Development of synthetic aminopeptidase N/CD13 inhibitors to overcome cancer metastasis and angiogenesis

Article abstract of DOI:10.1021/ml3000758

Cancer metastasis is a major barrier to its treatment and an important cause of patient death. Antimetastatic agents hold promise for patients with advanced metastatic tumors. Aminopeptidase N/CD13 (APN) is being pursued by many as an important target against cancer metastasis and angiogenesis, but there are few reports on the in vivo evaluation of synthetic APN inhibitors. Herein, a series of compounds targeting APN were synthesized and evaluated for their antimetastasis and antiangiogenesis potency both in vitro and in vivo. Excitingly, compounds 4m, 4t, and 4cc, with the most potent APN inhibitory activities, displayed significant antimetastasis and antiangiogenesis effects in vitro and in vivo, suggesting that those synthetic APN inhibitors have the potential to overcome cancer metastasis and angiogenesis.

Full text of DOI:10.1021/ml3000758

Letter
pubs.acs.org/acsmedchemlett
Development of Synthetic Aminopeptidase N/CD13 Inhibitors
to Overcome Cancer Metastasis and Angiogenesis
Li Su, Jiangying Cao, Yuping Jia, Xiaonan Zhang, Hao Fang,* and Wenfang Xu*
Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 250012 Ji’nan, Shandong, PR China
S
* Supporting Information
ABSTRACT: Cancer metastasis is a major barrier to its treatment and
an important cause of patient death. Antimetastatic agents hold
promise for patients with advanced metastatic tumors. Aminopeptidase
N/CD13 (APN) is being pursued by many as an important target
against cancer metastasis and angiogenesis, but there are few reports
on the in vivo evaluation of synthetic APN inhibitors. Herein, a series
of compounds targeting APN were synthesized and evaluated for their
antimetastasis and antiangiogenesis potency both in vitro and in vivo.
Excitingly, compounds 4m, 4t, and 4cc, with the most potent APN
inhibitory activities, displayed significant antimetastasis and antiangio-
genesis effects in vitro and in vivo, suggesting that those synthetic APN
inhibitors have the potential to overcome cancer metastasis and
angiogenesis.
KEYWORDS: APN/CD13, inhibitors, anticancer, metastasis, angiogenesis
n enduring problem in cancer treatment is the high fail-
high APN expression solid tumors were reported to be signi-
1
Aure rates in patients with advanced metastatic cancers. ficantly lower than those of patients with APN-negative solid
tumors.15 So far, reported APN inhibitors can be divided into
Cancer metastasis is considered as a key cause of operation
failure, postoperative relapse, and ultimately death.2 Metastasis
natural products and synthetic compounds. Among them,
bestatin (Ubenimex16) may be the most studied one. It is
is a complex, multistep process, in which cancer cells spread
from primary sites to new places.3 Cancer cell invasion is the
isolated from a culture filtrate of Streptomyces olivoreticuli and is
a cancer chemotherapeutic drug in some countries (Figure 1A).17
key stage, and angiogenesis is the prerequisite.4 The extra-
cellular matrix (ECM) is the main barrier of malignant cell
dissemination. The ECM is a substrate of aminopeptidase N
(APN/CD13, EC 3.4.11.2) and matrix metalloproteinases
(MMPs). Targeting MMPs in the treatment of cancer
metastasis has yielded unsatisfactory results.5 Consequently,
in recent years APN has become one of the most studied
cancer therapeutic targets.6
APN is a zinc-dependent integral peptidase,7 belonging to
the M1 family of the MA clan of peptidases.8 As an exo-
peptidase, APN preferentially cleaves neutral amino acids from
the N-terminal of various oligopeptides, including enkephalins,
neurokinins,9 angiotensins, and some cytokines.10 Human APN
exists in many tissues, organs, and cell types, including kidney
proximal tubule cells, stem cells, epithelial cells, endothelial
cells, fibroblast cells, leukemia cells, and immune cells, etc.7,11
APN degrades ECM to promote malignant cell invasion into
the bloodstream.11 In the angiogenesis process, neo-endothelial
cells invading through the ECM also need APN.12 Therefore,
APN would be a key target in cancer metastasis and angio-
genesis.13 Meanwhile, the dysregulated expression and high-
level exopeptidase activity of APN are detected in various mam-
malian cancer cell lines14 such as melanoma, prostate, ovarian,
colon, renal and pancreas carcinomas, etc., as well as neo-
endothelial cells. The statistical survival rates for patients with
Figure 1. (A) Chemical structure of bestatin; (B) chemical structure of
D24; (C) target compounds.
Bestatin is a potent APN inhibitor with a Ki value of 3.03 μM.18
Saiki et al. found that bestatin significantly inhibited can-
cer cell invasion in a dose-dependent manner.19 However,
research on synthetic compounds only aimed at evaluating
their APN and cell proliferation inhibition. There are few re-
ports on their in vivo antimetastatic and antiangiogenic effects.
Received: April 2, 2012
Accepted: September 21, 2012
Published: September 21, 2012
© 2012 American Chemical Society
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Therefore, in this paper, potent synthetic small-molecule APN
inhibitors were designed and synthesized, and their antimetastasis
and antiangiogenesis effects were evaluated in vitro and in vivo.
In 2006, Yoshimoto et al. reported the crystal structure of
APN from Escherichia coli with the active site occupied by
bestatin.20 Recently, Gu et al. engineered Thermoplasma
acidophilum factor F3 (a homologous protein of human APN)
with two-point directed mutation, which obtained the protein
with an identical active site to human APN. Structural biology
studies were then performed on the cocrystal structure of
this protein and one active APN inhibitor (D24, Figure 1B)
developed by our group. The result suggested that engineered
factor F3 mutant has a larger binding pocket than E. coli APN.21
According to the chemical structures of bestatin and D24,
both of them have a zinc-binding group (ZBG) and a neutral
amino acid residue. In our ongoing studies, hydroxamic acid
as strong ZBG in D24 was kept and five neutral amino acids
(L-leucine, L-phenylglycine, L-isoleucine, L-phenylalanine, and
L-methione) were introduced to the new target structures.
The nonpeptide structure in D24 was replaced by various
aromatic rings or an aromatic fragment with an alkyl linker so
as to increase interaction with the larger pocket of human APN
(Figure 1C).
The target compounds, 4a-4cc, 7, and 9 were first examined
for their inhibitory activities toward APN from porcine kidney.
Most compounds exhibited more potent inhibitory activities
than bestatin, some of which had 10-fold or more improve-
ment (Table 1). The substituents greatly influenced the
compounds potency. Replacement of the ureido linker provides
a serious potency decrease, such as for 7 and 9. With the same
a
Table 1. Structures and IC50 Values of the Target Compounds
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Table 1. continued
a
All compounds were assayed three times, and the results are expressed with standard deviations.
R2 substituent, L-leucine and L-phenylglycine residues con-
tributed more to the potency than the other residues. As for R2
substituents, a long side chain between the aromatic ring and
the ureido group decreased the potency, such as for 4i, 4j, 4d,
and 4h. Their potencies increased along with the chain length
decline, while not so regularly in 4k, 4a, and 4e. Most of the
L-leucine based compounds with substituted phenyl or benzyl
on the R2 position were much more potent. 4zb with phenyl
was less potent than 4i with benzyl, which could also be seen
in 4b versus 4za with fluorine, and in 4p versus 4x with an
L-leucine residue. But the opposite phenomenon was seen in 4q
versus 4w and in 4o versus 4v, with methyl or methoxy. As
heterocycle R2 substituents, thiophene (4r) contributed more
to the potency than furan (4l). For the L-leucine-based com-
pounds with substituted benzyl at R2, electron donating groups
seemed better than electron withdrawing groups. For instance,
4t was more potent than 4u, as well as 4w versus 4x, 4o and 4q
versus 4p, also seen in 4y and 4z versus 4b, except 4v. This
might also demonstrate that methyl or methoxy fit into the
pocket better than fluorine. The substituted position on benzyl
also influenced the potency. With methoxy substituted benzyl,
4t (meta-) seemed better than 4s (ortho-) and 4v (para-), while
this is not so obvious in the fluorine substituted ones 4u and 4x.
The most potent compounds were 4m (IC50 = 99 nM) with
1-naphthyl and 4cc (IC50 = 50 nM) with 1-naphthylmethyl,
suggesting that a larger group was better here, though 4n with a
biphenyl group was much less potent.
Table 2. IC50 Values and Inhibition Rates of the
a
Compounds
growth inhibition
rates toward ES-2
cells
IC50
(MMP-2)/
IC50 (APN
from
IC50 (μM)
toward APN
on the surface
IC50 (μM)
toward
MMP-2
procine
compd
of ES-2 cells 5 μg/mL 50 μg/mL
kidney)
4m
4o
0.48 0.05
6.4 0.6
<1.0%
<1.0%
<1.0%
<1.0%
<1.0%
<1.0%
<1.0%
<1.0%
<1.0%
<1.0%
<1.0%
3.63%
6.85%
1.78%
3.29%
<1.0%
2.81%
<1.0%
1.83%
11.9%
6.67%
2.89%
>1000
>106
>106
1740
>106
>106
>106
>106
>106
>106
10900
30.2
>1000
4q
0.94 0.08
1.1 0.1
573 35
>1000
4s
4t
0.79 0.07
3.0 0.3
>1000
4u
>1000
4v
6.9 0.6
>1000
4w
4x
1.5 0.1
>1000
0.91 0.09
0.42 0.03
16.0 1.6
>1000
4cc
bestatin
545 30
276 19
a
The compounds were assayed three times, and the results are
expressed with standard deviations.
Table 2 showed that at low concentration all compounds had
almost no influence on ES-2 cell survival. At high concen-
tration, there was only a slight antiproliferative effect for most
of the compounds, except 4x. Matrix metalloproteinase-2 (MMP-2)
is also a zinc-dependent metalloproteinase responsible for cancer
invasion, so MMP-2 inhibitory activity was also tested. 4m, 4q,
4t, and 4cc exhibited very high selectivity for APN over MMP-2
(Table 2).
ES-2 cells with high APN expression were used as human
APN, which makes more sense to the following in vitro and
in vivo results. We only assessed the ability of the nanomolar
range compounds to inhibit human APN on cultured ES-2 human
ovarian clear cell carcinoma cells (Table 2). Those compounds
were still better than bestatin, some even with more than
10-fold improvement. The effects of the more potent ones (4m,
4q, 4t, 4x, and 4cc) on ES-2 cell survival were evaluated, and
Cell migration is an important step during the invasion pro-
cess. Agents blocking cell motility would also exhibit anti-invasion
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effects. A cell migration assay was performed on transwell
chambers without Matrigel coating. Figure 2A showed that all
4t, and 4cc on tumor-induced angiogenesis and experimental
metastasis in vivo. 4m, 4t, and 4cc effectively inhibited murine
APN activity and had quite low antiproliferative effects on B16BL6
(Figure 3A). In 2004, Saiki et al. demonstrated a significant
Figure 2. (A) ES-2 cell migration. (B) ES-2 cell invasion inhibition:
(a) control; (b) bestatin 150 μM; (c) 4m, 150 μM; (d) 4q, 150 μM;
(e) 4t, 150 μM; (f) 4cc, 150 μM; (g) statistical chart. Each column
represents the mean values with SD for three independent
experiments.***, P < 0.001; **, P < 0.01; *, P < 0.05, versus the
control. ###, P < 0.001; ##, P < 0.01; #, P < 0.05, versus Bestatin
treated groups.
the tested compounds (150 μM) had only a slight inhibitory
tendency to ES-2 cell migration, without statistical significance.
An anti-invasion assay was performed on transwell chambers
coated with Matrigel. According to the result, ES-2 cells could
freely invade and pass through Matrigel, but this was signi-
ficantly blocked by bestatin, 4m, 4q, 4t, and 4cc. In 1989
Azuma et al. reported the tumor cell invasion block effects of
bestatin in various conditions to mouse B16BL6 and 3LL cells.
The result indicated that bestatin had approached 50−80% in-
hibitory rates at the 100 mg/mL level, with a dose-dependent
tendency.22 According to our results on human ES-2 cell invasion
inhibition, 4m and 4cc were more potent than bestatin at the
concentrations of 15 μM and 150 μM (Figure 2B(g)). Figure 2
shows that 4m, 4q, 4t, and 4cc were able to significantly inhibit
ES-2 cell invasion in a dose-dependent tendency without obvious
influence on ES-2 cell survival and migration, which indicated their
potential therapeutic application in overcoming cancer metastasis.
The ECM gel (Matrigel)-induced human umbilical vascular endo-
thelial cell (HUVEC) capillary tubular structure formation assay
was used as an in vitro measurement of angiogenesis.23 Bestatin,
4m, 4t, and 4cc decreased the number of branch points formed by
HUVECs (data not shown), which demonstrated the potential
antiangiogenesis effects of the APN inhibitors.
Figure 3. (A) The IC50 and GI50 values of 4m, 4t, and 4cc. (B) 4m, 4t,
and 4cc inhibit B16BL6 induced angiogenesis in vivo. **, P < 0.01,
versus the control. ##, P < 0.01, versus bestatin treated groups. (C)
B16BL6 pulmonary colonies. (a) Control, (b) bestatin, (c) 4m, (d) 4t,
(e) 4cc, (f) statistical chart. Each column represents the mean values
with SD *, P < 0.05, versus the control, **, P < 0.01, versus the
control.
antiangiogenesis effect of bestatin (100 or 200 mg/kg, p.o.) on
the C57BL6 mouse dorsal air sac model with the B16BL6 cell
line.25 In our assay, angiogenesis was induced by the implantation
of B16BL6 cells into the intradermal site of C57BL/6 mouse
backs,26 and it quantified by counting the number of vessels
oriented toward the tumor mass27 under a dissecting microscope.
B16BL6 cells, which represent an advanced malignant murine
tumor more metastatic than B16,24 were selected to examine 4m,
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Figure 3B showed that the intraperitoneal administration of
4m, 4t, and 4cc at the 100 mg/kg level led to significant anti-
angiogenesis effects, which were significantly better than those
for bestatin (P < 0.01). In 2006, our group reported a murine
H22 liver cancer experimental metastasis on Kunming mice, and
bestatin showed potency (60−70% inhibitory rate) at 50 mg/kg
orally.28 At this time, further study on APN inhibitor in vivo
antimetastasis effects was performed through choosing a much
higher advanced metastatic cancer cell line, B16BL6. The result
showed that intravenous inoculation of B16BL6 resulted in the
establishment of lung metastatic sites in C57BL/6 mice (as high
as 170 nodes in the control group), much more serious than
H22 (only 33 nodes). A significant decrease of the B16BL6
pulmonary colonies was detected in 4m, 4t, and 4cc treated
groups (Figure 3C).
phenacetyl chloride, and the hydroxamic acid was obtained by
the same method as above.
We have designed and synthesized a series of small-molecule
synthetic APN inhibitors. The APN inhibitory activity demon-
strated that most compounds were more potent than bestatin.
The three most potent candidates, 4m, 4t, and 4cc, with a
nanomolar range IC50, exhibited significant anti-invasion effects
to ES-2 cells in vitro without obvious cytotoxicity, which is
better than bestatin. In the in vivo evaluation including murine
B16BL6 induced angiogenesis and experimental lung metastasis
assays, 4m, 4t, and 4cc exhibited significant antiangiogenesis
and antimetastasis effects without obvious cytotoxicity toward
B16BL6 cells. Such results suggest that those three synthetic
APN inhibitors are potential candidates which can be developed
to overcome cancer metastasis and angiogenesis.
The synthetic methods for compounds 4a−4cc are shown
ASSOCIATED CONTENT
* Supporting Information
Synthesis of compounds, experimental procedures, character-
ization of new compounds, 1H NMR, 13C NMR, and biological
experiments. This material is available free of charge via the
Internet at http://pubs.acs.org.
in Scheme 1A. The amines listed were transformed into
■
S
Scheme 1
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +86-531-88382264 (W.X.); +86-531-88382731 (H.F.).
Fax: +86-531-88382264. E-mail: xuwenf@sdu.edu.cn (W.X.);
haofangcn@sdu.edu.cn (H.F.). W.X. is also associated with the
Key Laboratory of Molecule Design and Novel Drug Research,
Shandong province.
Funding
This work was supported by a National Natural Science
Foundation Research Grant (Grant No. 21172134), the Ph.D.
Programs Foundation of the Ministry of Education of China
(Grant No. 20110131110037), Major Programs of the Chinese
Academy of Sciences during the 12th Five-Year Plan Period
(Grant No. 2011ZX09401-015), the Shandong Province
Natural Science Foundation (Grant No. ZR2010HM028), the
Natural Science Foundation for Young Scholars of Shandong
Province (2006BS03021), and the Independent Innovation
Foundation of Shandong University, IIFSDU (2012JC003).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Jiong Jia for 1H NMR and 13C NMR of the target
compounds.
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