Discovery of Mechanism-Based Inactivators for Human Pancreatic Carboxypeptidase A from a Focused Synthetic Library

Article abstract of DOI:10.1021/acsmedchemlett.7b00346

Metallocarboxypeptidases (MCPs) are involved in many biological processes such as fibrinolysis or inflammation, development, Alzheimer's disease, and various types of cancer. We describe the synthesis and kinetic characterization of a focused library of 22 thiirane- and oxirane-based potential mechanism-based inhibitors, which led to discovery of an inhibitor for the human pro-carboxypeptidase A1. Our structural analyses show that the thiirane-based small-molecule inhibitor penetrates the barrier of the pro-domain to bind within the active site. This binding leads to a chemical reaction that covalently modifies the catalytic Glu270. These results highlight the importance of combined structural, biophysical, and biochemical evaluation of inhibitors in design strategies for the development of spectroscopically nonsilent probes as effective beacons for in vitro, in cellulo, and/or in vivo localization in clinical and industrial applications.

Full text of DOI:10.1021/acsmedchemlett.7b00346

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Letter
Discovery of mechanism-based inactivators for human
pancreatic carboxypeptidase A from a focused synthetic library
Sebastian A. Testero, Carla Granados, Daniel Fernandez, Pablo Gallego, Giovanni Covaleda,
David Reverter, Josep Vendrell, Francesc X. AVILÉS, Irantzu Pallarès, and Shahriar Mobashery
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00346 • Publication Date (Web): 22 Sep 2017
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Discovery of mechanism-based inactivators for human pancreatic
carboxypeptidase A from a focused synthetic library
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Sebastián A. Testero,† Carla Granados,‡ Daniel Fernández,‡ Pablo Gallego,‡ Giovanni Covaleda,‡
David Reverter, ‡ Josep Vendrell,‡ Francesc X. Avilés, ‡ Irantzu Pallarès, *‡ Shahriar Mobashery*†
†Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, United States
‡Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, and Institut de Biotecnologia i de Bio-
medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
The first two authors contributed equally to this work
KEYWORDS: Carboxypeptidase A, mechanism-based inactivators, thiiranes, X-ray crystallography.
ABSTRACT: Metallocarboxypeptidases (MCPs) are involved in many biological processes such as fibrinolysis or inflam-
mation, development, Alzheimer’s disease and various types of cancer. We describe the synthesis and kinetic characteri-
zation of a focused library of 22 thiirane- and oxirane-based potential mechanism-based inhibitors, which led to discovery
of an inhibitor for the human pro-carboxypeptidase A1. Our structural analyses show that the thiirane-based small-
molecule inhibitor penetrates the barrier of the pro-domain to bind within the active site. This binding leads to a chemi-
cal reaction that covalently modifies the catalytic Glu270. These results highlight the importance of combined structural,
biophysical and biochemical evaluation of inhibitors in design strategies for the development of spectroscopically non-
silent probes as effective beacons for in vitro, in cellulo, and/or in vivo localization in clinical and industrial applications.
The human genome is reported to have about 600 prote-
ases of which approximately 150 are believed to be zinc-
dependent.1 These can cleave within a stretch of a poly-
peptide (endopeptidases) or release amino acids from
either termini of the polypeptides (exopeptidases). Metal-
locarboxypeptidases (MCPs) excise one amino acid at a
time from the C-terminal end of the polypeptide sub-
strates. These enzymes had been historically considered
important for digestive functions, as exemplified by the
now-classical examples of the bovine pancreatic carboxy-
peptidases A and B. This limited role for these enzymes
has been refuted on the basis of the genomic information
Small-molecule inhibitors of various types have been
developed to target MCPs specifically; among them, there
has been considerable interest in irreversible mechanism-
based enzyme inhibitors.12-14 These compounds are inert
species that bind to their respective target enzymes based
on inherent affinity designed into the molecular scaffold-
ing. Upon binding, a reaction leading to covalent inhibi-
tion irreversibly modifies the enzyme active site limiting
the opportunity for unwanted cross-reactions. The pro-
longed longevity of the covalently inhibited species con-
fers to these compounds desirable attributes as pharma-
ceuticals, imaging reagents, mechanistic tools and diag-
nostic agents 15, 16
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and biochemical discoveries in recent years.2, Now,
MCPs are subdivided into the A/B subfamily (M14A ac-
cording to MEROPS), the N/E subfamily (M14B), the γ-D-
glutamyl-meso-diaminopimelate peptidase I (M14C) and
the complex cytosolic carboxypeptidases, CCPs (M14D).4, 5
These enzymes are currently known to be expressed in all
tissues and likely have functions in maturation of neuro-
peptides, hormones and cytokines, in blood fibrinolysis,
in anaphylaxis, among others. A variety of MCPs have
been linked to human diseases such as acute pancreatitis,
type 2 diabetes, Alzheimer’s Disease, and various types of
cancer.6-11 Due to their proteolytic nature, MCP functions
are strictly regulated by means of controlled secretion,
compartmentalization, substrate specialization or expres-
sion as inactive enzymes (zymogens), typically seen in the
M14A subfamily.
A few strategies for mechanism-based inhibition of zinc
proteases have been described in the literature.13, 17-19 Two
are based on the chemistries of oxiranes and thiiranes,
whose structural scaffolds dock into the S1’ specificity
pocket, typically through a single amino acid side chain.
The terminal carboxylate, as a recognition element for the
MCPs, is matched by a positively charged amino acid on
the part of the enzyme. These interactions bring the
heteroatoms in the thiirane or oxirane into the coordina-
tion sphere of the zinc ion, predisposing the compounds
for covalent modification by an active-site nucleophile,
typically the catalytic glutamate of MCPs (Scheme 1).20, 21
The thiirane-based mechanism-based inhibitors may also
lead to a reaction that generates a thiolate within the
active site, which results in tight-binding inhibition of the
target enzyme.14, 19
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Inspired by the known inhibitors BEBA and BTBA, we treatment of derivatives 26 with thiourea, followed by
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report herein the preparation of a focused library of 22
thiirane- and oxirane-based potential MCP irreversible
inhibitors (Figure 1). This library was first screened for
inhibition of the activated bovine caboxypeptidases A
(bCPA) and B (bCPB). Then, promising inhibitors were
screened against the human pancreatic CPA1 proenzyme
(hproCPA1). In hproCPA1, the prodomain is loosely cap-
ping the active site of the enzyme and small molecules
can gain access to the active site. We identified one com-
pound, the racemic mixture of trans-4-methyl-2-(thiiran-
2-yl)pentanoic acid (11), as a covalent modifier of the
enzyme. We also report the X-ray structure of the
hproCPA1-inhibitor 11 complex at 1.8-Å resolution, which
represents the first for a covalently inhibited zymogen.
This study provides for the first time structural evidence
on the capacity of low-molecular-weight compounds to
diffuse into the active site of a zymogen, as previously
inferred from the intrinsic activity of pro-CPs.22 It is im-
portant to note that the inhibitor was prepared as a race-
mate, but only one enantiomer was seen within the active
site (discussed below).
hydrolysis afforded the corresponding cis thiiranes 4, 8,
and 12. It is worth noting that conversion of the epoxide
to the corresponding thiirane occurs with inversion of
configuration.21 Similarly, syntheses of the cis oxiranes 2,
6, 10 and 14 and the trans thiiranes 3, 7, 11, 16, 18, 20 and
22 were accomplished from the derivatives 27 by the same
strategy. All compounds were prepared in racemic forms.
Cognizant of the fact that only the trans oxiranes and
thiiranes have been shown to be active against carboxy-
peptidases,25 we synthesized all the trans analogs of these
two families. Regardless, we also synthesized some cis
derivatives (from compounds 26 and 27) to test their
activity and to confirm that they are less active than the
trans ones.
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Scheme 1. Recognition of a polypeptide substrate within
the MCPs active site (top panel; boxed), mimicked by that
of a thiirane- or oxirane-based inhibitors. The metal-ion
coordination chemistry is necessary for the onset of cova-
lent modification.
Scheme 2 shows the general synthetic method for the
preparation of the library. The synthesis of oxiranes and
thiiranes began with alkylation of vinyl acetic acid with
eight different alkyl halides in moderate to good yields.23
The racemic α-substituted β,γ-unsaturated carboxylic
acids were esterified as methyl or benzyl ester by standard
methodologies. Epoxidation of the substituted 2-vinyl
propanoate esters 25 provided the separable mixture of
trans (26; major) and cis (27; minor) epoxides.24 With the
derivatives 26 in hand, the trans oxiranes 1, 5, 9, 13, 15, 17,
19 and 21 were obtained by treatment with lithium hy-
droxide or hydrogenolysis over Pd/C. On the other hand,
Figure 1. The library of synthetic oxiranes and thiiranes.
Each compound was synthesized as a racemic mixture.
Scheme 2. The general synthetic scheme for the preparation of
compound libraries.
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statistics details are presented in Table 1. The structure
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reported herein represents the first crystal structure of
any zymogen of the carboxypeptidase family in complex
with a small-molecule mechanism-based inhibitor.
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Figure 2A shows a stereoview of the hpro-CPA-inhibitor 11
complex. The pro-domain, albeit capping the entrance to
the active site, displays a flexibility that allows for a
measurable intrinsic activity due to the diffusion of small-
molecular-weight substrates.27 The presence of contigu-
ous electron density clearly indicated that the atoms con-
stituting the thiirane moiety of the inhibitor were cova-
lently attached to the side chain of Glu270, producing an
ester bond with the newly formed C-O bond length at 1.3
Å. The attendant thiirane ring opening results in the thio-
late of the covalently linked inhibitor as the fourth ligand
to the tetrahedral catalytic zinc ion, along with three
ligands from the protein (His69, His 196 and Glu72), as
stipulated by design paradigms. The position of the thio-
late as a zinc ligand was previously occupied by a water
molecule in the apo enzyme. Furthermore, the inhibitor
establishes a salt bridge to Arg145 via its carboxylate
group and hydrogen bonds to Tyr248 and Asn144 side
chains. Its isobutyl side chain is ensconced in the center
of the hydrophobic pocket of the active site. The hydro-
phobic pocket is defined by approximately 9 Å of separa-
tion from top to bottom (Thr268 to Ala250) and left to
right (Ser253 to Ile247), with Ile243 and Ile255 pair at the
deep end. The conserved, CPA-type specificity-
determinant residue Ile255 lies at 6.3 Å from the inhibitor,
the longest separation when comparing the bound and
unbound CPA structures (hproCPA4, 5.2 Å; hproCPA2, 5.7
Å; hCPA4 (peptide-bound) 5.6 Å; bCPA (inhibitor-bound)
5.8 Å). A schematic representation of the binding of in-
hibitor 11 is shown in Figure 2C. An analysis of the contact
areas between the catalytic domain and the pro-domain is
given in Figure S2.
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The library was first screened against the commercially
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available bovine CPA and CPB (see supporting infor-
mation, Table S1). The two aforementioned oxirane inhib-
itors for bCPA, compounds designated as BEBA and
BTBA,21 were also included as positive controls. The ob-
servation of time-dependence for inhibition by mecha-
nism-based inhibitors is a diagnostic criterion. As such,
the screening comprised of a short (2 min) and a long (3
h) pre-incubation of the compounds with the enzyme
prior to activity assays. This criterion identified 15 com-
pounds that inhibited bCPA activity (of which, incidental-
ly, none inhibited bCPB; data not shown). Based on the
IC50 values that we evaluated (Table S1), six compounds of
the 15 (3, 11, 19, 20, 21 and 22) were selected for further
analysis with the human pancreatic CPA (hCPA1; Table
S2).
Table 1. Data Collection and Refinement Statistics.
The inactivation reaction of hCPA1 by the above men-
tioned inhibitors could be represented by equation 1 in
the supporting information. The values of kobs were calcu-
lated from plots of ln(v/vo) vs incubation time (see sup-
porting information). The parameters Kiapp and kinact for
the process were obtained from the plot of kobs vs [I] in a
double-reciprocal fashion.26 The kinetic parameters for
hCPA1 inactivation for all the selected inhibitors are in-
cluded in Table S2. All efforts in crystallization of the
activated human CPA1 (lacking the pro domain) in com-
plex with the selected inhibitors were not successful.
Hence, we focused our attention on the crystallization of
human pro-CPA1 (the zymogen).
Data Collection
Inhibitor:hCPA1
Space group
Cell dimensions
a, b, c (Å)
P 1 21 1
53.90, 52.54, 133.97
90.00°, 95.00°, 90.00°
44.50–1.77 (1.86–1.77)
0.07 (0.13)
α, β, γ (°)
Resolution (Å)a
b
Rmerge
I/σI
10.2 (4.2)
Completeness (%)
Redundancy
Refinement
94.1 (66.0)
3.1 (2.2)
All six compounds were tried in crystallographic charac-
terization with the human enzyme; only inhibitor 11 pro-
duced results for a complex. The crystals diffracted to 1.8-
Å resolution and the crystallographic and refinement
Resolution (Å)
No. of reflections
43.68–1.80
63,800
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Rwork/Rfreec
No. of atoms
protein
15.45/21.18
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6751
6283
443
Water
R.m.s. deviations
Bond lengths (Å)
Bond angles
0.022
1.852°
5OM9
PDB code
9
a Statistic for highest resolution shell is shown in parentheses.
bRmerge = Σ || Ii – < I >| / Σ Ii , where Ii is the ith mesurement of the
intensity of an individual reflection or its symmetry-equivalent
reflections and < I > is the average intensity of that reflection and its
symmetry-equivalent reflections.
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cRwork = Σ ||Fo| - |Fc|| / Σ |Fo|, for all reflections and Rfree = Σ ||Fo| -
|Fc|| / Σ |Fo|, calculated based on the 5% of data excluded from
refinement.
Inhibitor-induced conformational changes have been
studied, both in the active and zymogenic forms of
MCPs.20, 28 As compared to “unliganded” pro-CPA crystal
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structures,27,
the most striking difference is that of
Tyr248, which undergoes an inhibitor-induced conforma-
tional change, whereupon the ring switches its position
120° between the surface-located “open” conformation to
the “close” conformation, where the phenolic hydroxyl of
the amino acid establishes a hydrogen bond to the car-
boxylate of the inhibitor (2.7 Å).20, 30 As indicated earlier,
the terminal carboxylate of the inhibitor makes additional
hydrogen bonds to Arg145 and Asn144 (Nδ1). All these
contacts are important for the proper sequestration of the
inhibitor within the active site. Geometric details of in-
hibitor-enzyme interactions are listed in Table S3.
Figure 2. (A) Stereoimage of human proCPA1 in complex
with compound 11. The backbone of the pro and the catalytic
domains are shown in gray and blue, respectively. The inhibi-
tor is shown in capped sticks. (B) 2Fobs-Fcalc electron-density
map showing the hCPA1 active site covalently bound inhibi-
tor 11, calculated deleting the Glu270 side chain and inhibitor
coordinates, is contoured at 1.5σ level. The inhibitor and
residues important for binding (labeled) are shown in capped
sticks (green and yellow, respectively). The catalytic zinc ion
is in magenta, while the sulfur is in yellow. Oxygen and ni-
trogen are colored red and blue, respectively. A continuous
electron density is clearly observed along the bond linking
Glu270 side chain and the inhibitor. (C) Schematic represen-
tation of the binding of inhibitor 11.
As with the benzyl moiety in phenylalanine in a typical
peptide substrate of CPA, the isobutyl group of the inhibi-
tor makes close contacts with the residues that configure
the hydrophobic pocket, namely Ala250 (3.4 Å), Ile243
(3.7 Å) and Thr268 (3.8 Å). In the present structure, we
can observe substantial conformational changes, com-
pared to other CPA1 (the activated enzyme) structures
with bound ligands.31, 32 Thr268 re-orients its side chain to
contact the inhibitor via its hydroxyl group, changing its
most common rotamer conformation of the χ1 dihedral
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angle from gauche to anti These rearrangements would
reflect the ability of the enzyme to adapt the size and the
shape of the hydrophobic pocket to the characteristics of
the ligand molecule, despite it being different from that of
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Interestingly, the small-molecule thiirane is able to nego-
the natural peptide substrate. We add here that the basis
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for lack of inhibition of the related CPB by compound 11
and its analogues is likely the presence of an acidic resi-
due at the distal end of the hydrophobic pocket. This
acidic residue interacts with a positively charge amino
acid from the substrate, which penetrates the recognition
pocket; hence, inhibitor 11 discriminates against the CPB.
tiate the physical barrier of the pro-domain in human
pro-CPA1. This illustrates the feasibility of employing
such a technology for tracing the fate and the location of
the target enzymes using imaging technology, as exempli-
fied by the fluorinated derivative (Fig. 3). The fact that
these compounds are agents that are converted to the
inhibitory species only within the active site of the target
enzyme, holds the promise for new tools in biomedical
research.
The crystal structure of thiirane-bound hpro-CPA1 has
been used for molecular modeling of the spectroscopically
non-silent inhibitor 22 as an example of compounds that
may act as tracking or imaging probes. As can be seen in
Figure 3, such fluorinated inhibitor molecule would fit
into the active site of hCPA1 with the fluoro-substituted
phenyl ring anchoring in the hydrophobic S1’ pocket of
the enzyme. No steric clashes are observed in the model,
consistent with the favorable kinetic results that we
measured.
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ASSOCIATED CONTENT
Supporting Information. Experimental details, synthe-
sis, kinetic studies, X-ray crystallographic statistics. The
crystallographic coordinates are deposited in the Protein
Data Bank (PDB code 5OM9). This material is available
free of charge via Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +34 935 812 154. E-mail: irantzu.pallares@uab.cat
*Tel: +1 574 631-2933. E-mail: mobashery@nd.edu
Present Addresses
†SAT present address: Instituto de Química Rosario−IQUIR
(CONICET), Facultad de Ciencias Bioquímicas y Farma-
céuticas, Universidad Nacional de Rosario, Suipacha 531,
Rosario S2002LRK, Argentina.
Author Contributions
Figure 3. Molecular modeling of the hCPA1-fluorine inhibi-
tor complex structure. The inhibitor and residues important
for binding are shown in capped sticks, in green and yellow,
respectively. The catalytic zinc ion is in magenta, the sulfur is
in yellow and the fluorine in light blue. Oxygens and nitro-
gens are in red and blue, respectively. The model has been
constructed with PyMOL using PDB file 5OM9.
SAT designed and synthesized compound, CG purified pro-
teins, performed the enzymatic analysis and the crystalliza-
tion trials, DF made preliminary enzymatic analysis, refined
the X-ray structure, GC purified proteins, performed enzy-
matic analysis, PG and DR worked on complex crystallization
and determined the 3D structure, IP calculated kinetic data,
determined the X-ray structure, prepared the figures, SAT,
CG, SM, I.P., JV and FXA wrote the paper with input from
coauthors.
We have documented that our thiirane- and oxirane-
based library of aliphatic and substituted aromatic com-
pounds were able to inhibit a well-studied zinc proteases,
CPA. We can conclude that the inhibitory activity of these
compounds was CPA-specific, as opposed to CPB, which
was not inhibited. Per design paradigms, the time-
dependence of kinetics and the high-resolution X-ray
structure of 11 in complex to human CPA1 zymogen sup-
port it as a mechanism-based inactivator. The structure
presented in this work represents the first human carbox-
ypeptidase zymogen in complex with a mechanism-based
inhibitor. Although all inhibitors were prepared as race-
mic mixtures, the crystal structure of the enzyme inhibit-
ed by compound 11 reveals that only the 2R,3R configura-
tion, the one that corresponds to the D-amino-acid se-
ries,25 could fit to the electron-density map. This indicates
that the enzyme is enantioselective in its interaction with
the inhibitor, coincident with what was reported for an
oxirane inhibitor.25
Funding Sources
Support from MINECO, grants BIO2013-44973R and
BIO2016-78057R is gratefully acknowledged.
Notes
The authors declare no competing financial interest
ACKNOWLEDGMENT
The authors wish to thank Prof. Maya Chávez for helpful
discussions
REFERENCES
1. Perez-Silva, J. G.; Espanol, Y.; Velasco, G.; Quesada, V., The
Degradome database: expanding roles of mammalian proteases
in life and disease. Nucleic Acids Res. 2016, 44 (D1), D351-D355.
2. Tanco, S.; Tort, O.; Demol, H.; Aviles, F. X.; Gevaert, K.; Van
Damme, P.; Lorenzo, J., C-terminomics Screen for Natural
Substrates of Cytosolic Carboxypeptidase 1 Reveals Processing of
Acidic Protein C termini. Mol. Cell Proteomics. 2015, 14 (1), 177-
190.
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3. Sapio, M. R.; Fricker, L. D., Carboxypeptidases in disease:
Inhibited by a Thiirane Mechanism-Based Inhibitor. Chem. Biol.
1
2
3
4
5
6
7
8
Insights from peptidomic studies. Proteomics Clin Appl. 2014, 8
(5-6), 327-337.
4. Rawlings, N. D.; Barrett, A. J.; Bateman, A., MEROPS: the
database of proteolytic enzymes, their substrates and inhibitors.
Nucleic Acids Res. 2012, 40 (D1), D343-D350.
Drug Des. 2010, 75 (1), 29-34.
21. Kim, D. H.; Chung, S. J., Inactivition of Carboxypeptidase-A
by 2-Benzyl-3,4-Epithiobutanoic acid. Bioorg. Med. Chem. Lett.
1995, 5 (15), 1667-1672.
22. Vendrell, J.; Cuchillo, C. M.; Aviles, F. X., The tryptic
activation pathway of monomeric procarboxypeptidase A. J. Biol.
Chem. 1990, 265 (12), 6949-6953.
23. Rajendra, G.; Miller, M. J., Intramolecular electrophilic
additions to olefins in organic syntheses. Stereoselective
synthesis of 3,4-substituted .beta.-lactams by bromine-induced
oxidative cyclization of O-acyl .beta.,.gamma.-unsaturated
hydroxamic acid derivatives. J. Org. Chem. 1987, 52 (20), 4471-
4477.
24. Kim, Y. M.; Kim, D. H., Convenient preparation of all four
possible stereoisomers of 2-benzyl-3,4-epoxybutanoic acid,
pseudomechanism-based inactivator for carboxypeptidase a via
alpha-chymotrypsin-catalyzed hydrolysis. Bull. Korean Chem.
Soc. 1996, 17 (10), 967-969.
25. Ryu, S. E.; Choi, H. J.; Kim, D. H., Stereochemistry in
inactivation of carboxypeptidase A. Structural analysis of the
inactivated carboxypeptidase A by an enantiomeric pair of 2-
benzyl-3,4-epoxybutanoic acids. J. Am. Chem. Soc. 1997, 119 (1),
38-41.
26. Kitz, R.; Wilson, I. B., Ester of Methanesulfonic Acid as
Irreversible Inhibitors of Acetyl Cholinesterase. J. Biol. Chem.
1962, 237 (10), 3245-3249.
5. de la Vega, M. R.; Sevilla, R. G.; Hermoso, A.; Lorenzo, J.;
Tanco, S.; Diez, A.; Fricker, L. D.; Bautista, J. M.; Aviles, F. X.,
Nna1-like proteins are active metallocarboxypeptidases of a new
and diverse M14 subfamily. FASEB J. 2007, 21 (3), 851-865.
6. Zhao, X. J.; Lu, C. P.; Chu, W. W.; Zhang, Y. X.; Zhang, B.;
Zeng, Q.; Wang, R. F.; Li, Z.; Lv, B. L.; Liu, J. B., microRNA-214
Governs Lung Cancer Growth and Metastasis by Targeting
Carboxypeptidase-D. DNA Cell Biol. 2016, 35 (11), 715-721.
7. Huang, S. F.; Wu, H. D. I.; Chen, Y. T.; Murthy, S. R. K.; Chiu,
Y. T.; Chang, Y.; Chang, I. C.; Yang, X. Y.; Loh, Y. P.,
Carboxypeptidase E is a prediction marker for tumor recurrence
in early-stage hepatocellular carcinoma. Tumour Biol. 2016, 37
(7), 9745-9753.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8. Denis, C. J.; Lambeir, A. M., The potential of carboxypeptidase
M as a therapeutic target in cancer. Expert Opin. Ther. Targets
2013, 17 (3), 265-279.
9. Lyons, P. J.; Sapio, M. R.; Fricker, L. D., Zebrafish Cytosolic
Carboxypeptidases
1 and 5 Are Essential for Embryonic
Development. J. Biol. Chem. 2013, 288 (42), 30454-30462.
10. Vovchuk I.L.; Petrov S.A., Role of carboxypeptidases in
carcinogenesis. Biomed. Khim. 2008, 54 (2), 167-178.
11. Fan, S. L.; Li, X.; Li, L. M.; Wang, L. G.; Du, Z. Z.; Yang, Y.;
Zhao, J. S.; Li, Y., Silencing of carboxypeptidase E inhibits cell
proliferation, tumorigenicity, and metastasis of osteosarcoma
cells. Onco Targets Ther 2016, 9, 2795-2803.
12. Fernandez, D.; Pallares, I.; Covaleda, G.; Aviles, F. X.;
Vendrell, J., Metallocarboxypeptidases and their Inhibitors:
Recent Developments in Biomedically Relevant Protein and
Organic Ligands. Curr. Med. Chem. 2013, 20 (12), 1595-1608.
13. Kim, D. H.; Mobashery, S., Mechanism-based inhibition of
zinc proteases. Curr. Med. Chem. 2001, 8 (8), 959-965.
27. Garcia-Saez, I.; Reverter, D.; Vendrell, J.; Aviles, F. X.; Coll,
M.,
The
three-dimensional
structure
of
human
procarboxypeptidase A2. Deciphering the basis of the inhibition,
activation and intrinsic activity of the zymogen. EMBO J. 1997, 16
(23), 6906-6913.
28. Fernandez, D.; Boix, E.; Pallares, I.; Aviles, F. X.; Vendrell, J.,
Analysis of a New Crystal Form of Procarboxypeptidase B:
Further Insights into the Catalytic Mechanism. Biopolymers
2010, 93 (2), 178-185.
29. Guasch, A.; Coll, M.; Aviles, F. X.; Huber, R., Three-
14. Forbes, C.; Shi, Q. C.; Fisher, J. F.; Lee, M.; Hesek, D.; Llarrull,
L. I.; Toth, M.; Gossing, M.; Fridman, R.; Mobashery, S., Active
Dimensional
structure
of
Porcine
Pancreatic
Procarboxypeptidase-A. A Comparison of the A-Zymogen and B-
Zymogen and their Determinants for Inhibition and Activation.
J. Mol. Biol. 1992, 224 (1), 141-157.
30. Cho, J. H.; Kim, D. H.; Chung, S. J.; Ha, N. C.; Oh, B. H.; Choi,
K. Y., Insight into the stereochemistry in the inhibition of
Site Ring-Opening of
a Thiirane Moiety and Picomolar
Inhibition of Gelatinases. Chem. Biol. Drug Des. 2009, 74 (6),
527-534.
15. Ruiz-Cabello, J.; Barnett, B. P.; Bottomley, P. A.; Bulte, J. W.
M., Fluorine (F-19) MRS and MRI in biomedicine. NMR Biomed.
2011, 24 (2), 114-129.
16. Kruger, A.; Arlt, M. J. E.; Gerg, M.; Kopitz, C.; Bernardo, M.
M.; Chang, M.; Mobashery, S.; Fridman, R., Antimetastatic
activity of a novel mechanism-based gelatinase inhibitor. Cancer
Res. 2005, 65 (9), 3523-3526.
17. Kim, D. H., Chemistry-based design of inhibitors for
carboxypeptidase A. Curr. Top. Med. Chem. 2004, 4 (12), 1217-
1226.
18. Brown, S.; Bernardo, M. M.; Li, Z. H.; Kotra, L. P.; Tanaka, Y.;
Fridman, R.; Mobashery, S., Potent and selective mechanism-
based inhibition of gelatinases. J. Am. Chem. Soc. 2000, 122 (28),
6799-6800.
19. Testero, S. A.; Lee, M.; Staran, R. T.; Espahbodi, M.; Llarrull,
L. I.; Toth, M.; Mobashery, S.; Chang, M., Sulfonate-Containing
Thiiranes as Selective Gelatinase Inhibitors. ACS Med. Chem.
Lett. 2011, 2 (2), 177-181.
carboxypeptidase
A
with
N-(hydroxyaminocarbonyl)
phenylalanine: Binding modes of an enantiomeric pair of the
inhibitor to carboxypeptidase A. Bioorg. Med. Chem. 2002, 10 (6),
2015-2022.
31. Mangani, S.; Carloni, P.; Orioli, P., Crystal Structure of the
Complex Between Carboxypeptidase A and the Biproduct Analog
Inhibitor L-benzylsuccinate at 2.0 Angstroms Resolution. J. Mol.
Biol. 1992, 223 (2), 573-578.
32. Fernández, D.; Boix, E.; Pallarès, I.; Avilés, F. X.; Vendrell, J.,
Structural and Functional Analysis of the Complex between
Citrate and the Zinc Peptidase Carboxypeptidase A. Enzyme Res.
2011, 2011:128676.
33. Pallares, I.; Fernandez, D.; Comellas-Bigler, M.; Fernandez-
Recio, J.; Ventura, S.; Aviles, F. X.; Bode, W.; Vendrell, J., Direct
interaction between
a human digestive protease and the
mucoadhesive poly(acrylic acid). Acta Crystallogr., Sect. D: Biol.
Crystallogr. 2008, 64, 784-791.
20. Fernandez, D.; Testero, S.; Vendrell, J.; Aviles, F. X.;
Mobashery, S., The X-Ray Structure of Carboxypeptidase A
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