Phosphonic Analogs of Alanine as Acylpeptide Hydrolase Inhibitors

Article abstract of DOI:10.1002/cbdv.202001004

Acylpeptide hydrolase is a serine protease, which, together with prolyl oligopeptidase, dipeptidyl peptidase IV and oligopeptidase B, belongs to the prolyl oligopeptidase family. Its primary function is associated with the removal of N-acetylated amino acid residues from proteins and peptides. Although the N-acylation occurs in 50–90 % of eukaryotic proteins, the precise functions of this modification remains unclear. Recent findings have indicated that acylpeptide hydrolase participates in various events including oxidized proteins degradation, amyloid β-peptide cleavage, and response to DNA damage. Considering the protein degradation cycle cross-talk between acylpeptide hydrolase and proteasome, inhibition of the first enzyme resulted in down-regulation of the ubiquitin-proteasome system and induction of cancer cell apoptosis. Acylpeptide hydrolase has been proposed as an interesting target for the development of new potential anticancer agents. Here, we present the synthesis of simple derivatives of (1-aminoethyl)phosphonic acid diaryl esters, phosphonic analogs of alanine diversified at the N-terminus and ester rings, as inhibitors of acylpeptide hydrolase and discuss the ability of the title compounds to induce apoptosis of U937 and MV-4-11 tumor cell lines.

Full text of DOI:10.1002/cbdv.202001004

DOI: 10.1002/cbdv.202001004
FULL PAPER
Phosphonic Analogs of Alanine as Acylpeptide Hydrolase Inhibitors
Maciej Walczak,a Agnieszka Chryplewicz,a Sandra Olewińska,a Mateusz Psurski,b Łukasz Winiarski,a
Karolina Torzyk,a Józef Oleksyszyn,a and Marcin Sieńczyk*a
a
Wroclaw University of Science and Technology, Department of Organic and Medicinal Chemistry, Faculty of
Chemistry, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland, e-mail: marcin.sienczyk@pwr.edu.pl
Department of Experimental Oncology, Hirszfeld Institute of Immunology and Experimental Therapy,
b
Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland
Acylpeptide hydrolase is a serine protease, which, together with prolyl oligopeptidase, dipeptidyl peptidase
IV and oligopeptidase B, belongs to the prolyl oligopeptidase family. Its primary function is associated with the
removal of N-acetylated amino acid residues from proteins and peptides. Although the N-acylation occurs in 50–
90% of eukaryotic proteins, the precise functions of this modification remains unclear. Recent findings have
indicated that acylpeptide hydrolase participates in various events including oxidized proteins degradation,
amyloid β-peptide cleavage, and response to DNA damage. Considering the protein degradation cycle cross-talk
between acylpeptide hydrolase and proteasome, inhibition of the first enzyme resulted in down-regulation of
the ubiquitin-proteasome system and induction of cancer cell apoptosis. Acylpeptide hydrolase has been
proposed as an interesting target for the development of new potential anticancer agents. Here, we present the
synthesis of simple derivatives of (1-aminoethyl)phosphonic acid diaryl esters, phosphonic analogs of alanine
diversified at the N-terminus and ester rings, as inhibitors of acylpeptide hydrolase and discuss the ability of the
title compounds to induce apoptosis of U937 and MV-4-11 tumor cell lines.
Keywords: acylpeptide hydrolase, protease inhibitors, apoptosis, 1-aminoalkylphosphonates, anticancer agents..
Introduction
DNA damage,[10] cleaves amyloid β-peptide,[11,12] while
Acylpeptide hydrolase (APH; EC 3.4.19.1), also known its overexpression results in crystallin degradation
as acylaminoacyl-peptidase (AAP), is a serine protease, facilitating cataract development and it regulates the
which, together with prolyl oligopeptidase (POP), activity of proteasome.[13,14] Since APH is involved in
dipeptidyl peptidase IV (DPPIV) and oligopeptidase B the protein degradation cycle, its cellular communica-
(OB), belongs to the prolyl oligopeptidase family (clan tion with proteasome allows the cell to discard
SC, family S9).[1–3] APH primarily removes N-acetylated cytotoxic denatured proteins.[15] Interestingly, APH was
amino acids residues and thus regulates the cellular found to be more sensitive than acetylcholinesterase
level of N-terminal acetylation of proteins.[4] Although to some organophosphorus compounds, which sug-
this post-translational modification occurs in 50–90% gests its potential role in cognitive functions of the
of eukaryotic proteins and N-acetylated proteins show brain.[16–19] Some researchers have demonstrated that
higher stability in vivo as compared to their non- the deficiency of human APH is associated with
acetylated counterparts,[5–7] the precise function of various carcinomas malignancies.[20,21] On the other
this process has not been fully understood. APH also hand, it has been shown that inhibition of APH
displays a peptidase activity on oxidized and glycated induces apoptosis.[22,23] Recent findings have demon-
proteins,[8,9] it participates in the cellular response to strated that APH inhibition induces down-regulation
of the ubiquitin-proteasome system (UPS).[14,24]
Although the molecular relationship between APH and
UPS is yet to be discovered, APH has been considered
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.202001004
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Chem. Biodiversity 2021, 18, e2001004
as an interesting target for new anticancer molecules toward substrates containing N-acetylated terminal
development.
alanine residue,[49,50] we have synthesized and eval-
Since diaryl esters of 1-aminoalkylphosphonates (1- uated the inhibitory potential of phosphonic alanine
AAPs) were obtained about 40 years ago,[25] which was derivatives containing structurally diverse N-terminal
followed by the discovery of their ability to inhibit serine acyl functions and different phenyl ester rings against
proteases,[26,27] they have been widely used to target APH isolated from bovine liver (APHPL) and U937 cell
many human (e.g., DPPIV,[28] thrombin,[29] proteinase lines-derived APH (APHU937) and suggested their
3,[30] neutrophil elastase,[31] cathepsin G,[32] matriptase- potential to induce the apoptosis in selected cancer
2[33]), bacterial (e.g., SufA,[34] SplA,[35] GluC[36]), and viral cell lines.
proteases (e.g., WNV,[37] HCV,[38] ZIKV[39]). The most
essential attribute of diaryl esters of 1-aminoalkylphosph-
onates as inhibitors is their complete lack of reactivity
with cysteine, threonine, aspartyl and metallo-
Results and Discussion
proteases.[40] The selectivity and reactivity of these Considering the substrate specificity of APH toward N-
compounds with the target protease can be increased acetylalanine, we have synthesized a series of diaryl
by introducing a peptide chain or by modifying the ester esters of 1-aminophosphonic acid derivatives
ring structures to change the electrophilicity of the (Scheme 1; 1–32), analogs of alanine having different
phosphorus atom.[41] They also have a few disadvan- N-acyl groups and phenyl ester rings (Table 1). Target
tages, e.g., relatively low solubility, cell membrane phosphonates were synthesized via an amidoalkyla-
permeability or bioavailability.[42] Despite these limita- tion reaction applying the method originally devel-
tions these compounds are very stable in plasma and oped by Oleksyszyn et al.[25] Hydrobromide salts of the
buffers. They were found useful in catalytic antibodies obtained 1-aminoalkylphosphonate diaryl esters were
development.[43] Due to such functional and structural next acylated with different carboxylic acids using
diversity, these compounds can serve as the activity- HOBT as the coupling agent in the presence of DIPEA.
based probes for an in vivo study of protease function or All of the compounds were further analyzed in order
for cellular localization of proteases in living cells.[44,45]
to evaluate their inhibitory potential toward APH. APH
The method for the 1-APPs synthesis originally of two origin was used (APHPL, isolated from porcine
developed by Oleksyszyn et al. relies on the α- liver and APHU937, form U937 cell lines lysate). Most of
amidoalkylation reaction of triphenyl phosphite with synthesized compounds displayed low potency of
benzyl carbamate and appropriate aldehyde.[25] Since action toward both APHs. The highest activity was
then, several new synthetic methods – among them observed for a series of N-acetylated phosphonic
the use of Lewis or Brønsted acids – have been alanines, where Ac-AlaP(OC6H4-4-SCH3)2 (14) displayed
developed.[46,47,48] The Cbz group can be easily the lowest IC50 values of 26 μM and 20 μM toward
removed either with HBr/AcOH or via hydrogenolysis APHPL and APHU937, respectively. Replacing the meth-
(Pd/C), allowing for further coupling with amino acids, ylthio group in 14 by a chlorine atom resulted in an
peptides, or proteins. Although the application of approximately ten-fold decrease in the inhibitor’s
these methods leads to 1-APP obtained as racemic potency of action (12). From the group of Cbz-
mixtures, various chromatographic techniques have protected alanine derivatives, Cbz-AlaP(OC6H4-4-
been found useful in separation of single di- OCH3)2 (IC50 =250 μM) and Cbz-AlaP(OC6H4-4-Cl)2
astereoisomers of 1-APP peptidyl derivatives.[29,31]
(IC50 =190 μM) showed the highest activity toward
The development of APH inhibitors is not very APHU937 while none of Cbz-analogs showed any
advanced, which is quite surprising considering a activity toward APHPL. As the highest activity was
broad spectrum of APH’s biological function. Only few observed for 4-methylthio-substituted esters, this
APH inhibitors have been described so far including group was selected for further derivatives synthesis.
chloromethylketones, peptides and linoleic acid APH from porcine liver was significantly (with minor
isomers.[14,22–24] Although a number of 1-APPs have exceptions) less susceptible to inhibition by synthe-
been designed toward many serine proteases includ- sized inhibitors than APHU937. Since the crystal struc-
ing DPPIV of the prolyl oligopeptidase family, there tures of both, porcine and human APHs have not been
was no account on their application to block the solved yet, it could only be speculated that the
activity of APH. Here we report the inhibitory activity structural differences between two APHs tested here
of simple phosphonic analogs of alanine as APH result in different inhibitors accessibility. Although
inhibitors. Considering the highest activity of APH synthesized compounds display relatively low potency
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Chem. Biodiversity 2021, 18, e2001004
Scheme 1. General synthesis of 1-aminophosphonic acid derivatives (1–32).
of action, their activity is comparable to that of the indicator of apoptosis induction (Figure 1). Both com-
most potent APH inhibitors reported up to date such pounds induced caspase-3 activity in a dose-depend-
as chloromethyl ketones (Ac-L-Leu-CMK; IC50 (APHU937 -
)
ent manner and the observed activity was statistically
=15 μM),[23] peptides (SsCEI 4; IC50 (APHPL)=84� higher in comparison to control starting from 50 μM
16 μM)[14] or linoleic acid isomers (t10,c12-CLA; IC50 concentrations. Compound 14 showed significantly
(APHPL)=105�23 μM).[14]
higher activity in U937 than in MV-4-11, which
The selected compounds most active in enzymatic corresponds to the results of their antiproliferative
assays were further evaluated for their antiproliferative activity. Compounds 5 and 12 showed limited influ-
activity in vitro using two cancer cell lines – U937 and ence on caspase-3 activity. The observed discrepancies
MV-4-11 (Table 2). In comparison to cisplatin and two in compounds’ activity as APH inhibitors and antipro-
known inhibitors of proteasome – MG-132 (Z-Leu-Leu- liferative agents might indicate that APH might not be
Leu-CHO) and Bortezomib (Pyz-Phe-boroLeu) the com- their major intracellular target.
pounds exhibited weak (12) to moderate (7, 14)
antiproliferative activity. The most pronounced differ-
ences between cell lines were observed for com-
pounds 6 for which the IC50 value on MV-4-11 was
Conclusions
over two times higher than for U937.
The reported compounds which belong to a class of
Further studies showed that compounds 7 (IC50 serine protease inhibitors showed ability to inhibit
values of 26.8�7.4 μM and 23.1�1.9 μM toward U937 APH activity. Although the inhibitory potency of
and MV-4-11, respectively) and 14 (IC50 =20.1�8.6 μM synthesized compounds is relatively low (IC50 values in
for U937 and 51.5�6.7 μM for MV-4-11) are relatively a micromolar range), they were able to induce
potent inducers of caspase-3 activity, which is a direct apoptosis in U937 and MV-4-11 tumor cell lines. This
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preliminary data might induce further, more advanced
Table 1. Inhibitory activity of obtained (1-aminoethyl)
phosphonic acid diaryl esters toward APHPL and APHU937
.
research on the development of novel specific APH
inhibitors of this class with potential anticancer
properties.
Compound APHPL
APHU937
inhibition
[%]a
inhibition
IC50
[μM]
IC50
[μM]
[%][a]
1
2
3
4
5
6
7
8
<5#
<5#
<5#
<5#
<5#
5
–
–
–
–
–
–
–
–
–
–
–
–
–
26
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
29
34
30
23
50
65
47
21
22
33
37
48
34
99
36
39
57
36
43
49
48
49
40
46
34
39
69
46
40
37
32
44
–
–
–
Experimental Section
Chemical Synthesis
–
250
190
–
–
–
–
–
–
–
20
–
–
219
–
–
–
–
–
–
–
–
–
180
–
–
–
–
All reagents were purchased from Sigma-Aldrich
(Poznan, Poland). The solvents were from POCh
(Gliwice, Poland). Triaryl phosphites were synthesized
as described previously.[41] Briefly, phosphorus trichlor-
ide (10 mmol) was added to a solution of substituted
phenol (30 mmol) in acetonitrile (50 mL) and the
mixture was refluxed for 8 h. The volatile elements of
the mixture were removed in vacuum and the
resulting crude product was used directly in the next
step for the synthesis of Cbz-protected 1-aminoalkyl-
phosphonate diaryl esters. The crude triaryl phosphite
was dissolved in glacial acetic acid (25 mL) followed by
the addition of an aldehyde (11 mmol) and benzyl
carbamate (11 mmol). The mixture was heated at 80–
<5
6.5
12
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
<5#
9
48
16
99
17
<5
18
<5
13
<5
<5
<5
9
<5
6
<5
17
°
90 C for 2 h. Volatile elements were removed under
reduced pressure, the resulting oil was dissolved in
[25]
°
methanol and left at –20 C for crystallization. Next,
the Cbz-group was removed with 33% HBr in acetic
acid solution. Cbz-protected 1-aminoalkylphosphonate
diaryl ester (10 mmol) was dissolved in a 33% HBr/
AcOH solution (4 mL). The reaction was performed at
room temperature for 2 h. The volatile components of
the mixture were removed under reduced pressure
and the product was crystallized from diethyl ether.
For the preparation of N-acyl derivatives of 1-amino-
alkylphosphonate diaryl esters, HBTU was used as a
coupling agent. The hydrobromide salt of 1-amino-
alkylphosphonate diaryl ester (10 mmol) was sus-
<5
<5
<5
<5
<5
–
[a] Percentage of inhibition after 15 min incubation of APH with
tested inhibitor (250 or 50 μM#).
class of inhibitors has never been examined toward pended in acetonitrile (25 mL) followed by the
their ability to block APH’s activity thus the presented addition of N,N’-diisopropylethylamine (25 mmol), ap-
Table 2. Antiproliferative activity of most active compounds assessed in vitro on two cancer cell lines after 72 h of incubation.
Compound
IC50 �SD [μM], n=3
U937
MV-4-11
12
14
7
5
[32]%[a]
20.1
26.8
42.1
0.49
0.031
1.1
�3
88.7
51.5
23.1
41.4
0.13
0.006
1.4
�5.5
�8.6
�7.4
�9.8
�0.18
�0.008
�0.2
�6.7
�1.9
�2.4
MG-132
Bortezomib
Cisplatin
�0.07
�0.001
�0.3
[a]
Reported values are mean cell proliferation inhibition �SD at highest concentration used [100 μM].
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Figure 1. Caspase-3 activity assessed in vitro on two cancer cell lines after 24 h of incubation with most active compounds and are
presented as fold change in caspase-3 activity �SD; * – p<0.05, one-way ANOVA with Dunnett’s post-test to untreated control,
n=4.
propriate carboxylic acid (10 mmol) and HBTU mL streptomycin (both Polfa-Tarchomin, Warsaw, Po-
(12 mmol). The reaction was performed at room land). All cell lines were cultured in a humidified
°
temperature for 24 h. Next, the solvent was evapo- atmosphere at 37 C with 5% CO2 and passaged twice
rated and the resulting oil was dissolved in ethyl a week.
acetate (100 mL) and washed with 5% NaHCO3, 5%
KHSO4 and brine. The final compounds were purified
by HPLC (Varian ProStar HPLC System, Palo Alto,
Enzymatic Studies
California, USA) equipped with C8 column 250× The APH (APHPL) was isolated in house from porcine
21.2 mm, 10 μm (Supelco, Bellefonte, USA) using 5– liver based on the method described by Kiss et al.[51]
100% in 20 min gradient of A in B, where A: H2O with Fresh porcine liver obtained from a local butcher
0.05% TFA, B: CH3CN with 0.05% TFA. The purity and (2 kg) was homogenized in 50 mM phosphate buffer,
mass of compounds was confirmed using UltiMate pH 7.2, containing EDTA (1 mM), 2-mercaptoethanol
3000 HPLC System with ISQ EC Mass Spectrometer (5 mM) and Na2SO3 (5 mM). Next, the homogenate
°
(Thermo Scientific, Waltham, Massachusetts, USA) was heated to 64 C for 7 min, cooled on ice and
°
equipped with Uptisphere Strategy C18HQ column centrifuged (4750 g, 1 h, 4 C). The obtained super-
250×4.6 mm, 5 μm (Interchim, Montluçon, France) natant was supplemented with ammonium sulfate up
°
using 5–95% in 20 min gradient of A in B, where A : to 20% and stirred for 30 min at 4 C. The precipitate
H2O with 0.1% HCOOH, B: CH3CN with 0.1% HCOOH was centrifuged (4750 g, 1 h, 4 C) and dissolved in
°
(Supporting Material).
50 mM phosphate buffer, pH 7.2, containing EDTA
(1 mM) and 2-mercaptoethanol (5 mM). The purifica-
tion was performed on DEAE-Sepharose column (CL6B;
AKTAprime plus, Pittsburgh, USA) equilibrated with
Cell Lines
The MV-4-11 (human biphenotypic B myelomonocytic 50 mM phosphate buffer, pH 7.2, containing EDTA
leukemia) and U937 (human histiocytic lymphoma) (1 mM) and 2-mercaptoethanol (5 mM). The enzyme
were purchased from the American Type Culture was eluted with the linear salt gradient (0–1 M NaCl
Collection (ATCC; Rockville, USA) and are maintained supplemented with 0.1 M ammonium acetate). The
at Hirszfeld Institute of Immunology and Experimental enzymatic activity at each step of isolation was
Oncology (HIIET, Wroclaw, Poland). The MV-4-11 and evaluated in 10 mM PBS buffer (pH 7.4) using Ac-Ala-
U937 cell lines were cultured in RPMI-1640 medium w/ pNA as the substrate (synthesized in house). Fractions
GlutaMAX® (Thermo Fisher Scientific, Warsaw, Poland) displaying enzyme activity were combined, dialyzed
supplemented with 10% fetal bovine serum (FBS; GE against 20 mM phosphate buffer, pH 7.2. and concen-
Healthcare HyClone, Logan, USA), additionally supple- trated in Amicon Ultra-15 unit (MWCO 30 kDa; Merck
mented with 1 mM sodium pyruvate (Sigma-Aldrich, Millipore, Darmstadt, Germany). The enzymatic activity
Poznań, Poland) for MV-4-11. All culture media con- was assayed with Ac-Ala-pNA (4 mM) using 5 μg/mL of
tained antibiotics – 100 U/mL penicillin and 100 μg/ APH in 10 mM PBS buffer (pH 7.4).
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The activity of the obtained inhibitors toward
Apoptosis evaluation by caspase3/7 activity assay
APHU937 was performed according to the procedure was performed as previously described.[52] 24 h after
described by Yamaguchi et al.[23] U937 cells were seeding the cells (105 cells/well) on 24-well plates
solubilized with PBS supplemented 0.05% Triton X- (Sarstedt, Nümbrecht, Germany), the tested com-
100 followed by centrifugation (13,000 g, 5 min. The pounds at various concentrations were applied for
obtained supernatant was assayed for APHU937 activity 24 h. Then, cells were lysed applying ice-cold lysis
with Ac-Ala-pNA (4 mM, synthesized in house) and buffer (50 mM HEPES, 10% (w/v) sucrose, 150 mM
50 μg/mL of cell lysate (calculated for the total protein NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, pH 7.3). Next,
content; protein content was determined by the micro 25 μL of each sample was transferred to an opaque
BCATM assay (Pierce, Gdańsk, Poland) containing U937- 384-well plate (Corning, NY, USA) containing 75 μL of
derived APH in 10 mM PBS buffer (pH 7.4) at room the reaction buffer (10 μM Ac-DEVD-AMC, 20 mM
temperature.
HEPES, 10% (w/v) sucrose, 100 mM NaCl, 1 mM EDTA,
10 mM DTT, pH 7.3) and the fluorescence was contin-
°
uously recorded at 37 C for 2 h with a Biotek Synergy
H4 Hybrid Reader (λex 350 nm, λem 460 nm) and used
Inhibition Studies
The inhibitory potency of synthesized compounds to calculate the maximal velocity of the reaction.
toward APHs was evaluated in PBS buffer, using Ac- Compounds at each concentration were tested in
Ala-pNA (4 mM) as a substrate and 5 μg/mL of APH duplicates in a single experiment and each experiment
(isolated from porcine liver) or 50 μg/mL (calculated was repeated at least three times. Results were
for the total protein content) of U937-derived APH. All normalized by means of the MTT method and are
tested inhibitors were dissolved in DMSO (10 mM). reported as mean relative caspase-3/7 activity com-
Final DMSO concentration in the assay did not exceed pared to the untreated control.
2.5%. The progress of the enzymatic reaction was
monitored at 405 nm at room temperature for 60 min.
The IC50 values were calculated from the plot of the
percent of inhibition vs. inhibitor concentration. For
Acknowledgments
inhibitors of low potency of action, the percent of APH This work was supported by National Science Center
inhibition at 50 μM or 250 μM inhibitor concentration NCN – Preludium 5 DEC-2013/09/N/ST5/02472. The
was calculated in relation to control sample without funder had no role in the study design, data collection
inhibitor.
and analysis, decision to publish, or preparation of the
manuscript. K.T., Ł.W. and M.S. are thankful to the
Compound’s antiproliferative activity by MTT assay Wrocław University of Science and Technology for
was performed as previously described.[52] 24 h after support (Subsidiary Funds).
seeding the cells (104 cells/well) in 96-well plates
(Sarstedt, Nümbrecht, Germany), tested compounds at
concentrations ranging from 100 to 1 μM were added.
Cisplatin (Ebewe, Unterach am Attersee, Austria) as
Author Contribution Statement
well as two proteasome inhibitors – MG-132 and All the authors have contributed significantly to the
Bortezomib (Sigma-Aldrich, Poznan, Poland) were used submitted work. J.O. and M.S. conceived and designed
as the reference compounds. After 72 h treatment, the experiments. M.W., A.C., S.O., M.P., Ł.W., and K.T.
plates underwent MTT protocol and the absorbance performed the experiments and analyzed the data.
was recorded at 570 nm with a Biotek Hybrid H4 M.W. and M.S. analyzed the data and wrote the article.
Reader (Biotek Instruments, Bad Friedrichshall,
Germany).[53] Compounds at each concentration were
tested in triplicate in a single experiment, and each
experiment was repeated at least three times inde-
pendently. The results are presented as the mean cell
proliferation inhibition or IC50 (half-maximum inhib-
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Received December 7, 2020
Accepted January 8, 2021
[43] Y. Nishiyama, S. Planque, Y. Mitsuda, G. Nitti, H. Taguchi, L.
Jin, J. Symersky, S. Boivin, M. Sieńczyk, M. Salas, C. V.
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