Chemically synthesized LYRM03 could inhibit the metastasis of human breast cancer MDA-MB-231 cells in vitro and in vivo

Article abstract of DOI:10.1016/j.bmcl.2019.05.027

Aminopeptidase N (APN) belongs to the aminopeptidase family, which is widely distributed throughout the animal and plant kingdoms. APN is thought to be a very important target for cancer therapy as it is linked to cancer progression and metastasis. Howeve

Full text of DOI:10.1016/j.bmcl.2019.05.027

Accepted Manuscript
Chemically synthesized LYRM03 could inhibit the metastasis of human breast
cancer MDA-MB-231 cells in vitro, in vivo
Yun-kai Yang, Da-dong Shen, Peng He, Liang-dong Du, Ding-jian Wan, Pu
Wang, Tao Wang, Mei-qing Feng
PII:
S0960-894X(19)30326-9
https://doi.org/10.1016/j.bmcl.2019.05.027
BMCL 26447
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 March 2019
9 April 2019
15 May 2019
Please cite this article as: Yang, Y-k., Shen, D-d., He, P., Du, L-d., Wan, D-j., Wang, P., Wang, T., Feng, M-q.,
Chemically synthesized LYRM03 could inhibit the metastasis of human breast cancer MDA-MB-231 cells in vitro,
in vivo, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.027
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chemically synthesized LYRM03 could inhibit the metastasis of
human breast cancer MDA-MB-231 cells in vitro and in vivo
Yun-kai Yanga, Da-dong Shenb, Peng Hea, Liang-dong Duc, Ding-jian Wanc,
Pu Wangb, Tao Wangc*, Mei-qing Fenga*
a Department of microbiology and biochemical pharmacy, School of Pharmacy,
Fudan University, Shanghai 201203, P. R. China
b college of Pharmaceutical Science, Zhejiang University of Technology. Hangzhou
310032, Zhejiang P. R. China
c Shanghai Laiyi Center for Biopharmaceuticals R&D, 5B, Building 8 200 Niudun
Road Pudong District, Shanghai, 201203, P. R. China
 These authors contributed equally to this work.
* Corresponding authors:
Mei-qing Feng, Tel: +86-21-51980035; E-mail: fmq@fudan.edu.cn;
Tao Wang, Tel: +86-21-50275322; E-mail: wangtao823@hotmail.com;

Abstract
Aminopeptidase N (APN) belongs to the aminopeptidase family, which is widely
distributed throughout the animal and plant kingdoms. APN is thought to be a very
important target for cancer therapy as it is linked to cancer progression and metastasis.
However, bestatin (Ubenimex) is the only approved drug that targets various
aminopeptidases for the treatment of acute myelocytic leukemia and lymphedema. A
compound 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine (also known as
LYRM03), isolated from a Streptomyces strain HCCB10043, exhibited more potent
inhibitory activity than bestatin. In this work, we applied a chemical synthesis strategy
to generate LYRM03 to overcome the low yields typically achieved from
fermentation. Finally, we explored a suite of experiments to determine the bioactivity
of LYRM03 and revealed that the metastasis of MDA-MB-231 cells was significantly
restrained with LYRM03 treatment or injection both in vitro and in vivo. Because of
its anti-metastasis capacity, further structure modifications of LYRM03 will be of
interest for its use alone or in combination as a therapy in cancer.
Keywords: LYRM03; APN/CD13; breast cancer; MDA-MB-231; metastasis.
Actinomycetes are an important source of active natural biological products. It has
been found that many secondary metabolites derived from Streptomyces are very
important for the treatment of various diseases1-4. In 2002, Zeeck and his colleagues
proposed the concept of “OSMAC” (one strain/many compounds), which emphasizes
that primary and secondary metabolites can be produced by changing culture
conditions, adoption of co-culture and other methods5,6. According to the literature,
Streptomyces parvus could produce actinomycin D7 and a daptomycin analogue8 (Fig.
1). Valistatin and bestatin have been reported to be produced by Streptomyces sp.
SL202099-11 and Streptomyces olivoreticuli FERM-P no. 2590 (ATCC31159)12,13
,
respectively. Streptomyces parvus HCCB10043 is stored in the China General
Microbiological Culture Collection Center (CGMCC no. 4027). Culture extracts of
Streptomyces parvus HCCB10043 had high antibacterial activity against Gram-

positive bacteria in previous studies14,15
.
O
H3C
C Thr-Dval-Pro-MeGly-MeVal
O
O
N
H3C
C Thr-Dval-Pro-MeGly-MeVal
O
NH2
Actinomycin D
NH2
O
H
N
O
NH2
O
H
O
N
N
H
OH
NH
O
NH
O
H
O
O
O
N
O
N
H
N
H
O
NH
OH
HN
O
O
O
O
OH
O
HN
NH
O
O
O
O
NH2
NH
N
H
O
HO
OH
Daptomycin
Fig. 1. Chemical structures of Actinomycin D and Daptomycin
A new compound isolated from Streptomyces parvus HCCB10043 was reported
2011. Its structure was identified as 3-amino-2-hydroxy-4-
in
phenylbutanoylvalylisoleucine (1), also known as LYRM03, a three-peptide
compound consisting of one non-natural amino acid and two natural amino acids (Fig,
2), which was found to exhibit greater bioactivity than valistatin or bestatin in an
aminopeptidase N (APN) inhibition assay14. However, the content of the compound in
a bacterial fermentation broth is very low (about 20 mg/L). Thus, this method does
not supply sufficient material for larger scale activity testing and drug development.
Therefore, a synthetic approach is urgently needed to improve the availability of this
compound to support its characterization.

H2N
O
H
N
NH2
O
OH
O
OH
O
HN
N
O
O
NH
H
OH
NH
HO
3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine(1)
O
NH2
O
OH
Valistatin
Bestatin
Fig. 2. Chemical structures of 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine (1), valistatin
and bestatin
APN, also known as CD13, is a membrane-associated enzyme that catalyzes the
cleavage of amino acids from the N-terminus of proteins or peptides16. APN
expression was detected in many different human cells (including macrophages,
stromal cells, smooth muscle cells and fibroblasts17) and overexpression of APN in
miscellaneous cancers was shown, such as breast18, ovarian19, pancreatic20, and
colorectal21,22. In 2012, Liliana and her colleagues published an article concluding that
APN played an important role in cancer metastasis23. Additionally, targeting APN
was verified to lead dephosphorylation of EPHA2, a mediator of BRAF inhibitor
resistance, and induces growth arrest in melanoma cells24. Taken together, these
observations support the idea that APN may serve as a therapeutic target for treatment
of various cancers25-27
.
Herein, we described a chemical synthetic approach to produce sufficient
amounts of LYRM03 to support mechanistic studies, which will help to further our
understanding of how APN contributes to cancer development. Furthermore, we
found that LYRM03 inhibited the enzymatic activity of APN leading to restrained
migration and metastasis of a human breast cancer cell line MDA-MB-231 in vitro
and in vivo.
Results and Discussion
Chemical synthesis of LYRM03
Peptides were generally synthesized using an amino acid condensation reaction. First,
coupling of L-isoleucine benzyl ester 4-toluenesulphonate (2) with N-Boc-Val-OH (3)
by means of TBTU and DIEA in DCM at 0°C gave the protected dipeptide (4). The

N-Boc dipeptide was then deprotected using dry hydrogen chloride in ethyl acetate to
produce the dipeptide hydrochloride (5), which was easily purified by reslurrying in
ethyl acetate at 35°C and isolation by filtration. Next, condensation of the amines with
a common optically active precursor, (2S,3R)-3-[(N-t-butoxycarbonyl) amino]-2-
hydroxy-4-phenylbutanoic acid (7), easily obtained from (2S, 3R)-3-amino-2-
hydroxy-4-phenylbutanoic acid by means of TBTU and DIEA in DCM at 0°C, gave
the tripeptide derivative (8). The N-Boc tripeptide was then deprotected using dry
hydrogen chloride in ethyl acetate to produce the tripeptide hydrochloride (9). This
was also easily purified by reslurrying in ethyl acetate to obtain the tripeptide
hydrochloride (9) as a white solid. The purity of the product from this step by HPLC
was over 99.5%. Finally, the target product was obtained by hydrogenation of
palladium to remove the benzyl group (Fig. 3).
O
O
O
O
O
Boc
a
b
O
NH
O
NH
OH
O
HN
+
NH2
TosOH
O
3
NH2
HCl
NH
Boc
2
4
5
+
Boc
NH2
O
NH
O
c
OH
OH
OH
OH
7
6
d
Boc
NH
O
NH2
O
O
O
NH2
O
N
H
N
H
O
OH
NH
OH
NH
N
H
f
e
OH
NH
HO
O
O
O
O
O
1
9
8
Fig. 3 a) TBTU, DIEA, DCM, 0°C. b) HCl, EA, rt, 82.7 % (for two steps); c) (Boc)2O, NaOH,
THF/H2O, rt, 99 %; d) TBTU, DIEA, DCM, 0°C, 87.4%; e) HCl, EA, rt, 96.7%; f) Pd/C, H2,
MeOH, rt, 97.8 %.

By following the synthetic method described above, we prepared 100 g of
LYRM03. The structure of the product was determined by NMR and confirmed to be
in agreement with the standard NMR spectra previously reported14 (Table 1). Using
this material, we studied the bioactivity of the compound as described below.
Table 1 NMR data of compound 1
Group
Ile
1H NMRa
1HNMRb
13C NMRa
174.4
58.2
13C NMRb
173.18
57.31
qc
2``-CH
3``-CH
4.29 (d, J=6.0)
4.15 (d, J=6.0)
1.84 (m)
1.75(s)
38.2
36.54
4``-CH2 1.47 (m), 1.21 (m) 1.44-1.39 (m)
26.2
24.8
1.14-1.08 (m)
5``-CH3 0.87 (dd, J=6.8, 0.85-0.80 (m)
16.0
11.8
15.56
11.44
1.44)
6``-CH3 0.84 (dd, J=6.8, 0.88 (d, J=6.5)
1.44)
qc
Val
173.5
60.1
31.9
172.05
57.31
31.02
2`-CH
3`-CH
4.19 (d, J=8.0)
4.01 (d, J=6.0)
2.10 (dd, J=5.2, 2.09-2.05 (m)
13.2)
4`-CH3
5`-CH3
0.97 (t, J=6.8)
0.95 (t, J=6.8)
0.85-0.80 (m)
0.85-0.80 (m)
19.6
18.8
173.2
69.6
56.3
19.21
17.89
170.45
71.10
57.16
36.54
AHPA qc
2-CH
4.09 (d, J=3.6)
3.90 (d, J=2.5)
3-CH
3.72 (m)
3.35-3.34 (m)
4-CH2
3.07 (dd, J=7.8, 2.69 (dd, J=13.5, 36.5
13.5) 7.5)
2.89 (dd, J=7.8, 2.85 (dd, J=13.5,
13.5)
7.5)
4a-qc
136.6
130.5
130.1
128.6
130.1
130.5
138.64
129.25
128.24
126.13
128.24
129.25
4b-CH
4c-CH
4d-CH
4e-CH
4f-CH
7.25 (m)
7.35 (m)
7.24 (m)
7.35 (m)
7.25 (m)
7.29-7.21(m)
7.32-7.30(m)
7.29-7.21(m)
7.32-7.30(m)
7.29-7.21(m)
a NMR data from reference 14
b NMR data from this article
Anti-metastasis activity of LYRM03
LYRM03 inhibited APN enzymatic activity of MDA-MB-231 cells without
affecting its expression.

Breast cancer causes the majority of deaths in women who have cancer28. Since APN
expression in breast tumors was confirmed18 and LYRM03 is a derivative of bestatin29,
a APN inhibitor approved in Japan, it was of interest to determine if LYRM03 can
inhibit the APN enzymatic activity in MDA-MB-231 cells. As expected, LYRM03
suppressed the enzymatic activity of APN by more than 50% at concentrations greater
than 50 μg/ml, which was better than bestatin (Fig. 4A). To exclude any discrepancy
in the results attributed to the APN expression level, the protein level of APN in
MDA-MB-231 cells was determined using western blot. As shown in Fig. 4B, there
was no difference in APN expression between cells treated with or without LYRM03.
These results are consistent with a previous report where incubation with bestatin did
not result in a change in the protein level of APN in renal cell carcinoma cells30.
Fig. 4 APN enzymatic activity of MDA-MB-231 cells was inhibited by LYRM03. A) The
overnight cultured cells were collected and treated with the indicated concentrations of LYRM03
and bestatin. After 1 h incubation, the supernatants were measured and calculated. P < 0.05 was
considered as significant. B) The cells were cultured under different concentrations of LYRM03
and then lysed with RIPA buffer. The CD13 level was assessed by western blot and blots were
clipped with Photoshop CS6. Tubulin was used as the loading control.
LYRM03 induced minor cell death of the human breast cancer cell line MDA-

MB-231 but not apoptosis.
According to previous reports31,32, APN is associated with tumor growth. Thus in this
work, we examined the cell viability and apoptosis in MDA-MB-231 cells under
treatment with LYRM03 or bestatin. However, LYRM03 caused no more than 20%
cell death in MDA-MB-231 cells at 24 h and 48 h, even at a concentration of 100
μg/mL (Fig. 5A). Similar to LYRM03, bestatin did not cause cell death of MDA-MB-
231 except that when the cells were exposed to bestatin for 48 h at 100 μg/mL (data
not shown).
A previous report demonstrated that NGR-LDP-AE, an APN targeting peptide,
displayed cytotoxicity toward human liver cancer cells32. To test whether LYRM03
could induce apoptosis of MDA-MB-231 cells, we carried out western blot and flow
cytometry analyses. We found that no significant apoptosis was detected at the
indicated concentrations and time points compared with untreated cells (Fig. 5B-C).
These results indicated that LYRM03 was not significantly cytotoxic to MDA-MB-
231 cells.
Fig. 5 LYRM03 did not induce apoptosis of MDA-MB-231 cells. (A-C) The cells were exposed
to various concentrations of LYRM03 for 6, 12, 24, and 48 h. Cell viability was analyzed by MTT
assay. Apoptosis was predicted by cleaved PARP and Annexin V / PI using western blot and flow
cytometry, respectively.
LYRM03 reduced migration in vitro and lung metastasis in vivo of MDA-MB-231

cells.
The human cancer cell line MDA-MB-231 was investigated as a common metastatic
cell model33,34 that was found to express APN (Fig. 4B). APN might also influence
cell motility because of its association with tumor-associated antigen L6 in human
lung cancer cells35. Thus on the basis of these observations, we performed a series of
experiments to study the association between LYRM03 and metastasis in MDA-MB-
231 cells and found that the migration ability of MDA-MB-231 cells was inhibited by
LYRM03 in a dose-dependent manner (Fig. 6). As shown in more details, the scratch
healed about 50% in MDA-MB-231 cells after 24 h incubation, whereas cells treated
with LYRM03 migrated much more slowly, especially in the 100 μg/mL group (Fig.
6A). In line with the wound healing assay result, we drew the same conclusions from
the colony formation assay and transwell experiment. The number of colonies was
less in the LYRM03 treated groups (Fig. 6B). Additionally, there were fewer cells
migrating through the transwell membrane when the cells were exposed to LYRM03
(Fig. 6C). By means of these three different methods, we ascertained that LYRM03 is
associated with the motility of MDA-MB-231 cells and this association may rely on
its inhibitory function of APN enzymatic activity (Fig. 4A).
To verify the results in vivo, female SCID mice were used to establish a lung
metastasis model of MDA-MB-231 cells. Compared with normal lungs, lungs from
mice injected with cells exhibited metastasis foci (Fig 6D). By comparison, much
fewer metastasis foci were found in lungs from LYRM03 and bestatin injected mice.
Surprisingly, LYRM03 performed better at inhibiting the lung metastasis of MDA-
MB-231 cells (Fig. 6D). These findings are supported by a previous study revealing
that APN could affect lung metastatic foci formation23. In conclusion, LYRM03 not
only suppressed migration in vitro, but also inhibited lung metastasis in vivo in the
human breast cancer cell line MDA-MB-231.

Fig. 6 LYRM03 impairs the metastasis of MDA-MB-231 cells in vitro and in vivo. A) The cells
were treated with the indicated concentrations of LYRM03 and the photos were obtained at 0, 6,
12, and 24 h. The comparative distances of the scratches are summarized as a histogram (mean±
SD). B) One hundred cells were exposed to LYRM03 for 10 days. After fixation and staining, the
colonies are shown. C) Migration of MDA-MB-231 cells during LYRM03 treatment was
performed using a transwell model. The numbers of migrated cells were quantified and are shown
as a histogram (mean±SD). D-E) Lungs from female SCID mice administered with and without
LYRM03 or bestatin are shown in D (4 of 6 mice per group). The representative images of lungs
stained with hematoxylin-eosin are displayed in E. The black arrows represent metastatic nodules.
Breast cancer is regarded as the most common cancer type occurring in women
worldwide. Of the various forms of breast cancer, the subtype lacking the expression

of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal
growth factor receptor 2 (HER2), termed as triple-negative breast cancer (TNBC), is
found in about 15% of cases. Because of the absence of therapeutic targets,
chemotherapy is still the only choice for the treatment of TNBC patients, which may
be one of the reasons why the median survival is less than one year36. Metastasis also
contributes to the low median survival in TNBC patients. Thus, we explored the
cytotoxicity and anti-metastasis potency of LYRM03 synthesized in this study in a
TNBC cell line MDA-MB-231. Even though the LYRM03 did not cause apoptosis of
the MDA-MB-231 cells, the metastatic ability of this cell type was seriously inhibited
by LYRM03 in vitro and in vivo. These results indicate that the synthesized LYRM03
may be used in the early stage of treatment of TNBC patients combined with other
chemotherapy agents.
The successful metastasis needs several steps, failure to complete any of which
will terminate the process37. There are plenty of factors involved in metastasis. Firstly,
tumor cells must invade surrounding tissues through modifying cell-to-cell adhesion,
in which cadherin family plays an important role38. For example, cadherin-6 which is
a structural protein and associated with morphogenesis of central nervous system and
kidney39-41, restrained cancer metastasis by inhibiting autophagy42. Metalloproteinases
(MMPs) are thought to be participants in breast cancer metastasis for a long time43.
However, no significant alteration of MMP2, MMP7 and MMP9 was found in MDA-
MB-231 cells treated with or without LYRM03 (data not shown). Thus, there must be
another mechanism between APN and metastasis in TNBC, which will be of interest
to investigate the mRNA profile when the cells are stimulated with LYRM03 in the
future.
Acknowledgments
This study was funded by National Key Basic Research Program of China
(2015CB931800) and MHHFDU-SPFDU Joint Research Fund (RO-MY2018**). We
thank Renee Mosi, PhD, from Liwen Bianji, Edanz Group China
(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

References
1
Jeong, S. Y. et al. Apoptosis induction of human leukemia cells by Streptomyces sp. SY-103
metabolites through activation of caspase-3 and inactivation of Akt. International journal of
molecular medicine 25, 31-40 (2010).
2
3
Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs over the last 25
years. Journal of natural products 70, 461-477 (2007).
Martinez-Montiel, N., Rosas-Murrieta, N. H., Martinez-Montiel, M., Gaspariano-Cholula, M.
P. & Martinez-Contreras, R. D. Microbial and Natural Metabolites That Inhibit Splicing: A
Powerful Alternative for Cancer Treatment. BioMed research international 2016, 14 pages
(2016).
4
5
Leiros, M. et al. The Streptomyces metabolite anhydroexfoliamycin ameliorates hallmarks of
Alzheimer's disease in vitro and in vivo. Neuroscience 305, 26-35 (2015).
Onaka, H., Mori, Y., Igarashi, Y. & Furumai, T. Mycolic acid-containing bacteria induce
natural-product biosynthesis in Streptomyces species. Applied and environmental
microbiology 77, 400-406 (2011).
6
Bode, H. B., Bethe, B., Hofs, R. & Zeeck, A. Big effects from small changes: possible ways to
explore nature's chemical diversity. Chembiochem : a European journal of chemical biology 3,
619-627 (2002).
7
Hitchcock, M. J. & Katz, E. Actinomycin biosynthesis by protoplasts derived from
Streptomyces parvulus. Antimicrobial agents and chemotherapy 13, 104-114 (1978).
Shuang, Z. et al. Preliminary identification of Streptomyces sp. DT-08 producing a novel
lipopeptide antibiotic. J China Pharm Univer 40, 94-96 (2009).
8
9
Nagai, M. et al. Phebestin, a new inhibitor of aminopeptidase N, produced by Streptomyces sp.
MJ716-m3. The Journal of antibiotics 50, 82-84 (1997).
10
Rao, M., Feng, L., Ruan, L., Ge, M. & Sheng, X. UPLC-MS-Based Metabolomic Study of
Streptomyces Strain HCCB10043 Under Different pH Conditions Reveals Important
Pathways Affecting the Biosynthesis of A21978C Compounds. Analytical Letters 46, 2305-
2318 (2013).
11
Nguyen, J. T. et al. Design and synthesis of several small-size HTLV-I protease inhibitors
with different hydrophilicity profiles. Bioorganic & medicinal chemistry letters 21, 2425-2429
(2011).
12
13
Umezawa, H., Aoyagi, T., Suda, H., Hamada, M. & Takeuchi, T. Bestatin, an inhibitor of
aminopeptidase B, produced by actinomycetes. The Journal of antibiotics 29, 97-99 (1976).
Tello-Aburto, R., Hallada, L. P., Niroula, D. & Rogelj, S. Total synthesis and absolute
stereochemistry of the proteasome inhibitors cystargolides A and B. Organic & biomolecular
chemistry 13, 10127-10130 (2015).
14
15
Rao, M. et al. A new aminopeptidase inhibitor from Streptomyces strain HCCB10043 found
by UPLC-MS. Analytical and bioanalytical chemistry 401, 699-706 (2011).
Rao, M., Wei, W., Ge, M., Chen, D. & Sheng, X. A new antibacterial lipopeptide found by
UPLC-MS from an actinomycete Streptomyces sp. HCCB10043. Natural product research 27,
2190-2195 (2013).
16
Taylor, A. Aminopeptidases: structure and function. FASEB journal : official publication of
the Federation of American Societies for Experimental Biology 7, 290-298 (1993).

17
18
Dixon, J. et al. Expression of aminopeptidase-n (CD 13) in normal tissues and malignant
neoplasms of epithelial and lymphoid origin. Journal of clinical pathology 47, 43-47 (1994).
Ranogajec, I., Jakic-Razumovic, J., Puzovic, V. & Gabrilovac, J. Prognostic value of matrix
metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and aminopeptidase
N/CD13 in breast cancer patients. Medical oncology (Northwood, London, England) 29, 561-
569 (2012).
19
20
Surowiak, P. et al. Expression of aminopeptidase N/CD13 in human ovarian cancers.
International journal of gynecological cancer : official journal of the International
Gynecological Cancer Society 16, 1783-1788 (2006).
Ikeda, N. et al. Clinical significance of aminopeptidase N/CD13 expression in human
pancreatic carcinoma. Clinical cancer research : an official journal of the American
Association for Cancer Research 9, 1503-1508 (2003).
21
22
Hashida, H. et al. Aminopeptidase N is involved in cell motility and angiogenesis: its clinical
significance in human colon cancer. Gastroenterology 122, 376-386 (2002).
Xu, H., Sun, J. & Zhang, Z. CD13 overexpression promotes rectal cancer cell proliferation by
increasing VEGF expression. International journal of clinical and experimental medicine 11,
4688-4694 (2018).
23
24
25
Guzman-Rojas, L. et al. Cooperative effects of aminopeptidase N (CD13) expressed by
nonmalignant and cancer cells within the tumor microenvironment. Proc Natl Acad Sci U S A
109, 1637-1642 (2012).
Azimi, A. et al. Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of
EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in
melanoma cells. 8, e3029 (2017).
Kessler, T. et al. Aminopeptidase N (CD13): Expression, Prognostic Impact, and Use as
Therapeutic Target for Tissue Factor Induced Tumor Vascular Infarction in Soft Tissue
Sarcoma. Translational oncology 11, 1271-1282 (2018).
26
27
Schmidt, L. H. et al. CD13 as target for tissue factor induced tumor vascular infarction in
small cell lung cancer. Lung cancer (Amsterdam, Netherlands) 113, 121-127 (2017).
Carrascal, M. A. et al. A functional glycoproteomics approach identifies CD13 as a novel E-
selectin ligand in breast cancer. Biochimica et biophysica acta. General subjects 1862, 2069-
2080 (2018).
28
29
Scully, O. J., Bay, B. H., Yip, G. & Yu, Y. Breast cancer metastasis. Cancer genomics &
proteomics 9, 311-320 (2012).
Hui-qiong et al. LYRM03, an ubenimex derivative, attenuates LPS- induced acute lung injury
in mice by suppressing the TLR4 signaling pathway. Acta Pharmacological Sinica 38, 342-
350 (2017).
30
Kehlen, A., Göhring, B., Langner, J. & Riemann, D. Regulation of the expression of
aminopeptidase A, aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 in renal
carcinoma cells and renal tubular epithelial cells by cytokines and cAMP-increasing mediators.
Clinical and experimental immunology 111, 435-441 (1998).
31
32
Zheng, Y. B., Gong, J. H., Liu, X. J., Li, Y. & Zhen, Y. S. A CD13-targeting peptide
integrated protein inhibits human liver cancer growth by killing cancer stem cells and
suppressing angiogenesis. Molecular Carcinogenesis 56 (2017).
Pasqualini, R. et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target

for inhibiting angiogenesis. Cancer research 60, 722-727 (2000).
33
34
35
Zhu, S. et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell
Research 18, 350-359 (2008).
Chen, Y. et al. ZMYND8 acetylation mediates HIF-dependent breast cancer progression and
metastasis. Journal of Clinical Investigation, 1937-1955 (2018).
Chang, Y. W. et al. CD13 (aminopeptidase N) can associate with tumor-associated antigen L6
and enhance the motility of human lung cancer cells. International Journal of Cancer 116,
243–252 (2005).
36
Abramson, V. G., Lehmann, B. D., Ballinger, T. J. & Pietenpol, J. A. Subtyping of triple-
negative breast cancer: implications for therapy. Cancer 121, 8-16 (2015).
Poste, G., . & Fidler, I. J. The pathogenesis of cancer metastasis. Nature 283, 139-146 (1980).
Dong-Mei, L. & Yu-Mei, F. Signaling mechanism of cell adhesion molecules in breast cancer
metastasis: potential therapeutic targets. Breast Cancer Res Treat 128, 7-21 (2011).
Inoue, Y. U., Asami, J. & Inoue, T. Cadherin-6 gene regulatory patterns in the postnatal
mouse brain. Molecular & Cellular Neurosciences 39, 95-104 (2008).
Cho, E. A. et al. Differential expression and function of cadherin-6 during renal epithelium
development. Development (Cambridge, England) 125, 803-812 (1998).
Inoue, T., Chisaka, O., Matsunami, H. & Takeichi, M. Cadherin-6 expression transiently
delineates specific rhombomeres, other neural tube subdivisions, and neural crest
subpopulations in mouse embryos. Dev Biol 183, 183-194 (1997).
37
38
39
40
41
42
43
Gugnoni, M. et al. Cadherin-6 promotes EMT and cancer metastasis by restraining autophagy.
Oncogene 36, 667 (2016).
Kelly, T. et al. Proteolysis of extracellular matrix by invadopodiafacilitates human breast
cancer cell invasion and ismediated by matrix metalloproteinases. Clinical & Experimental
Metastasis 16, 501 (1998).
Material and Methods
Chemical synthesis
General. Unless otherwise mentioned, all commercial reagents and solvents were
used directly as purchased. Flash chromatography was performed using silica gel with
petroleum ether/ethyl acetate or dichloromethane/methanol as the eluent. Melting
points were uncorrected. Optical rotations were measured with a sodium lamp. NMR
spectra were recorded on a spectrometer at 500 MHz (1H NMR), 126 MHz (13C
NMR). Mass spectrometry data were recorded on a high-resolution mass spectrometer
in the electrospray ionization (ESI) mode. Chemical shifts (δ) are reported in parts per
million and referenced to the residual solvent peak, and J values are given in hertz
(Hz).
Preparation of (2S,3R)-benzyl-2-{(S)-2-[(tert-butoxycarbonyl) amino]-3-methyl-

butanamido]-3-methylpentanoate (4)
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (24.5g, 76.2
mmol) was added to a mixture of (2S,3R)-benzyl 2-amino-3-methylpentanoate 4-
methylbenzenesulfonate (30 g, 76.2 mmol) and (S)-2-[(tert-butoxycarbonyl)- amino]-
3-methylbutanoic acid (16.6g, 76.2mmol) in dichloromethane at room temperature.
Then N,N-Diisopropylethylamine (17.5 ml, 190.5 mmol) was added dropwise to this
o
o
solution at 0 C. The reaction mixture was stirred at 0 C for 2 h until completion of
the reaction. the solution was successively with water, 1% solution phosphoric acid,
water, 2 % solution Potassium carbonate and with water again. The organic layer was
dried and evaporate to give the crude product 4 (27.7 g) as an oil, which was used to
next step without further purification.
Preparation of (2S,3R)-benzyl-2-[(S)-2-amino-3-methylbutanamido]-3-methyl-
pentanoate hydrochloride (5)
To a solution of 4 (27.7 g, 65.9 mmol) in ethyl acetate (200 ml) was purged with dry
o
hydrogen chloride to saturate the solution at 0 C, the reaction mixture was stirred at
room temperature for 12 h until completion of the reaction. The precipitate was
isolated by filtration, washed with ethyl acetate and dried to afford compound 5 as a
white solid (22.5 g), the two step total yields: (82.7 %), purity: 98 % (HPLC).1H
NMR (500 MHz, DMSO) δ 8.66 (d, J = 8 Hz, 1H), 8.15 (s, 3H), 7.38 – 7.34 (m, 5H),
5.14 (d, J = 1.5 Hz, 2H), 4.3 (t, J = 11.5 Hz, 1H), 3.70 (s, 1H), 2.05 – 2.03 (m, 1H),
1.84 – 1.82 (m, 1H), 1.40 – 1.38 (m, 1H), 1.23 – 1.21 (m, 1H), 0.91 – 0.82 (m, 12H).

13C NMR (126 MHz, CDCl3) δ 170.68, 168.26, 135.65, 128.37, 128.22, 128.17, 66.05,
56.79, 56.75, 36.18, 29.84, 24.67, 18.16, 17.61, 15.39, 11.22.
Preparation of (2S,3R)-3-[(tert-butoxycarbonyl) amino]-2-hydroxy-4-phenyl-
butanoic acid (7)
(Boc)2O (12.3 g, 56.3 mmol) was added to a solution of (2S,3R)-3-amino-2-hydroxy-
4-phenylbutanoic acid (10 g, 51.2 mmol) in H2O (100 ml) and THF (50 ml) at room
temperature, then Sodium hydroxide solution (128 ml, 1mol/L) was added dropwise
to the mixture at 0 oC. The reaction mixture was stirred at room temperature for 24 h
until completion of the reaction. The pH of the solution was adjusted to1 by adding
saturated Citric acid monohydrate solution, and the mixture was extracted with ethyl
acetate (3× 200ml). The combined organic phases are washed with water, dried over
anhydrous magnesium sulphate, filtered, the solvent was removed under vacuum, and
the residue was further purified by flash column chromatography on silica gel
(DCM/MeOH = 20/1) to afford compound 7 as a white solid (15 g), yield: 99 %,
purity: 99 % (HPLC).1H NMR (500 MHz, DMSO) δ 7.45 – 7.05 (m, 5H), 6.53 (s, 1H),
4.99 (s, 1H), 4.12 (d, J = 44 Hz, 2H), 2.99 – 2.95 (m, 2H), 1.37 (s, 9H). 13C NMR
(126 MHz, CDCl3) δ 158.07, 137.90, 129.52, 129.38, 128.58, 126.59, 81.66, 70.95,
69.72, 56.25, 54.56, 38.38, 38.11, 28.09.
Preparation of (6R,7S,10S,13S)-benzyl-6-benzyl-13-[(R)-sec-butyl]-7-hydroxy-
10-isopropyl-2,2-dimethyl-4,8,11-trioxo-3-oxa-5,9,12-triazatetradecan-14-oate (8)
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (16.1 g, 50.1

mmol) was added to a mixture of 7 (15 g, 50.1 mmol) and 5 (18.1 g, 50.1 mmol) in
dichloromethane at room temperature. Then N,N-Diisopropylethylamine (11.5 ml,
o
125.3 mmol) was added dropwise to this solution at 0 C. The reaction mixture was
o
stirred at 0 C for 2 h until completion of the reaction. the solution was successively
with water, 1% solution phosphoric acid, water, 2 % solution Potassium carbonate
and with water again. The organic layer was dried over anhydrous magnesium
sulphate, filtered, the solvent was removed under vacuum, and the residue was further
purified by flash column chromatography on silica gel (DCM/MeOH = 60/1) to afford
1
compound 8 as a white solid (26.2 g), yield: 87.4 %, purity: 99 % (HPLC). H NMR
(500 MHz, DMSO) δ 7.47 (s, 1H), 7.36-7.33 (m, 5H), 7.31-7.28 (m, 2H), 7.26-7.21
(m, 3H), 6.48 (s, 1H), 5.86 (s, 1H), 5.20 (d, J=10 Hz, 1H), 5.10 (d, J=10 Hz, 1H), 5.04
(d, J=5 Hz, 1H), 4.62 (dd, J= 10, 5 Hz, 1H), 4.20 (t, J=6 Hz, 1H), 4.15 (dd, J= 6, 2.5
Hz, 1H), 3.96 (s, 1H), 3.22-3.18 (m, 1H), 3.05-3.02 (m, 1H), 2.19-2.15 (m, 1H), 1.93-
1.87 (m, 1H), 1.40-1.32 (m, 11H), 1.13-1.11 (m, 1H), 0.91 (dd, J= 15, 5 Hz, 6H),
0.86-0.84 (m, 6H). 13C NMR (126 MHz, DMSO) δ 172.89, 171.51, 171.0, 157.43,
138.16, 135.24, 129.34, 128.57, 128.49, 128.46, 128.41, 126.53, 80.35, 74.26, 67.03,
58.40, 56.57, 55.72, 37.72, 36.24, 20.86, 28.22, 25.03, 19.32, 18.15, 15.49, 11.53.
Preparation of (2S,3R)-benzyl-2-{(S)-2-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-
butanamido]-3-methylbutanamido}-3-methylpentanoate (9)
To a solution of 8 (26.2 g, 43.8 mmol) in ethyl acetate (200 ml) was purged with dry
o
hydrogen chloride to saturate the solution at 0 C, the reaction mixture was stirred at
room temperature for 12 h until completion of the reaction. The solid was isolated by
filtration, washed with ethyl acetate, the solid was dissolved in H2O (200 ml), the pH
of the solution was adjusted to 11 by adding ammonium hydroxide and extracted with
ethyl acetate (3× 200ml). The combined organic phases are washed with water, dried
over anhydrous magnesium sulphate, filtered, the solvent was removed under vacuum

to afford compound 9 as a white solid (21.1 g), yield: 96.7 %, purity: 99.5 %
(HPLC).1H NMR (500 MHz, DMSO) δ 7.94 (d, J=9 Hz, 1H), 7.36-7.30 (m, 7H),
7.26-7.23 (m, 3H), 6.53 (d, J=9 Hz, 1H), 5.21 (d, J=12 Hz, 1H), 5.12 (d, J=12 Hz, 1H),
4.65 (dd, J= 8.5, 4.5 Hz, 1H), 4.21 (t, J=7.5 Hz, 1H), 3.96 (d, J=2.5 Hz, 1H), 3.62 (m,
1H), 2.97 (dd, J= 13.5, 4.5 Hz, 1H), 2.97 (dd, J=13.5, 10.5 Hz, 1H), 2.53-2.48 (m, 1H),
2.20-2.19 (m, 1H), 1.91-1.80 (m, 1H), 1.40-1.28 (m, 2H), 1.21-1.12 (m, 1H), 0.95-
0.92 (m, 6H), 0.87-0.83 (m, 6H). 13C NMR (126 MHz, DMSO) δ 173.52, 171.53,
170.79, 138.30, 13.5.24, 129.27, 128.73, 128.59, 128.47, 128.42, 126.68, 72.63, 67.06,
59.12, 56.51, 54.12, 38.42, 37.80, 30.53, 24.99, 19.35, 18.23, 15.54, 11.53. MS (ESI)
m/z: 498.2977[M + H]+.
Preparation of 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine (1)
9 (21.1 g, 42.4 mmol) was mixed with palladium on carbon (2.1g, 10 % palladium,
50 % water) and methanol (300 ml), this mixture was treated with hydrogen until the
reaction was deemed to be complete. The catalyst was removed by filtration and
washed methanol, the solvent was removed under vacuum to afford compound 1 as a
white solid (16.9 g), yield: 97.8 %, purity: 99.5 % (HPLC).1H NMR (500 MHz,
DMSO+D2O) δ 7.32-7.30 (m, 2H), 7.29-7.21 (m, 3H), 4.15 (d, J= 6.0 Hz, 1H), 4.01
(d, J= 6.0 Hz, 1H), 3.90 (d, J=2.5 Hz, 1H), 3.35-3.34 (m, 2H), 2.85 (dd, J= 13.5, 7.5
Hz, 1H), 2.69 (dd, J= 13.5, 7.5 Hz, 1H), 2.09-2.05 (m, 1H), 1.75 (s, 1H), 1.44-1.39 (m,
13
1H), 1.14-1.08 (m, 1H), 0.88 (d, J=6.5 Hz, 3H), 0.85-0.80 (m, 9H). C NMR (126
MHz, DMSO) δ 173.18, 172.05, 170.45, 138.64, 129.25, 128.24, 126.13, 71.10, 57.31,
57.16, 36.54, 31.02, 24.80, 19.21, 17.89, 15.56, 11.44. MS (ESI) m/z: 408.2501[M +
H]+.

Anti-metastasis analysis
Cells and reagents
LYRM03 was synthesized as described in this work. The human breast cancer cell
line MDA-MB-231 was purchased from the Cell Bank of Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The primary
antibodies for PARP, α-tubulin, APN and secondary antibody were from Cell
Signaling Technology (Danvers, Massachusetts, USA). The annexin V-FITC/PI
apoptosis detection kit is a product of KeyGEN BioTECH (Jiangsu, China). The L-
leucine-p-nitroanilide was over 98% purity and purchased from Sigma-Aldrich
Corporation (Missouri, USA). The MTT reagent and bestatin powder were acquired
from Meilun Biotechnology (Dalian, China).
Western blot
The MDA-MB-231 cells stimulated with LYRM03 at the indicated concentrations
and time points were lysed in RIPA buffer (Beyotime, Shanghai, China) containing 1
mM PMSF. The lysates were mixed with 5×loading buffer (Meilun Biotechnology,
Dalian, China) and boiled for 10 min. Then equal amounts of proteins were applied to
12% SDS-PAGE gels, transferred to a PVDF membrane (Merck Millipore,
Massachusetts, USA) and incubated with shaking in 5% non-fat milk at 37°C for 2 h.
After washing, different primary antibodies were added and the membranes were
incubated overnight at 4 °C. After incubation with secondary antibody, protein bands
were detected using a chemiluminescence substrate (ThermoFisher Scientific,
Massachusetts, USA) and imaged by Image Lab software (Bio-Rad, California, USA).
Cell viability
The human breast cancer MDA-MB-231 cells were seeded in 96-well plates and
incubated overnight. Then LYRM03 was added with indicated concentrations. After
certain times, cells were exposed to MTT for 4 h at 37°C. To dissolve the formazan,
the supernatant was discarded and 150 μL DMSO were added to each well. After
shaking for several minutes, the absorbance was measured at 570 nm using a
MultiskanTM FC microplate reader (ThermoFisher Scientific).

Flow cytometric analysis
MDA-MB-231 cells stimulated with LYRM03 were stained with FITC-conjugated
Annexin V and PE-conjugated PI in accordance with the manufacturer’s instructions.
The samples were analyzed on a CytoFlex S flow cytometer (Beckman Coulter,
California, USA).
Inhibition of APN activity
The APN enzymatic activity was determined spectrophotometrically using L-leucine-
p-nitroanilide as a substrate. First, 0.6 million cells (210 μL), 60 μL LYRM03 or
bestatin and 30 μL substrate were added successively into a 1.5 mL tube and mixed
gently. Tubes without cells or compound were chosen as the blank and control,
respectively. Then the samples were incubated at 37°C for 1 h. APN enzymatic
activity was measured at 405 nm. The inhibition ratios of LYRM03 and bestatin were
calculated with the formula:
Inhibition ratio (%) = (ODcontrol – ODcompound) / (ODcontrol - ODblank) * 100%
In vitro migration assays
Wound healing assay and transwell model were applied to conduct the cell migration
assay in vitro. Briefly, MDA-MB-231 cells were seeded in a 6-well plate for the
wound healing assay (1 × 106 cells/well) and the upper chamber of a transwell plate (5
× 104 cells/chamber). For the wound healing assay, 10 μL pipette tips were used to
scratch the wound. Then LYRM03 or bestatin at the indicated concentrations were
added and incubated at 37°C for 6, 12, and 24 h (wound healing assay) and 24 h
(transwell). At each time point, images were acquired using a microscope.
Colony formation assay
Two hundred cells were counted and planted in a 6-well plate followed by overnight
incubation. After washing with PBS, cells were stimulated with different
concentrations of LYRM03 for 10 days. The colonies were recorded and the images
were saved as photos.
Animal study
The animal study was performed as previously described24. Briefly, 1×106 cells in 100
μL PBS were injected through the tail vein of SCID mice. LYRM03 and bestatin were

injected intraperitoneally in 100 mg/kg for 3 times a week. Three weeks later, the
mice were killed and the lungs were collected and fixed in 4% paraformaldehyde for
several days. Then the samples were analyzed by hematoxylin-eosin staining.
Statistics
All data are presented as the mean±SD. Significance analyses were performed by the
two-tailed Student’s t test. A P < 0.05 was defined as significant (*P < 0.05, **P <
0.01, ***P < 0.001 versus control).
Highlights
1. This work applied a chemical synthesis strategy to generate LYRM03 for the first time.
2. The chemically synthesized LYRM03 overcomes the low yields typically achieved from
fermentation.
3. We firstly found that the metastasis of human breast cancer MDA-MB-231 cells was
restrained by LYRM03.