lambeir A.-M.; Grellier
P.; Croft S.L.; Augustyns K.; Haemers A. Prolylisoxazoles: potent
inhibitors of prolyloligopeptidase with antitrypanosomal activity.
Bioorg. Med. Chem. Lett. 2003, 13, 2875-2878.
24. Saito, M.; Hashimoto, M.; Kawaguchi, N.; Shibata, H.; Fukami,
H.; Tanaka, T.; Higuchi, N. Synthesis and inhibitory activity of acyl-
peptidyl-pyrrolidine derivatives toward post-proline cleaving enzyme;
a study of subsite specificity. J. Enzyme Inhib. Med. Chem. 1991, 5, 51-
75.
25. Jarho, E. M.; Venäläinen, J. I.; Huuskonen, J.; Christiaans, J. A.
M.; Garcia-Horsman, J. A.; Forsberg, M. M.; Järvinen, T.; Gynther, J.;
Männistö, P. T.; Wallén, E. A. A. A cyclopent-2-enecarbonyl group
mimics proline at the P2 position of prolyl oligopeptidase inhibitors. J.
Med. Chem. 2004, 47, 5605-5607.
26. Kaszuba, K.; Róg, T.; Danne, R.; Canning, P.; Fülöp, V.; Juhász,
T.; Szeltner, Z.; St. Pierre, J. -.; García-Horsman, A.; Männistö, P. T.;
Karttunen, M.; Hokkanen, J.; Bunker, A. Molecular dynamics,
crystallography and mutagenesis studies on the substrate gating
mechanism of prolyl oligopeptidase. Biochimie. 2012, 94, 1398-1411.
27. Van Der Veken, P.; Fülöp, V.; Rea, D.; Gerard, M.; Van Elzen,
R.; Joossens, J.; Cheng, J. D.; Baekelandt, V.; De Meester, I.; Lambeir,
A.; Augustyns, K. P2-substituted N-acylprolylpyrrolidine inhibitors of
prolyl oligopeptidase: Biochemical evaluation, binding mode
determination, and assessment in a cellular model of synucleinopathy.
J. Med. Chem. 2012, 55, 9856-9867.
28. Venäläinen, J. I.; Garcia-Horsman, J. A.; Forsberg, M. M.;
Jalkanen, A.; Wallén, E. A. A.; Jarho, E. M.; Christiaans, J. A. M.;
Gynther, J.; Männistö, P. T. Binding kinetics and duration of in vivo
action of novel prolyl oligopeptidase inhibitors. Biochem. Pharmacol.
2006, 71, 683-692.
29. Venäläinen, J. I.; Juvonen, R. O.; Forsberg, M. M.; Garcia-
Horsman, A.; Poso, A.; Wallen, E. A. A.; Gynther, J.; Männistö, P. T.
Substrate-dependent, non-hyperbolic kinetics of pig brain prolyl
oligopeptidase and its tight binding inhibition by JTP-4819. Biochem.
Pharmacol. 2002, 64, 463-471.
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ACS Medicinal Chemistry Letters
For Table of Contents Use Only
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Tetrazole as a Replacement of the Electrophilic Group in Characteristic Prolyl Oligopeptidase Inhibitors
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Tommi P. Kilpeläinen, Jonna K. Tyni, Maija K. Lahtela-Kakkonen, Tony S. Eteläinen, Timo T.
Myöhänen and Erik A. A. Wallén
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Effect on Syn
dimeriz. at 10 M
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IC50 (nM)
O
Xaa
Xaa
HN
N
Pro
Ala
Sar
MeAla
Gly
12
129
10 640
27 180
205 400
82 %
75 %
75 %
75 %
77 %
N
N
N
7
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