Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain

Article abstract of DOI:10.1016/j.bmcl.2011.12.079

Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10⁴ M^-1 s^-1 and IC₅₀ values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.

Full text of DOI:10.1016/j.bmcl.2011.12.079

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1340–1343
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal
asparaginyl endopeptidase, legumain
Jiyoun Lee a,b, , Matthew Bogyo a,
⇑
⇑
a Department of Pathology and Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Dr. Stanford, CA 94305-5324, USA
b Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Hawolgok-dong 39-1, Seongbuk-gu, Seoul 136-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of spec-
ificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after
aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity
is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation,
yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors
of legumain would not only be valuable for studying the functional roles of legumain in these conditions,
but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation
of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-
peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and char-
acterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled
inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second
order rate constants of up to 5 ꢀ 104 Mꢁ1 sꢁ1 and IC50 values as low as 4 nM against recombinant mouse
legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-
reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that
can be used to study legumain function in multiple disease models.
Received 8 November 2011
Revised 13 December 2011
Accepted 14 December 2011
Available online 21 December 2011
Keywords:
Legumain
Protease inhibitors
Covalent inhibitors
Cysteine protease
Asparaginyl endopeptidase
Activity-based probes
Ó 2012 Elsevier Ltd. All rights reserved.
Legumain is a lysosomal cysteine protease that is conserved in
diverse cell types including, plants, invertebrate parasites and
mammals. It has a high propensity to cleave protein substrates
on the C-terminal side of asparagine residues.1 Mammalian legu-
main is known to have a role in antigen processing,2,3 albumin
maturation4 and matrix degradation5,6 and it is also implicated in
various pathological conditions including parasitic infection,7,8
atherosclerosis,9 inflammation10 and tumorigenesis.11,12 In addi-
tion, legumain is found to be over-expressed in the majority of
human solid tumors such as carcinomas of the breast, colon and
prostate,11 and knock-down of legumain in mouse models of can-
cer results in a marked decrease in tumor growth and metastasis.12
Based on these recent findings, legumain may be a therapeutically
important enzyme, especially in tumor progression and metastasis.
Therefore, highly selective and potent legumain inhibitors will be
valuable for studying the roles of legumain in diseases but also
potentially useful for the treatment of these diseases. A number
of legumain inhibitors have been developed and tested in vitro.
13–16 Notably, Powers and coworkers have developed highly selec-
tive and potent inhibitors of legumain in blood flukes and hard
ticks.17 However, only a few of these inhibitors have been tested
in mammalian cells or disease models that have therapeutic
impact.18
Previously, we have reported the development of irreversible
inhibitors and active site probes of legumain in intact cells and
mice.19 A unique feature of these inhibitors is that they contain
both an aza-asparagine and a proline moiety in the P2 position that
minimizes cross-reactivity towards other cysteine proteases such
as the cathepsins. In an effort to optimize selectivity and potency
of these inhibitors, we decided to extend our dipeptidyl aza-
asparaginyl scaffold by adding various non-natural amino acids
in the P3 position. We also wanted to test several different electro-
philic reactive groups (Fig. 1 and Table 1). Based on the previously
published study from our group,20,21 we selected 12 non-natural
amino acids that were found to prefer legumain over the caspases
in a P3 positional scan of a caspase inhibitor library. We initially
synthesized a group of compounds containing the core aza-aspar-
agine and proline from our previously reported lead compound19
linked to each of the selected non-natural amino acids in the P3
position and an aza Michael acceptor electrophile. In addition,
we also prepared the original dipeptidyl scaffold found in our ori-
ginal lead compound to determine the effects of using this electro-
phile in place of the original epoxide functional group. We
synthesized all compounds using the solid phase synthetic
⇑
Corresponding authors.
E-mail addresses: jlee@kist.re.kr (J. Lee), mbogyo@stanford.edu (M. Bogyo).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.079

J. Lee, M. Bogyo / Bioorg. Med. Chem. Lett. 22 (2012) 1340–1343
1341
Figure 1. Structures of the aza-peptidyl inhibitors of legumain.
methods previously reported by our laboratory22 with a slight
modification in order to attach the third amino acid (Scheme 1
and Supplementary data).
We carried out a simple IC50 determination of each compound
against recombinant mouse legumain and the results are shown
in Table 1. Within this compound series, we found that when the
P3 side chains were small alkyl groups (i.e., R = NN1, NN4, NN6),
these compounds showed excellent inhibitory effect. If these R
groups were relatively bulky groups such as aromatic or piperazine
groups (R = NN7, NN9, NN10, NN12), these compounds showed a
substantial drop in activity. It should be noted that all the previous
work done by other groups mainly focused on modifications of the
P10 site,17 however our results suggest that additional specificity
and potency may be achieved by modifications at sites distal to
the active site cysteine.
Table 1
IC50 values and second-order rate constants for inhibition of legumain by aza-peptidyl
inhibitorsa
Next, we chose three Michael acceptor inhibitors (R = NN1,
NN4, NN6) with the most optimal IC50 values and replaced the
Michael acceptor electrophile with the epoxide group from our ini-
tial lead compound. All six compounds, both with Michael acceptor
and epoxide electrophiles had similar potency with IC50 values in
the low nano molar range. Interestingly, the second-order inhibi-
tion rate constants (kobs/[I]), indicated that the epoxide-containing
compounds were approximately 1.5–2-fold more potent than their
Michael acceptor counterparts. These data suggest that the epoxide
electrophile reacts with the active site cysteine faster than the
Michael acceptor electrophile. However, this faster reactivity may
lead to an increase in overall off-target modification. In agreement
with this idea, we found that while all the compounds had weak
inhibition of cathepsin L (>100 M), all inhibitors containing the
epoxide electrophile showed slightly higher reactivity toward
cathepsin compared to the Michael acceptor compounds.
In order to further assess the potential cross reactivityof the com-
pounds, we generated activity based probes of the most potent com-
pounds for use in cells. This was accomplished by attachment of a
Cy5 fluorophore followed by labeling of RAW264.7 macrophages.
We chose to focus on the compounds containing NN1 with the
Michael acceptor electrophile and NN4 with the epoxide
electrophile since these two inhibitors showed the highest potency
in each series. The results of the cellular labeling study are shown
Inhibitor
R0
Legumain
kobs/[I] (Mꢁ1 sꢁ1
Cathepsin L
R
IC50 (nM)
)
IC50 (lM)
NN1
NN2
NN3b
NN4
NN5
NN6
NN7b
NN8
NN9
NN10
NN11
NN12b
Ac
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Michael Acceptor
Epoxide
6.5
36,169
3450
964
ND
ND
>1 mM
ND
855
ND
ND
ND
ND
ND
ND
>1 mM
370
890
106
358
141
148
9.3
198
17
638
167
>1000
>1000
267
>1000
8.3
4994
24,412
3622
14,480
1757
5376
ND
ND
3621
ND
10,802
27,397
53,674
46,467
25,713
Ac
9.3
8.1
4.4
8.6
NN1
NN4
NN6
Epoxide
Epoxide
Epoxide
a
Inhibition assays for legumain were performed in 0.1 M citrate-phosphate,
4 mM DTT, pH 5.8, <0.1% DMSO with a final concentration of 10 M Cbz-Ala-Ala-
Asn-AMC as substrate. Second-order rate constants were determined by linear or
non-linear regression analysis as described in the Supplementary data. All mea-
surements were triplicated and the average values were reported. Detailed proce-
dures for the enzyme assays can be found in the Supplementary data.
l
b
These compounds (NN3, NN7 and NN12 with Michael acceptor) were isolated
and tested as hydrolyzed fumaric acid.

1342
J. Lee, M. Bogyo / Bioorg. Med. Chem. Lett. 22 (2012) 1340–1343
Scheme. 1. Synthesis of legumain inhibitors (a) DIC, NMP, 1 h; (b) Fmoc-hydrazide, DIEA, NMP; (c) allyl chloroformate, DIEA, DMF; (d) 20% piperidine in DMF then Fmoc-Pro-
OH, DIC, HOBT, DMF; (e) 20% piperidine in DMF then Fmoc-NN1ꢂ12-OH, HATU, DIEA, DMF; (f) 20% piperidine in DMF then acetic anhydride, DIEA, DMF; (g) phenylsilane,
Pd(PPh3)4, DCM; (h) fumaric acid monoethyl ester (for Michael acceptor series) or ethyl
95%TFA.
L-trans-epoxysuccinic acid (for epoxide series), HBTU, DIEA, DMF, overnight then
had significantly reduced activity. In addition, inhibitors contain-
ing the aza-epoxide electrophile have faster inhibition kinetics
compared to the Michael acceptor compounds, but also showed
more cross-reactivity toward cathepsins. We also attached a Cy5
fluorophore to the selected inhibitors to test specificity and reac-
tivity inside living cells and all the synthesized probes selectively
labeled legumain in intact RAW264.7 macrophages. Currently we
are moving forward to scale up some of these inhibitors to investi-
gate the therapeutic applications of legumain inhibitors in various
mouse models of inflammation and cancer.
Acknowledgments
We thank Andrew Guzzetta and Juan Valderramos for HR-MS
analysis and Drs. Victoria Albrow and Fangfang Yin for helpful dis-
cussions about the project.
Supplementary data
Figure 2. Intact cell labeling of legumain with fluorescently labeled inhibitors. (a)
Structures of Cy5-labeled legumain probes (b) RAW 264.7 macrophages were
treated with Cy5-labeled legumain inhibitors for 60 min at different concentrations.
After incubation, cells were lysed under hypotonic conditions and all labeled
proteins were separated by 12.5% SDS–PAGE and analyzed by scanning the gel with
a Typhoon flatbed scanner.
Supplementary data (the synthetic procedures and character-
ization data of all compounds and the experimental protocols for
enzyme assays and cellular labeling experiments) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.12.079.
in Figure 2. As expected, all four probes showed highly selective
labeling of legumain at nanomolar concentrations. However, the
epoxide containing probes (LP-1, LP-4) labeled cathepsins at high
probe concentrations indicating that higher reactivity probably
contributed to this off-target labeling. We did not observe
significant differences in labeling intensity for all four probes,
although it seems that epoxide probes were slightly more sensitive
compared to the Michael acceptor probes (LP-2, LP-3). Regardless,
all the synthesized probes were cell permeable and highly selective
toward legumain.
In conclusion, we have designed and synthesized aza epoxide
and aza Michael acceptor inhibitors for legumain. We incorporated
non-natural amino acids to improve potency and selectivity of the
previously developed dipeptidyl scaffold. We found that inhibitors
with small alkyl groups in the P3 position demonstrated slightly
enhanced inhibitory effect compared to the inhibitor without a
P3 amino acid, whereas inhibitors with bulkier aromatic groups
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