Peptide-cleaving agents for human islet amyloid polypeptide containing substrate recognition site based on quinoxaline: Cleavage efficiency enhanced by lowering substrate concentration

Article abstract of DOI:10.1016/j.bmcl.2012.01.008

Oligomers of human islet amyloid polypeptide (h-IAPP) are believed to be the pathogenic species for type 2 diabetes mellitus. Peptide-cleaving agents selective for oligomers of h-IAPP were synthesized by using quinoxaline derivatives as recognition sites

Full text of DOI:10.1016/j.bmcl.2012.01.008

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1533–1537
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Peptide-cleaving agents for human islet amyloid polypeptide containing
substrate recognition site based on quinoxaline: Cleavage efficiency
enhanced by lowering substrate concentration
⇑
Woosuk Chei, Heeyeon Ju, Junghun Suh
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Oligomers of human islet amyloid polypeptide (h-IAPP) are believed to be the pathogenic species for type
2 diabetes mellitus. Peptide-cleaving agents selective for oligomers of h-IAPP were synthesized by using
quinoxaline derivatives as recognition sites attached to the Co(III) complex of cyclen in this study. When
Received 26 October 2011
Revised 2 January 2012
Accepted 4 January 2012
Available online 10 January 2012
the initial concentration of h-IAPP was lowered from 4.0 to 0.20 lM, cleavage yield of the new agents was
enhanced by 3 times reaching 16–22 mol %. This shows that the agents would have significant activities
at subnano molar concentrations if the concentration of h-IAPP is lowered to the in vivo values. This fur-
ther indicates that the peptide-cleaving agents prepared previously in this laboratory possess sufficiently
high activity for application as a new therapeutic option for Alzheimer’s disease, type 2 diabetes mellitus,
and Parkinson’s disease.
Keywords:
Peptide-cleaving agent
Diabetes
h-IAPP
Amyloid
Ó 2012 Elsevier Ltd. All rights reserved.
Amyloid diseases
We have proposed target-selective peptide-cleaving agents as a
new therapeutic option for amyloid diseases. It is generally be-
lieved that the soluble oligomers of amyloidogenic oligopeptides
or proteins such as amyloid b-42 (Ab42), human islet amyloid poly-
and the plateau values of cleavage yields observed at the agent
concentrations of 1–50 M were 10–40 mol %.
l
To develop clinically safe peptide-cleaving agents for the path-
ogenic proteins of amyloid diseases, it is necessary to raise the
efficiency of the cleavage action, so that considerable activities
are observed at agent concentrations below 1 nM. In an attempt
to raise the cleavage efficiency, we have tested various analogs of
cyclen as the chelating ligand of the Co(III) center to enhance the
Lewis acidity of the Co(III) ion.14 Efforts to improve the cleavage
efficiency by varying the structure of the chelating ligand of the
catalytic center, however, were unsuccessful. Instead, we have
achieved considerable enhancement in the efficiency of the cleav-
age action of the peptide-cleaving agents by lowering substrate
concentration in the present study.
peptide (h-IAPP), and a
-synuclein are the pathogenic species1–3 of
amyloid diseases1 such as Alzheimer’s disease, type 2 diabetes
mellitus, and Parkinson’s disease, respectively. Those pathogenic
proteins for the amyloid diseases do not have active sites and,
therefore, conventional drugs targeting active sites cannot be ob-
tained. The peptide-cleaving agents selective for the soluble oligo-
mers of amyloidogenic oligopeptides or proteins have been
designed4–6 by using the Co(III) complex of 1,4,7,10-tetraazacy-
clododecane (cyclen) as the catalytic center for peptide hydrolysis.
The Co(III)-based peptide-cleaving agents were found to penetrate
animal cells readily.7 The Cu(II) complex of cyclen has been also
used as catalytic center for target-selective peptide-cleaving
agents,8–12but we prefer the Co(III) complex due to its exchange-
inertness13 and the consequent protection from metal abstraction
under biotic conditions.
The peptide-cleaving agents previously synthesized in this
laboratory for selective cleavage of oligomers of Ab42, h-IAPP,
and
a-synuclein contained 2-phenylbenzothiazole or 2-phen-
ylbenzooxazole as parts of organic pendants attached to the
Co(III) complex (Co(III)cyclen) of cyclen.4–6 The organic pendants
act as substrate recognition sites, that can selectively bind to the
aromatic domains of the oligomers of the amyloidogenic pep-
tides or proteins formed by assembly of the aromatic side chains
of the respective monomers. 2-Phenylbenzothiazole or 2-phen-
ylbenzooxazole was included in the substrate recognition site
to ensure complexation to the target pathogenic proteins of
amyloid diseases since their derivatives had been developed15
as agents for imaging b-amyloid plaque of Alzheimer’s disease
Efficiency of the peptide-cleaving agents for the oligomers of
Ab42, h-IAPP, and a-synuclein has been estimated in terms of cleav-
age yield after incubation with 4.0 lM substrate for 36 h at pH 7.5
and 37 °C at various concentrations of the agents.4–6 The agents
manifested activities at the concentrations as low as 10–100 nM
⇑
Corresponding author.
E-mail address: jhsuh@snu.ac.kr (J. Suh).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.008

1534
W. Chei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1533–1537
R1–NH–R2 and 32 primary or secondary amines as R3–NH–R4.
Compounds 1b were purified by column chromatograph whereas
compounds 1c were partially purified by short column chromatog-
raphy and used in the next steps.
The 288 compounds of the library were screened by the matrix-
assisted laser desorption/ionization (MALDI)-time of flight (TOF)
mass spectrometry for their activity to cleave Ab42 (4.0
lM) or h-
IAPP (4.0 M) at pH 7.50 and 37 °C for 48 h. Some Co(III)cyclen–
l
quinoxaline conjugates of the library showing activity to cleave
Ab42 or h-IAPP were synthesized on a larger scale, thoroughly puri-
fied, and tested again for the peptide-cleaving activity to confirm
the activity. Two compounds (Q1 and Q2) were identified as pep-
tide-cleaving agents selective for h-IAPP and none for Ab42
.
NH
NH
OH2
OH2
OH2
OH2
III
NH
NH
III
NH
NH
Co
Co
HN
H
H
N
H
N
N
N
N
NH
N
H
N
Figure 1. MALDI-TOF mass spectrum of the reaction mixture obtained after
incubation of h-IAPP (4.0 lM) with Q1 (1.0 lM) for 48 h at pH 7.50 and 37 °C:
O
O
N
N
N
Peaks with m/z of 1933 and 2076 corresponds to h-IAPP19–37, and h-IAPP1–19
,
respectively.
Q2
Q1
Unless noted otherwise, the action of Q1 or Q2 was initiated by
adding the alkaline stock solution of h-IAPP to a buffer solution
10
8
6
4
2
Figure 2. MALDI-TOF mass spectrum of the reaction mixture obtained after
incubation of h-IAPP (4.0 M) with Q2 (1.0 M) for 48 h at pH 7.50 and 37 °C:
0
l
l
-9
-8
-7
-6
0/M
-5
-4
-3
Peaks with m/z of 2076 and 2543 corresponds to h-IAPP1–19, and h-IAPP14–37
,
log
C
respectively.
Figure 3. Plot of cleavage yield against logC0/M for the cleavage of h-IAPP (4.0
with Q1 for 36 h at pH 7.50 and 37 °C.
lM)
with positron emission tomography. 2-Phenylbenzothiazole or 2-
phenylbenzooxazole moieties are, however, inevitably accompa-
nied by unproductive binding of the agents to amyloid plaques
and high molecular weights of the agents.
In this letter, we describe the results of our attempts to design
peptide-cleaving agents containing quinoxaline instead of 2-phen-
ylbenzothiazole or 2-phenylbenzooxazole as well as considerable
enhancement in the efficiency of the peptide-cleaving agents
achieved by lowering substrate concentration.
To design the peptide-cleaving agents that do not depend on 2-
phenylbenzothiazole or 2-phenylbenzooxazole moieties for sub-
strate recognition, quinoxaline was employed in the present study
as the core of the organic pendants attached to Co(III)cyclen
exploiting its bicyclic aromatic structure. Some quinoxaline deriv-
atives have antibiotic or antitumor activities, presumably due to
the bicyclic aromatic structure of quinoxaline.16,17
6
5
4
3
2
1
0
-8
-7
-6
log
-5
0/M
-4
-3
C
A chemical library of quinoxaline–Co(III)cyclen conjugates were
prepared as summarized in Scheme
Supplementary data. Nine cyclen derivatives were employed as
1 and as described in
Figure 4. Plot of cleavage yield against logC0/M for the cleavage of h-IAPP (4.0
with Q2 for 36 h at pH 7.50 and 37 °C.
lM)

W. Chei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1533–1537
1535
R1
N
N
N
R2
R3-NH-R4
N
N
Cl
Cl
R1-NH-R2
Cl
1b
1a
R1
N
N
removal of
CoCl2, O2
R2
R3
Co(III)cyclen-
quinoxaline
conjugate
protecting
groups
N
N
R4
1c
0
3
6
12 20 36 52 62 80
Scheme 1. Synthetic route for construction of the chemical library of candidates for
the peptide-cleaving agents.
preincubation period (hr)
Figure 6. Plot of cleavage yield against preincubation period for the cleavage of h-
IAPP (4.0 M) with Q1 (4.0 M) for 36 h at pH 7.50 and 37 °C.
l
l
containing Q1 or Q2 in the present study. The MALDI-TOF mass
spectra obtained after incubation of h-IAPP with Q1 or Q2 for
48 h at pH 7.50 and 37 °C are illustrated in Figures. 1 and 2, respec-
tively. Here, the h-IAPP fragments are named according to the
amino acid sequence of h-IAPP. The structures of h-IAPP1–19
,
h-IAPP14–37, and h-IAPP19–37 were positively identified by MALDI-
TOF/TOF LIFT mass spectrometry.18 This provides evidence not
only for cleavage sites but also for the hydrolytic nature of the
peptide cleavage. Some other oligopeptides formed by cleavage
of h-IAPP may not have been detected by MALDI-TOF mass spec-
trometry. The results indicate that His18–Ser19 and Ser19–Ser20 of
h-IAPP are among the cleavage sites for the action of Q1 and
Ala13–Asn14 and Ser19–Ser20 for the action of Q2.
After incubation of h-IAPP (4.0 lM) with Q1 or Q2 for 36 h at
Figure 7. Formation of various assemblies of amyloidogenic oligopeptides or
37 °C and pH 7.50, the product solution was filtered through a
membrane with a cutoff MW of 10,000 to remove aggregates of
h-IAPP. Then, the resulting mixture was separated with HPLC to
isolate the oligopeptide fragments formed by the cleavage of h-
IAPP and to ensure the exclusion of aggregated h-IAPP. The oligo-
peptide fragments were hydrolyzed under alkaline conditions,
and the resulting amino acids were quantified with fluorescamine
as described previously.5 The amounts of oligopeptide fragments
obtained by the action of Q1 or Q2 were estimated as mol % of
the initially added amount of h-IAPP and were defined as the cleav-
age yields. The cleavage yields measured after the reaction with
various initial concentrations (C0) of Q1 or Q2 are plotted against
logC0/M in Figures. 3 and 4, respectively. The cleavage yields re-
ported in this study are the average values resulting from 4 to 6
measurements carried out with different reaction mixtures. Since
isolation of the oligopeptide fragments inevitably involves some
proteins (AmP) and reduction of the assemblies by using cleavage agents.
losses, the cleavage yields are somewhat underestimated. The
cleavage yields were also measured for Q1 by incubating with h-
IAPP (4.0 lM) at 37 °C and pH 7.50 for various periods, and the re-
sults are summarized in Figure 5. The results reveal that incubation
for more than 36 hours does not raise the cleavage yield. Similar
results had been obtained previously for the action of peptide-
cleaving agents containing 2-phenylbenzothiazole or 2-
phenylbenzooxazole.5
The cleavage yields were also measured by incubating the solu-
tion of h-IAPP (4.0
lM) for various periods at 37 °C and pH 7.50 be-
fore adding Q1 (4.0
lM). After incubation for further 36 h following
addition of Q1, the amount of the cleavage products was measured.
The results are summarized inFigure 6. Our previous study showed
that more than half of h-IAPP is converted to the oligomeric or the
protofibrillar polymeric form (Fig. 7) after incubation for 3 h at pH
7.50 and 37 °C.5 The similar cleavage yields measured for preincu-
bation period of 0 and 3 h (Fig. 6) indicate that the monomeric
form of h-IAPP is not the major species cleaved by Q1. When the
preincubation period is longer than 20 h, h-IAPP is mostly con-
verted into polymeric forms. The diminished cleavage yields
shown in Figure 6 after extended period of preincubation demon-
strates that the polymeric form of h-IAPP is not the major species
cleaved by Q1. The results of Figure 6, therefore, reveal that Q1
cleaves the soluble oligomers of h-IAPP.
To demonstrate that Q1 and Q2 do not cleave non-target pro-
teins, Q1 or Q2 (10
l
M) was incubated with proteins (15
lM) such
as bovine serum albumin, bovine serum
c-globulin, chicken egg
white lysozyme, and horse heart myoglobin at pH 7.50 or 9.50
for 3 days at 37 °C. For those non-target proteins, no protein cleav-
age was observed by MALDI-TOF mass spectrometry.
0
20
40
60
80
reaction time (hr)
Cleavage yields for Q1 and Q2 were also measured by lowering
the initial concentrations (S0) of h-IAPP. Similar experiments were
Figure 5. Plot of cleavage yield against reaction time for the cleavage of h-IAPP
(4.0 M) with Q1 (4.0 M) at pH 7.50 and 37 °C.
l
l

1536
W. Chei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1533–1537
Table 1
have longer lifetime at lower S0 concentrations leading to higher
cleavage yields for the action of cleavage agents.
The degree of increase in the cleavage yield on reduction of S0
from 4.0 to 0.20 lM is considerably greater for Q1 and Q2 com-
Cleavage yields measured with various initial substrate concentrations in the
selective cleavage of h-IAPP by Q1, Q2, T1, or T2
Agenta
Cleavage yield (mol %) when S0=
pared with T1 and T2. The reason for this difference is not clear
at present, but one possible explanation is to assume that different
oligomers of h-IAPP are involved as the targets for different types
of cleavage agents.
4.0
l
M
0.40
12
8.5 0.9
—
—
l
M
0.20
l
M
Q1
Q2
T1
T2
7.5 0.6
5.1 0.5
19
15
1
22
16
27
19
2
1
3
2
3
2
The level of the amyloidogenic oligopeptides or proteins in the
bodies of patients is much lower than the S0 concentrations used in
this experiment. For correct estimation of the cleavage yields, it is
not possible to lower the S0 concentrations substantially. The con-
centration of Ab42 has been estimated as 0.2–150 fM in the cere-
brospinal fluid of the patients of Alzheimer’s disease and that of
h-IAPP as 2–30 pM in the blood of the patients of type 2 diabetes
mellitus.21,22 If S0 is lowered by 104–109 times from that used in
the present study, cleavage yields would be improved remarkably
compared with those shown in the plots of cleavage yields against
logC0/M such as Figures. 3 and 4. We have reported several
peptide-cleaving agents selective for the soluble oligomers of amy-
loidogenic oligopeptides or proteins for Alzheimer’s disease, type 2
diabetes mellitus, and Parkinson’s disease which manifested
activities at the concentrations as low as 10–100 nM. Those pep-
tide-cleaving agents are expected to have significant activities at
subnano molar C0 concentrations when the S0 concentration is
lowered to the in vivo values. The peptide-cleaving agents, there-
fore, possess sufficiently high activity for application as a new ther-
apeutic option for Alzheimer’s disease, type 2 diabetes mellitus,
and Parkinson’s disease.
a
The initial concentration of the agent is 26
T1, or 40 M for T3.
l
M for Q1, 66
l
M for Q2, 81 lM for
l
carried out for two compounds (T1, T2) containing 2-phenylbenzo-
thiazole moieties which had been discovered as peptide-cleaving
agents selective for h-IAPP in the previous5 study. The concentra-
tions of Q1, Q2, T1, and T2 employed in this experiment were cho-
sen from the range corresponding to the plateau values of cleavage
yields measured with 4.0 lM h-IAPP. Cleavage yields measured
with various S0 concentrations of h-IAPP are summarized in Table
1.
NH
NH
III
III
OH2
OH2
NH
NH
OH2
OH2
NH
NH
Co
N
Co
N
T2
T1
HN
HN
O
N
N
Acknowledgments
NH
N
N
HN
NH
N
This work was supported by National Research Foundation of
Korea (NRF) grant (No. 2010-0016023) funded by the Korea gov-
ernment (MEST).
O
N
N
S
N
N
S
N
Supplementary data
Supplementary data (procedures for preparation of the library
and for synthesis of Q1 and Q2 and the control MALDI-TOF mass
spectra) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmcl.2012.01.008.
The results summarized in Table 1 reveal that the cleavage
yields for Q1 and Q2 were enhanced by 3 times when S0 was low-
ered from 4.0 lM to 0.20 lM. For T1 and T2, the cleavage yield was
also increased when S0 was lowered, although the degree of
enhancement was smaller than Q1 or Q2. Thus, the cleavage yields
for Q1 and Q2 become comparable to those for T1 or T2 when S0
References and notes
1. Bitan, G.; Fradinger, E.; Spring, S.; Teplow, D. Amyloid 2005, 12, 88.
2. Lesné, S.; Koh, M. T.; Kotilinek, L.; Kayed, R.; Glabe, C. G.; Yang, A.; Gallagher,
M.; Ashe, K. H. Nature 2006, 440, 352.
was 0.20
lM although the yields were considerably higher for T1
or T2 when S0 was 4.0
lM.
Actions for cleavage agents such as T1 or T2 have been
explained with the scheme of Figure 7, where AmP stands for amy-
loidogenic oligopeptides or proteins such as Ab42, h-IAPP, and
3. Lin, C. Y.; Gurlo, T.; Kayed, R.; Butler, A. E.; Haataja, L.; Glabe, C. G.; Butler, P. C.
Diabetes 2007, 56, 1324.
4. Suh, J.; Yoo, S. H.; Kim, M. G.; Jeong, K.; Ahn, J. Y.; Kim, M.; Chae, P. S.; Lee, T. Y.;
Lee, J.; Lee, J.; Jang, Y. A.; Ko, E. H. Angew. Chem. Int. Ed. 2007, 46, 7064.
5. Suh, J.; Chei, W. S.; Lee, T. Y.; Kim, M. G.; Yoo, S. H.; Jeong, K.; Ahn, J. Y. J. Biol.
Inorg. Chem. 2008, 13, 693.
6. Lee, J.; Yoo, S. H.; Jeong, K.; Lee, T. Y.; Ahn, J. Y.; Suh, J. Bull. Korean Chem. Soc.
2008, 29, 882.
7. Chei, W. S.; Lee, J.-W.; Kim, J. B.; Suh, J. Bioorg. Med. Chem. 2010, 18, 5248.
8. Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Song, J. B.; Suh, J. Org. Lett. 2002, 4,
4155.
9. Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Suh, J. Bioorg. Med. Chem. 2003, 11,
901.
10. Wu, W.; Lei, P.; Liu, Q.; Hu, J.; Gunn, A. P.; Chen, M.; Rui, Y.; Su, X.; Xie, Z.; Zhao,
Y.; Bush, A. I.; Li, Y. J. Biol. Chem. 2008, 283, 31657.
11. Hu, J.; Yu, Y.-P.; Cui, W.; Fang, C.-L.; Wu, W.-H.; Zhao, Y.-F.; Li, Y.-M. Chem.
Commun. 2010, 46, 8023.
12. Jeong, K.; Chung, W. Y.; Kye, Y. S.; Kim, D. Bioorg. Med. Chem. 2010, 18, 2598.
13. Greenwood, N. N.; Earnshaw, A. Chemistry of the Elements, 2nd Ed.;
Butterworth-Heinemann: Oxford, 1997. 1123.
a
-synuclein.4,5 AmP forms various kinds of soluble oligomers
which are in equilibria with one another and undergo polymeriza-
tion to form protofibrils or fibrils.2,19,20 A peptide-cleaving agent
selective for a soluble oligomer removes the target oligomer
leading to reduction of levels of other oligomers or monomer
which are readily converted to the target oligomer.
The target oligomers of the cleavage agents are transient spe-
cies undergoing further oligomerization and polymerization.2,19,20
Disappearance of the target oligomers by further oligomerization
or polymerization becomes slower as S0 is lowered, increasing
the lifetime of the target. On the other hand, removal of the target
oligomer by the cleavage agent is a pseudo-first-order process
when the agent concentration is fixed and the corresponding
half-life is not affected by reduction of S0. The scheme of Figure
7, therefore, predicts that the transient target oligomers would
14. Chei, W. S.; Ju, H.; Suh, J. J. Biol. Inorg. Chem. 2011, 16, 511.
15. Cai, L.; Chin, F. T.; Pike, V. W.; Toyama, H.; Liow, J.-S.; Zoghbi, S. S.; Modell, K.;
Briard, E.; Shetty, H. U.; Sinclair, K.; Donohue, S.; Tipre, D.; Kung, M.-P.;

W. Chei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1533–1537
1537
Dagostin, C.; Widdowson, D. A.; Green, M.; Gao, W.; Herman, M. M.; Ichise, M.;
Innis, R. B. J. Med. Chem. 2003, 46, 2740.
19. Bitan, G.; Kirkitadze, N. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D.
B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 330.
16. Martin, D. A.; Mizsak, S. A.; Biles, C.; Meulman, P. A. J. Antibiotics 1975, 28, 332.
17. Gao, H.; Yamasaki, E. F.; Chan, K. K.; Shen, L. L.; Snapka, R. M. Mol. Pharmacol.
2003, 63, 1382.
18. Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle, A.
Anal. Bioanal. Chem. 2003, 376, 952.
20. Hull, R. L.; Westermark, G. T.; Westermark, P.; Kahn, S. E. J. Clin. Endocrinol.
Metab. 2004, 89, 3629.
21. Georganopoulou, D. G.; Chang, L.; Nam, J.-M.; Thaxton, C. S.; Mufson, E. J.;
Klein, W. L.; Mirkin, C. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2273.
22. O’Brien, T. D.; Butler, P. C.; Westermark, P.; Johnson, K. H. Vet. Pathol. 1993, 30,
317.