Engineered Thermoplasma acidophilum factor F3 mimics human aminopeptidase N (APN) as a target for anticancer drug development

Article abstract of DOI:10.1016/j.bmc.2011.03.028

Human aminopeptidase N (hAPN) is an appealing objective for the development of anti-cancer agents. The absence of mammalian APN experimental structure negatively impinges upon the progression of structure-based drug design. Tricorn interacting factor F3 (factor F3) from Thermoplasma acidophilum shares 33% sequence identity with hAPN. Engineered factor F3 with two point directed mutations resulted in a protein with an active site identical to hAPN. In the present work, the engineered factor F3 has been co-crystallized with compound D24, a potent APN inhibitor introduced by our lab. Such a holo-form experimental structure helpfully insinuates a more bulky pocket than Bestatin-bound Escherichia coli APN. This evidence discloses that compound D24 targetting the structure of E. coli APN cannot bind to the activity cleft of factor F3 with high affinity. Thus, there is a potential risk of inefficiency to design hAPN targeting drug while using E. coli APN as the target model. We do propose here now that engineered factor F3 can be employed as a reasonable alternative of hAPN for drug design and development.

Full text of DOI:10.1016/j.bmc.2011.03.028

Bioorganic & Medicinal Chemistry 19 (2011) 2991–2996
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Engineered Thermoplasma acidophilum factor F3 mimics human
aminopeptidase N (APN) as a target for anticancer drug development
Jing Su a, Qiang Wang b,c, Jinhong Feng b, Cong Zhang a, Deyu Zhu a, Tiandi Wei a,
a,
Wenfang Xu b, , Lichuan Gu
⇑
⇑
a State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
b Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Jinan 250012, China
c Department of Pharmaceutics, School of Life Sciences, South-Central University for Nationalities, Wuhan 430074, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Human aminopeptidase N (hAPN) is an appealing objective for the development of anti-cancer agents. The
absence of mammalian APN experimental structure negatively impinges upon the progression of struc-
ture-based drug design. Tricorn interacting factor F3 (factor F3) from Thermoplasma acidophilum shares
33% sequence identity with hAPN. Engineered factor F3 with two point directed mutations resulted in a
protein with an active site identical to hAPN. In the present work, the engineered factor F3 has been co-
crystallized with compound D24, a potent APN inhibitor introduced by our lab. Such a holo-form experi-
mental structure helpfully insinuates a more bulky pocket than Bestatin-bound Escherichia coli APN. This
evidence discloses that compound D24 targetting the structure of E. coli APN cannot bind to the activity
cleft of factor F3 with high affinity. Thus, there is a potential risk of inefficiency to design hAPN targeting
drug while using E. coli APN as the target model. We do propose here now that engineered factor F3 can be
employed as a reasonable alternative of hAPN for drug design and development.
Received 16 January 2011
Revised 11 March 2011
Accepted 12 March 2011
Available online 21 March 2011
Keywords:
Aminopeptidase N
Inhibitors
Tricorn interacting factor F3
Crystal structure
Drug design
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
kinds of Bestatin analogues have been well developed, such as
Probestin,6 Amatatin,7 Prebestatin,8 etc. It is gold finger that the
It needs to be spotlighted that cancer causes a fairly large
number of deaths in the world every year. Metastasis is one of
the intrinsic characteristics of malignant tumors, which often
results in the clinical treatment failure and patient mortality.1
Therefore, in the present climate, tumor metastasis in cancer
therapy is becoming a challenging predicament, and how to
efficiently restraint the invasion and metastasis of cancer cells is
of indispensability for cancer medication.
Although there are many elements regarding the transfer
process of the tumor cells’ metastasis, the interaction between
tumor cells and its surrounding micro-environment protein-
degrading enzymes performs a critical portrayal. These enzymes
comprise matrix metalloproteinases (MMPs), plasminogen and
aminopeptidases (AP). Aminopeptidase N (APN) is related to the
growth of primary tumor, second tumor and angiogenesis. It can
also encourage tumor cell proliferation and differentiation.2–5 All
these interesting evidences make this enzyme an important target
for drug design. The first known APN inhibitor, Bestatin (Ubeni-
mex) extracted from Streptomyces olivoreticuli, was marketed in
Japan in 1987 as an anti-neoplastic agent. In recent decades, some
design of small molecules to target a specific protein can be greatly
facilitated by experimental structure. However, the human
aminopeptidase N (hAPN) crystal structure has not been deter-
mined so far. This troublesome condition frustrates the develop-
ment of further specific drugs targeting hAPN. For that reason,
homologous proteins with available 3D structure would contribute
an applicable alternative.
Crystal structures of hAPN homologues, such as APN from Esch-
erichia coli and its complex structure with Bestatin,9,10 APN from
Neisseria meningitides,11 leukotriene A4 hydrolase (LTA4H) from
human12 and tricorn interacting factor F3 from Thermoplasma aci-
dophilum (T. acidophilum),13 have been determined recently. These
crystallographic results provide appropriate awareness for APN re-
search. At present time, the drug design targeting hAPN is gener-
ally depended on the three dimensional structures of these
homologous proteins and enzyme-inhibitor models. However,
structure-based sequence alignment analysis clearly exhibits that
none of these proteins has identical active site with hAPN. Accord-
ingly, drug design using these proteins’ structures is undeniably
not the best choice.
Tricorn interacting factor F3 (factor F3) from T. acidophilum has
33% sequence identity with hAPN. In particular, there are only two
different residues between the active sites of factor F3 and of hAPN
(Fig. 1). Point mutations of these two amino acids produce an
⇑
Corresponding authors. Tel./fax: +86 531 88382264 (W.X.); tel./fax: +86 531
88362039 (L.G.).
E-mail addresses: xuwenf@sdu.edu.cn (W. Xu), lcgu@sdu.edu.cn (L. Gu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.028

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J. Su et al. / Bioorg. Med. Chem. 19 (2011) 2991–2996
Figure 1. Sequence alignment of human aminopeptidase (hAPN) and homologous proteins. Structure-based sequence alignment of the hAPN, Susscrofa APN, T. acidophilum
factor F3, human leukotriene A4 hydrolase and E. coli APN. The five important residues in the activity site are presented in pane. Residues conserved in hAPN and factor F3 are
labeled in pink pane. Residues unconserved in hAPN and factor F3 are labeled in red pane.
active site for factor F3 identical to that of hAPN. Bestatin and other
synthesized APN inhibitors also have inhibitory effect to the mu-
tant factor F3 (Fig. S1). Thus, the 3D structure of such mutant pro-
Optimum temperature assay was performed as following proce-
dures. Purified factor F3 mutant was mixed with the substrate
Leu-p-nitroanilide then incubated for 30 min at 40, 50, 60, 65, 70,
80 and 90 °C, respectively. The activity was determined by measur-
ing the absorbance increase at 405 nm.
Optimum pH of the enzyme was measured by using 50 mM ace-
tic acid (pH 4–5.5), 50 mM MES (2-(N-morpholino) ethanesulfonic
acid, pH 6–6.5) and 50 mM Tris–HCl (hydroxymethyl aminometh-
ane hydrochloride, pH 7–9) then incubated at 37 °C for 30 min to
measure the absorbance increase at 405 nm. The remaining session
of the assay was done the same as the assay for enzyme optimum
temperature.
tein could present
a reasonable model for the downstream
molecular design. Moreover, the holo-form crystal structure of this
protein can be employed to determine the inhibitor-enzyme inter-
action as well as to design novel potent inhibitors.
Our group has long-term interest on the development of novel
APN inhibitors. For example, we reported various series of APN
inhibitors,14–17 such as B6: N6-[(benzyloxy) carbonyl]-N2-(2,4-dic-
hlorobenzoyl)-N1-hydroxy-L-lysinamide, a potent APN inhibitor
equivalent to Bestatin,14 and D24, which exhibits higher solubility
than B6. In the current study we crystallized engineered factor F3
in complex with compound D24 for deciding protein-inhibitor
interaction and designing new efficient inhibitors. It should be
noted that the crystal structure of such complex would demon-
strate a plausible exemplary for anti-cancer drug design. Further-
more, the thermostablility of this engineered protein adjudicates
that it can be the substitution for expensive and instable marketed
APN during biological assay.
2.2. Synthesis of D24
The synthetic scheme of target compound is depicted in Figure
2. Compound 5 was prepared as described by Wang et al.14 After
that, N-(tert-butoxycarbonyl)-L-phenylalanine (Boc-L-Phe) was
conjugated to the –COOH of compound 5 by amidation to provide
6. In the next step, the methyl ester 6 can be transformed to corre-
sponding hydroximic acid 7 as per the methods mentioned before.
Finally, the N-(tert-butoxycarbonyl)-protecting group of 7 can be
cleaved with 3 N HCl in ethyl acetate to give hydrochloride salt
of D24. White solid, yield 76.3%, mp 139–141 °C. MS (ESI) m/z
[M+H]+ 443.5; 1H NMR (600 MHz, DMSO-d6) d 10.72 (s, 1H), d
8.92 (s, 1H), d 8.81 (d, J = 8.4 Hz, 1H), d 8.11 (brs, 2H), d 7.34 (m,
10H), d 4.99 (s, 2H), d 4.16 (dd, J1 = 8.4 Hz, J2 = 14.4 Hz, 1H), d
3.79 (t, J = 7.2 Hz, 1H), d 2.95 (m, 2H), d 1.63 (m, 2H), d 1.38 (m,
2H), d 1.20 (m, 2H), d 0.90 (m, 2H).
2. Experimental
E. coli strains were cultured at 37 °C in lysogeny broth (LB)
and transformed as described previously.18 T. acidophilum
(ATCC25905TM) was cultured in according to ATCC protocol.
The plasmid pF3 native is the pET21b vector containing T. acido-
philum factor F3. All biochemical reagents were purchased from
Sigma (St. Louis, MO, USA).
2.3. Enzyme inhibition comparison between factor F3 and APN
2.1. Kinetics analytical method
In the measurement of compounds inhibition constant on factor
F3 mutants (E101Q, N261T), compounds were performed at the
concentration 1280, 320, 80, 20, and 5 lg/mL in ddH2O. The light
absorbance changing with time was measured with a 96-well plate
reader. To measure the inhibitory effect, the compounds and the
enzyme were preincubated at 37 °C for 30 min prior to the addition
The recombinant factor F3 mutant was purified by following the
standard procedure as previously described.10 Its APN activity was
determined by measuring the absorbance increase at 405 nm of
nitroanilide from the substrate (Leu-p-nitroanilide).19 The reaction
was incubated at 37 °C for 30 min. One unit of enzyme activity was
defined as the amount of enzyme that causes an increase absor-
bance at 405 nm of 1 u/h.
of the substrate. The inhibition constant was analyzed with IC50
.

J. Su et al. / Bioorg. Med. Chem. 19 (2011) 2991–2996
2993
Figure 2. Reagents and reaction conditions: (a) CuCO3Cu(OH)2, 1.2 N HCl, H2O, 80–90 °C, 2 h; (b) NaHCO3, CbzCl, 25 °C, 12 h; (c) EDTA SS, 25 °C, 24 h; (d) MeOH, HCl, 25 °C,
6 h; (e) Boc- -Phe, Et3N, TBTU, CH2Cl2, 25 °C, 5 h; (f) NH2OK, MeOH, 25 °C, 12 h; (g) HCl–EtOAc, 25 °C, 24 h.
L
2.4. Crystallization and data collection
Recombinant factor F3 mutant was concentrated to 10 mg/mL
before crystallization. Crystals were grown by sitting-drop, va-
por-diffusion method at 20 °C by mixing equal volume of protein
complex with reservoir solution containing 0.1 M Tris-HCl (pH
8.5), 18% (w/w) PEG 2000, 0.2 M Li2SO4 and 1 mM compound
D24. Diffraction data were collected at Shanghai Synchrotron
Radiation facility (SSRF) beam line BL17u1. The data set were pro-
cessed with HKL2000.20 The X-ray data statistics are summarized
in Table 1.
Table 1
X-ray data collection and refinement statistics
Parameters
F3-mutant with D24
Data collection
Space group
a (Å)
b (Å)
c (Å)
P21212
114.7
183.1
105.2
90
a
(°)
b (°)
(°)
90
90
c
Resolution (Å)
Total reflections
Unique observations (outer shell)
50–2.9
353272
49,142 (4287)
16.13 (2.7)
0.128 (0.529)
2.5. Structure determination and refinement
The crystal structure was solved by molecular replacement
methods using the program Phaser in the CCP4 program suite21
with T. acidophilum factor F3 (PDB entry: 1Z5H) as search model.
The initial phase obtained from molecular replacement was further
refined using PHENIX22 and the model was rebuilt using COOT.23
Data collection and structure refinement statistics are summarized
in Table 1. All the molecular graphics figures were generated use
PyMol (http://www.pymol.org). The structure has been deposited
in the Protein Data Bank, access code: 3Q7J.
I/r (outer shell)
Rsym (outer shell)a
Refinement
Resolution range (Å)
Number of reflections (|F| >0)
Data coverage (test set)
50–2.9
47,411
b
Rworking/Rfree
0.2024/0.2713
12,639
0.009
Total number of atoms
Root mean square deviation bond length (Å)
Bond angles (degree)
1.228
Ramachandran plotc
Most-favored region (%)
Favored region (%)
Generously allowed region (%)
Disallowed region (%)
90
3. Results and discussion
8.5
0.9
0.6
3.1. Engineered F3 mutant (E101Q, N261T) has normal activity
and is themostable
P
P
P
P
a
Rsym
=
i|I(h k l)i À <I(h k l)>|/
I<I(h k l)i> over i observations.
hkl
hkl
b
Value of Rfree for 1.91% of randomly selected reflections excluded from
Structure-based sequence alignment discloses the factor F3 of T.
acidophilum has only two amino acids different from the hAPN in
refinement.
As defined in PROCHEK.25
c

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J. Su et al. / Bioorg. Med. Chem. 19 (2011) 2991–2996
active site. One is E101 substituted by Q in hAPN, and the other is
N261 substituted by T. Site-directed mutations result in an engi-
neered protein with active site identical to hAPN. To explore
whether the engineered protein is druggable, biochemical charac-
terization of this enzyme and compared the IC50’s tendency be-
tween APN and engineered F3 mutant was well estimated. The
experiments responded the Km value of 62.453
was higher than that of native factor F3 (34.7
l
l
m (Fig. 3), which
m).24 The engi-
neered protein divulges the maximum enzyme activity at 75 °C
(Fig. 4) and pH 5.5 (Fig. 5) using Leu-NA as the substrate. The inhib-
itory tendency of four series of compounds (Bestatin, LYP,26 A6,27
D24) to engineered F3 mutant is accordance with APN. In another
word, these molecules can suppress the F3 mutant’s activity as
well (Fig. S1). Consequently, our biochemical data evidently
substantiated the feasibility of the substitution of hAPN by the
engineered F3 protein.
3.2. Synthesis of D24 and evaluation of inhibitor
Figure 5. Effects of pH on leucyl-p-nitroanilide hydrolysis by factor F3 mutant. The
buffers used were 50 mM acetic acid (pH 4–5.5), 50 mM MES (pH 6–6.5) and
100 mM tris-HCl (pH 7–9). The substrate and the enzyme were preincubated at
37 °C for 30 min to measure its absorbance at 405 nm.
Our lab has unveiled numerous APN inhibitors, for example, B6:
N6-[(benzyloxy) carbonyl]-N2-(2,4-dichlorobenzoyl)-N1-hydroxy-
L
-lysinamide that was equivalent to the positive control Bestatin.14
Based on the compounds mentioned above, we designed the target
compound D24 that has better solubility than B6. The synthetic
route of target compound is shown in Figure 2. The N6-amino
Figure 3. Enzyme assays of the factor F3 mutant with the substrate Leu-p-
nitroanilide. Factor F3 mutant exhibits typical Michaelis–Menten enzymatic
kinetics with a Km constant of 62.45 lM for this substrate.
Figure 6. Stereo representation of the zinc-binding residues and interacted amino
acids with compound D24. (A) The activity site of the factor F3 mutant with
compound D24. Zinc ion was co-ordinated by H265, H269, E288 and compound
D24 molecule. These amino acids are labeled in cyan stick model and the distances
are labeled with yellow dash. (B) Eight amino acids have interactions with the
inhibitors. They are Q101, A229, A231, E233, E288, T292, R316 and G352. The
compound D24 is labeled with yellow stick, the zinc is labeled with green sphere
and the eight amino acids are shown with cyan sticks.
group of
L-lysine was selectively protected by Cbz (carbobenzoxy).
Next, Boc-
L
-Phe was linked to the –COOH by amide bond to provide
6. Then, the methyl ester 6 can be transformed to corresponding
hydroximic acid 7 with methods mentioned before. Finally, the
Boc-protecting group of 7 can be easily removed with 3 N HCl in
ethyl acetate to give hydrochloride salt of D24 (Fig. 2).
Recombinant T. acidophilum factor F3 and the substrate of
Leu-p-nitroanilide were used to evaluate the IC50 of the
Figure 4. Effects of temperature on leucyl-p-nitroanilide hydrolysis by factor F3
mutant. The buffer used was 50 mM Tris–HCl (pH 8.0). The substrate and the
enzyme were preincubated at 40, 50, 60, 65, 70, 75, 80 and 90 °C for 30 min and
measured at 405 nm.

J. Su et al. / Bioorg. Med. Chem. 19 (2011) 2991–2996
2995
Figure 7. Structural comparison of factor F3 mutant with compound D24 and F3 native crystal forms (PBD codes: 1Z1W and 1Z5H). Green ribbon represents the native crystal
structure of 1Z1W; pink represents the native crystal structure of 1Z5H; red ribbon represents the factor F3 mutant (A, B). The amino acids are shown in different orientations
with stick model and the zincs are shown in sphere model. (A) E101 mutant has an obvious conformational discrimination between the three structures. (B) N261 mutant
increases the hydrophobicity of the surrounding environment.
Figure 8. Binding pocket comparison of E.coli with Bestatin (PDB code: 2hpt) and factor F3 mutant with compound D24. (A) and (C) represent the E. coli structure. (B) and (D)
represent the factor F3 mutant. Red represents negatively charged surfaces and blue represents positively charged surfaces, Bestatin is shown in yellow stick and compound
D24 is shown in purple stick model. (A) and (B) Electrostatic surface representation shows the clefts’ shape between these two structures. E. coli with Bestatin has a narrower
cleft than factor F3 mutant with compound D24. The cleft is narrower on the left where the N-terminus of the substrate binds and widens on the right to accommodate an
extended polypeptide substrate. (C) and (D) The five amino acids at the binding pocket are different between the two structures. The amino acids (E382, R293, M260, K319
and E121) in E. coli at the binding pocket are different with hAPN. The amino acids (G352, N287, F346, A229 and 101N) aligned with E. coli are identical to hAPN.
synthesized compound D24. It showed the IC50 value of 30
factor F3 mutants.
l
M for
D24 complex structure, there are eight amino acids interacting
with the inhibitor: Q101, A229, A231, E233, E288, T292, R316
and G352 (Fig. 6B). The inhibitor is stabilized mainly by the com-
bination of hydrophobic interaction and several hydrogen bonds.
Through superimposition of the two native structures (PDB
codes: 1Z1W and 1Z5H) onto our compound D24 complex struc-
ture, we found that the residue positions in active site exhibit dra-
matic change (Fig. 7A). In the two native structures, E101 adopts
distinctive conformations. In 1Z1W, it adopts an outside orienta-
tion while in 1Z5H it is oriented to the inside but not quite ex-
tended due to the same charge repulsion with E233 that stand
by. In our structure, the side chain of Q101 is buried inside deeply
due to the loose of negative charge and stabilized by the inhibitor.
The mutation of N261T does not cause conformational changes
3.3. Analysis of the engineered factor F3 mutant protein-
inhibitor interactions
3.3.1. The inhibitor occupies the active site of factor F3
The factor F3 is a zinc aminopeptidase. In the model of native
protein, the catalytic zinc ion is coordinated by the N 2 atoms of
e
H265 and H269, a carboxylate oxygen atom of E288 and a water
molecule.10 The zinc-coordinating atoms form a close to a perfect
tetrahedral coordination sphere with the zinc ion in the center.
In our model, the zinc ion is coordinated by H265, H269, E288
and the amino group of compound D24 (Fig. 6A). In the compound

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J. Su et al. / Bioorg. Med. Chem. 19 (2011) 2991–2996
except that it increases the hydrophobicity of its surrounding envi-
ronment (Fig. 7B).
work was supported by State Key Laboratory of Microbial Technol-
ogy, Shandong University and Grant of Hi-Tech Research and
Development Program of China (2006AA02A324). We thank the
support by National Nature Science Foundation of China (Grant
No. 30772654 and No. 90713041) and National High Technology
Research and Development Program of China (863 project; Grant
No. 2007AA02Z314). We also thank support by special fund for Ba-
sic Scientific Research of Central Colleges, South-Central University
for Nationalities (ZZQ10013).
3.3.2. Engineered factor F3 mutant has a bigger substrate
binding pocket than E. coli APN in complex with Bestatin
In the compound D24 complex structure, a part of the inhibitor
loses electron density. Thus the occupancy of compound D24 in
T. acidophilum factor F3 is not high. The compound D24 is designed
using E.coli aminopeptidase as a target model. This situation
provokes a very essential issue on whether the structural differen-
tation between E. coli APN and T. acidophilum factor F3 leads to a
less-effective inhibitor. A structural comparison of the active sites
of E. coli APN with Bestatin and factor F3 mutant with compound
D24 manifested that the former one (Fig. 8A) possesses narrower
pocket to accommodate the inhibitor, whereas the later one is
larger (Fig. 8B). This structural discrimination was mainly precipi-
tated by the distinctive amino acids around the binding cavity
(Fig. 8C, and D). At the substrate binding pocket, the replacement
of G352 of T. acidophilum factor F3 concluded a bulky binding
pocket. Furthermore, there are four additional different amino
acids within these two proteins. Consequently, the inhibitor de-
signed using E. coli APN as the model cannot tightly fasten the
engineered T. acidophilum factor F3. Structure-based sequence
alignment suggests that with these five amino acid substitutions,
the engineered T. acidophilum factor F3 could be the best mimic
of the hAPN as the drug design target.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.028.
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An engineered factor F3 from T. acidophilum was planned to mi-
mic hAPN as an anti-cancer drug target here now. A new protein
generated by two point mutations on the native protein reveals
an active site identical to the counterpart of hAPN. On the basis
of this engineered protein, an known APN inhibitor, compound
D24, exhibits inhibition activity. The protein-inhibitor interactions
were characterized by biochemical and structural methods as well.
According to our observation, the two residues (Q101 and T261) in
point mutations experienced significant conformational changes in
compound D24 complex structure. This interesting evidence pro-
poses that these two point mutations are necessary for the engi-
neered protein to mimic hAPN. Subsequently, the comparison
between E. coli APN with Bestatin and factor F3 mutant with com-
pound D24 demonstrated that the former has a slighter cleft than
the factor F3 mutants. That’s the reason that the inhibitor designed
based on E. coli coordinate cannot securely buckle up the F3 mu-
tant. Therefore, engineered factor F3 can be functioned as a satis-
factory alternation of hAPN for drug design and screening. In
conclusion, our structure would like to provide a brand new start
point for the development of potent anti-cancer leads targeting
hAPN.
Acknowledgements
26. Luan, Yepeng; Wang, Xuejian; Zhu, Huawei; Qu, Xianjun; Fang, Hao; Xu,
Wenfang Lett. Drug Des. Disc. 2009, 6, 420.
We thank the staff at beam line BL17u1 at the Shanghai Syn-
chrotron Radiation facility for support with data collection. This
27. Shang, L.; Fang, H.; Zhu, H.; Wang, X.; Wang, Q.; Mu, J.; Wang, B.; Kishioka, S.;
Xu, W. Bioorg. Med. Chem. 2009, 17, 2775.