Aminobenzosuberone scaffold as a modular chemical tool for the inhibition of therapeutically relevant M1 aminopeptidases

Article abstract of DOI:10.3390/molecules23102607

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays wer

Full text of DOI:10.3390/molecules23102607

molecules
Article
Aminobenzosuberone Scaffold as a Modular
Chemical Tool for the Inhibition of Therapeutically
Relevant M1 Aminopeptidases
Emmanuel Salomon 1,†, Marjorie Schmitt 1,†, Anil Kumar Marapaka 2,3,†
,
Athanasios Stamogiannos 4,†, Germain Revelant 1, Céline Schmitt 1, Sarah Alavi 1,
Isabelle Florent 5, Anthony Addlagatta 2,3, Efstratios Stratikos 4 , Céline Tarnus 1,‡ and
Sébastien Albrecht 1,
*
1
Laboratoire d’Innovation Moléculaire et Applications, Université de Haute-Alsace, Université de Strasbourg,
CNRS, 68093 Mulhouse, France; emmanuel.salomon@uha.fr (E.S.); marjorie.schmitt@uha.fr (M.S.);
germain.revelant@hotmail.fr (G.R.); celine.schmitt85@gmail.com (C.S.); SALAVI@esta-groupe.fr (S.A.);
celine.tarnus@uha.fr (C.T.)
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana,
India; anilmarapaka@gmail.com (A.K.M.); anthonyaddlagatta@gmail.com (A.A.)
Academy of Scientific and Innovative Research (AcSIR), Rafi Marg, New Dehli 110001, India
Protein Chemistry Laboratory, INRASTES, National Centre for Scientific Research Demokritos,
Agia Paraskevi, 15310 Athens, Greece; a.stamogiannos@gmail.com (A.S.); stratikos@gmail.com (E.S.)
Molécules de Communication et Adaptation des Micro-organismes, Muséum National d’Histoire Naturelle,
CNRS, 75231 Paris, France; isabelle.florent@mnhn.fr
2
3
4
5
*
†
‡
Correspondence: sebastien.albrecht@uha.fr; Tel.: +33-389-336-714
These authors contributed equally to this work.
Current address: Laboratoire Vigne, Biotechnologies et Environnement, Université de Haute-Alsace,
Université de Strasbourg, 68008 Colmar, France.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 September 2018; Accepted: 10 October 2018; Published: 11 October 2018
Abstract: The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one
hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to
investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of
proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases
and including eight members of the monometallic M1 family of aminopeptidases as well as two
members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold
indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested
protease families; it was particularly potent against mammalian APN and its bacterial/parasitic
orthologues EcPepN and PfAM1.
Keywords: M1 aminopeptidases; selective inhibitors; aminobenzosuberone scaffold
1. Introduction
Aminopeptidases (APs) constitute a group of exopeptidases with closely related activities, able to
remove amino-acids from unblocked N-termini of peptide and protein substrates. Among them,
metallo-aminopeptidases (metallo-APs) are widely distributed in organisms with representative
members in animal cells, plant cells, fungi, parasites and bacteria [
sorted into two important subgroups on the basis of the number of zinc ions in their active site that
are involved in catalysis as well as on their protein signatures and folding [ ]. While some of these
enzymes contain only one zinc, others possess a binuclear metal centre, usually referred as a co-catalytic
1,2]. Metallo-APs have been broadly
1,2
Molecules 2018, 23, 2607; doi:10.3390/molecules23102607
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2607
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unit [
3
,4]. Members of the M1 family belong to the subclan MA(E) of metallopeptidases [
1], also called
Glu-zincins and possess a single essential catalytic zinc ion and a common fold, related to their common
ancestry that is related to thermolysin. Present mainly as monomers or homo-dimers (in mammals,
mainly), metallo-APs occupy various cell compartments and although some are secreted, the vast
majority operates as membrane bound ectoenzymes or cytosolic catalysts [
history indicates that they are related to a complex and ancestral gene family that subsequently
yielded series of more divergent sequences [ ]. For this reason, their biochemical characterization and
2,5–7]. Their evolutionary
8
respective biological importance has, for a long time, been hindered by considerable confusion, due in
part to their often large and sometimes overlapping substrate specificities as well as their very close
biochemical properties. For the time being, we have a more precise picture of their highly specialized
roles depending on their localization with many related paralogous members in mammals (12 in
H. sapiens, 11 in rodents), whereas only a handful are known to be encoded in other species (3 or less;
only one in P. falciparum) [1,9].
Human M1 paralogous members [1] have been named Leucyl-cystinyl aminopeptidase (HsIRAP),
endoplasmic reticulum aminopeptidase 1 (HsERAP1), endoplasmic reticulum aminopeptidase
2 (HsERAP2), aminopeptidase Q (HsAPQ), puromycin-sensitive aminopeptidase (HsPSA),
thyrotropin-releasing hormone-degrading ectoenzyme (HsTRHDE), aminopeptidase A (HsAPA),
aminopeptidase N (HsAPN), aminopeptidase O (HsAPO), leukotriene A4 hydrolase (HsLTA4H),
arginyl aminopeptidase B (HsAPB) and arginyl aminopeptidase B like 1 (HsRNPL1) and their
orthologues in E. coli (EcPepN) and P. falciparum (PfAM1). Their so far recognized biological
implications are summarized in Table 1. Broadly, human M1 members play important biological
roles in the renin-angiotensin system, the immune system as well as during the inflammatory process,
while the bacterial and malarial members are involved in nutrient acquisition.
Table 1.
Current established biological functions for representative members of the M1
aminopeptidases [1,10,11].
Organism
M1 Family
Biological Roles
Associated Diseases
Metabolism of regulatory peptides of diverse cell types
Processing of peptide hormones (Angiotensin III and IV,
neuropeptides and chemokines
Regulation of angiogenesis, cell motility, cell-cell adhesion
Coronavirus receptor
Pain sensation, Inflammatory diseases, cancer
& upper respiratory tract infections
APN
Activation or inactivation of various components of the
angiotensin system
APA
Hypertension
LTA4H
Biosynthesis of the proinflammatory mediator LTB-4
Inactivation of Thyrotropin Releasing Hormone (brain)
Inflammatory & allergic diseases
TRHDE
Regulation of neuropeptide activity, digestion of
polyQ peptides
Antigen processing pathway for MHC class I molecules
hematologic cancer
Impedes the development of neuropathology
PSA
H. sapiens
Peptide hormone degradation (oxytocin, vasopressin,
angiotensin III)
cognitive impairment (Alzheimer’s disease,
head trauma, cerebral ischemia)
IRAP
Maintain homeostasis during pregnancy
Inactivation of neuronal peptide (enkephalin, dynorphin)
Generation of antigenic peptides in dendritic cells [12]
Inflammatory autoimmunity [13]
APB
ERAP1
ERAP2
APQ
Biosynthesis of the proinflammatory mediator LTB-4
Regulation of blood pressure
Antigen processing pathway for MHC class I molecules
Autoimmune diseases (ankylosing spondylitis,
psoriasis, type 1 diabetes, Crohn . . . )
Cancer [14–16]
Antigen processing pathway for MHC class I molecules
Placentation: regulation of biological activity of key
peptides at the embryo-maternal interface
Pre-eclampsia
Activation or inactivation of various components of the
angiotensin system
APO
PfAM1
PepN
Catabolism of host haemoglobin in the food vacuole
of Plasmodium
P. falciparum
E. coli
Malaria [17]
Cytosolic peptide catabolism and adaptation to nutritional
downshift and high temperature stress
As a consequence from the definition of the M1 family, all members (the MEROPS database
has so far identified 14718 amino acid sequences belonging to this M1 family) share critical
characteristics [18,19]. Sequences alignment of a selection of M1 aminopeptidases highlights the

Molecules 2018, 23, 2607
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conserved active site residues for substrate recognition and peptide bond hydrolysis that is, the zinc
binding motif Exx x18 , the substrate binding motif, including the GxMEN domain and an extra or
distal residue, responsible for the recognition and binding of the key free amino terminus of peptide
substrates, as well as the catalytic residue (part of H xxH motif) and a distal residue essential for
transition state stabilization in the closed conformations of M1 aminopeptidases (Figure S1) [1].
H
H
E
E
Q
E
E
Y
Although overall amino acid sequence similarity may fall down to a mean value of 20%, a highly
conserved 3D structure, reminding that of a sea-horse with 4 domains, has been found for all these
M1 APs. Domain I folds as a twisted
thermolysin-like fold, domain III is a beta sandwich built with 2
domain IV is a prominent bowl-like structure composed of -helices (330 aa) which contains the active
site channel entrance (about 40 Å long and 12 Å large) [20].
β-barrel of 200 aa, domain II corresponds to the ancestral
β-sheets (absent in HsLTA4H) and
α
Dynamic conformational ensembles, involving inter-domains movements, are crucial for catalysis
and various conformational states are particularly marked for mammalian enzymes. They include
active site opening and closure, particularly important for HsERAP1, HsIRAP and mammalian
APN [20–24], suggesting that the peptide-binding channel could accommodate variously exposed
N-termini of peptides and/or proteins. The influences of protein dimerization on active site accessibility
and specificity as well as on various moonlighting functions have still to be more extensively studied
for all M1 family of APs [23,25,26].
Substrate specificities have also been responsible for the confusion in biological roles attributed
to these enzymes; as illustrated in Table 2, these substrate specificities are rarely discriminant with
overlapping specificities being the rule and specific specificities the rare exceptions.
Table 2. Substrate specificity: preferred N-terminus amino-acid for a selection of M1 aminopeptidases [1].
M1 Aminopeptidase
Substrate Specificity: Favoured N-Terminus Amino-Acid
Ala, Phe, Tyr, Leu, most of aa including Pro (slow)
Glu and to a lesser extent Asp
APN
APA
LTA4H
TRHDE
PSA
Ala, Arg, Leu, Pro
pGlu (pyroglutamyl)
Ala, Leu, Lys, most of aa (except Gly and Pro)
Cys, Leu, Arg, Ala and most of aa (except Asp, Glu) including
cyclic peptides [27]
IRAP
APB
ERAP1
ERAP2
RNPL1
APQ
Arg, Lys
Leu and most of aa including Met, Cys, Phe
Arg, Lys
Ala, Lys, Ser, Ile, Met, most of aa
Leu, Arg, Lys, Met, most of aa
APO
Arg and to a lesser extent Asn
PfAM1
PepN
Ala, Leu, Lys, Arg and most of aa (except Pro, Asp, Glu)
Arg, Ala, most of aa including Pro
This common substrate specificity, together with a very conserved structure and catalytic
mechanism, makes the development of highly selective inhibitors of a given metallo-aminopeptidase
obviously challenging [11,28–31]. Thereby the most notable inhibitors, depicted in Figure 1, consist
in tetrahedral intermediate mimics, as for the widely used bestatin [32] and phosphinic [33]/
phosphonic [34] acid derivatives, or zinc-chelating group inserted in a peptide-like scaffold, as for
hydroxamic acids [35–37].

Molecules 2018, 23, 2607
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O
O
HO
O
HO
O
H
N
P
P
OH
NH2
COOH
H2N
N
H
NH2
O
NH2
Phosphinic acid
OH
O
Ph
(2)
(1)
Bestatin
(3)
Phosphinic acid
Ph
NH
O
OEt
N
O
CO2H
HS
N
N
Ph
NH2
NH2.HCl
(5)
Aminotetralone
(4)
1,2,5,6-Tetrahydropyridine
(6)
Aminothiol
O
F
OH
O
O
O
H
N
N
H
R
H
N
HO
OH
O
N
O
R'O
H
O
O
O
HN
OH
(7)
(9)
(10)
Hydroxamic acid
R = cyclobutyl, R' = H, CHR-2797
Actinonin
(8)
R = H, R' = H, CHR-79888
N
NH2
O
NH2
O
H
N
N
NH
Bn
CO2Et
O
O
HO
HFI-437
NH
O
O
N
H
O
(12)
PN-22
(11)
3,4-Diaminobenzoic acid
(13)
Figure 1. Representative inhibitors of M1 aminopeptidases (non-exhaustive list) For more detailed
structures see references [11,28–31].
In Table 3, we report inhibition values for several members of the M1 aminopeptidase
family and for three selected bi-metallic aminopeptidases, since selectivity towards these other
metallo-aminopeptidases remains of major concern and is indeed currently poorly documented.
Inhibitors targeting mammalian APN have been the most extensively developed, since mammalian
APN was one of the first metallo-aminopeptidase to be biochemically and enzymatically characterized
(although many of its first assigned biological functions were later demonstrated to involve other
related M1 paralogous enzymes). Among them, Bestatin
1 has been very often misused as a “specific”
inhibitor of this peculiar APN enzyme, since it turns out to be a highly potent inhibitor of bimetallic
enzyme families and a quite broad “unspecific” inhibitor of monometallic M1 aminopeptidases.
But overall, it appears obvious that the development of M1 aminopeptidase inhibitors remains mainly
in its infancy.

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Table 3. Ki or * IC50 values in micromoles reported in literature for representative M1 AP’s inhibitors.
M1 Aminopeptidases
‘Monometallic’
M17 & M28 Aminopeptidases
‘Bimetallic’
Compound
1
HsAPA
NI [38]
-
HsAPB
mAPN
HsERAP1 HsERAP2 HsIRAP
HsLTA4H
HsPSA
EcPepN
7.3 * [43]
-
PfAM1
AAP
mLAPc PfAM17
0.014
[39]–6.0
[38]
0.3 *
[37]–89.0
[40]
0.0006
38]–0.020 0.025 [45
[39]
0.2 * [37]
0.5 [42]
0.35 *
[37]
0.0016
[42]
11.2 [41]
0.19 [44]
[
]
]
2
-
0.002 [46
]
-
-
-
-
-
0.079 [47
]
-
-
0.066 [33
3.0
]
0.013 [45
3 [48]
4 [49]
5 [38]
6 [50]
0.043 *
0.037 *
0.002 *
>100
0.02
0.12
1.79 *
>100
>100
>1000
0.14
>1000
>1 *
7 [37]
8 [37]
>10 *
0.008 *
0.15 *
0.85 *
0.1 *
0.03 *
0.079 [36
]
-
0.22 *
0.3
>5 *
-
-
-
6 [36]
-
9
>100 [40] >100 [51] [52]–2.0 *
[40]
2.6 * [51]
10 [35,53]
11 [54]
1.37 *
0.006 *
0.92 *
1.6 *
0.105 *
0.03
12 [55]
>100
0.8 *
13 [56]
M1 monometallic enzymes: human aminopeptidase A (HsAPA), human arginyl aminopeptidase B (HsAPB), mammalian aminopeptidase N (mAPN), human endoplasmic reticulum
aminopeptidase 1 (HsERAP1), human endoplasmic reticulum aminopeptidase 2 (HsERAP2), human Leucyl-cystinyl aminopeptidase (HsIRAP), human leukotriene A4 hydrolase (HsLTA4H)
and human puromycine sensitive aminopeptidase (HsPSA); E. coli (EcPepN) and P. falciparum (PfAM1) M1 aminopeptidases; representative members of the bimetallic aminopetidases
from A. proteolytica (AAP), mammalian cytosolic leucine aminopeptidase (mLAPc) and P. falciparum M17 aminopeptidase (PfAM17). NI, no inhibition. * IC50
.

Molecules 2018, 23, 2607
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Our research group has previously reported the development of potent and selective inhibitors of
APN, a human M1 aminopeptidase involved in angiogenesis and tumour metastasis [57]. This work
allowed us to identify aminobenzosuberone moiety as a promising scaffold that demonstrates an
exceptional selectivity towards mono-metallic aminopeptidases [42,58–61]. In the present work, we have
closely considered the evaluation of these inhibitors on a number of potential interesting targets within
the M1 family of aminopeptidases and established their inhibition profiles. We have reported herein an
improvement of the aminobenzosuberone synthetic route and the biological evaluation of this family of
non-peptidic compounds against 6 human and 2 bacterial/parasitic M1 aminopeptidases, plus members of
the M17 and M28 families. We have also verified the observed selectivity on a panel of proteinases, which
includes representative members of aspartic, cysteine, metallo and serine endopeptidases.
2. Results
2.1. Chemistry
The aminobenzosuberone’s synthetic route, based on our previously described procedure [42
was optimized to improve reproducibility and avoid the appearance of side-products (Scheme 1).
Indeed, silyl enol ether 15, obtained in four steps from commercially available product 14
,58,60],
,
was submitted to a Rubottom oxidation to yield the key intermediate silyloxy-ketone 16. This reaction
was classically performed by action of m-CPBA but regular and unpredictable failure with this
reagent led us to consider alternative oxidizing agents. The best results were obtained with in situ
generated dimethyl dioxirane (DMDO) [62,63], which gave only the expected product 16 in a nearly
quantitative and reproducible yield (up to 95%). The second optimized step was the conversion of the
hydroxy-oxime 17 to its corresponding amino-alcohol 18. Our previously described method for this
reduction consisted of a hydrogenation over Raney nickel [42,58]. Although the procedure worked
efficiently, it was very dependent on the quality and supply of Raney nickel. Moreover, in the case
of brominated compounds it required a careful monitoring to avoid any aromatic debromination.
Thus, several conditions were investigated to obtain compound 18 as Mg/ammonium formate [64],
SmI2 [65], BH3
with this latter but a small amount of debrominated product was still observed. Therefore, addition of
NiCl2 6H2O was successfully replaced by addition of CoCl2 6H2O and the expected amino compound
·
THF [66], LiAlH4 [67] or NaBH4/NiCl2
·
6H2O [68]. Promising results were obtained
·
·
was solely obtained with no effect on aromatic bromine, even without strict monitoring of the reaction
progress and in acceptable yields (60–80%).
Scheme 1. Synthetic scheme of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one
hydrochloride salts. Reagents and conditions: (i) NBS, AIBN, CCl4, reflux, 3 h. (ii) Dimethyl-1,3-
◦
acetonedicarboxylate, NBu4Br, NaHCO3 1M aq., CH2Cl2, 40 C, overnight. (iii) H2SO4, CH3CN, reflux,
◦
◦
overnight. (iv) TBDMSOTf, NEt3, CH2Cl2, 25 C, 2 h. (v) Oxone®, NaHCO3, acetone, CH2Cl2, 25 C,
◦
◦
3 h. (vi) NH2OH. HCl, pyridine, 25 C, 5 h. (vii) TBAF, THF, 25 C, 2 h. Potential regioisomers may be
◦
◦
separated at this point. (viii) NaBH4, CoCl2
·
6H2O, MeOH,
−
30 C to 25 C, 2 h. (ix) Boc2O, Na2CO3,
◦
◦
MeOH, 25 C, overnight. (x) Dess-Martin Periodinane, CH2Cl2, 25 C, 2 h. (xi) PhB(OH)2 or FcB(OH)2,
Pd(PPh3)4, K2CO3, DME, H2O, MW 125 C, 30 min. (xii) 2M HCl in Et2O, 25 C, overnight or for 21d
◦
◦
:
◦
TFA, CH2Cl2, 0 C to RT, 20 min.

Molecules 2018, 23, 2607
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2.2. In Vitro Inhibition of A Representative Panel of M1 Aminopeptidases
We have confirmed and extended the outstanding selectivity of aminobenzosuberone derivatives
for M1 aminopeptidases over most other classes of proteinases (aspartic, cysteine, metallo and serine
endopeptidase) with IC50 value >> 30 µM in all testes cases (Table S1). As expected, the bimetallic
aminopeptidases from the M17 (PfAM17) and M28 (AAP) families were not or only very weakly
inhibited by the tested compounds (Table 4).
Among M1 aminopeptidases, the most potent inhibitory values remained on mammalian APN,
with a subnanomolar Ki value for compound 21i (Ki = 60 pM). Note that HsLTA4H was not inhibited by
this low-molecular weight scaffold, except for 21c (Ki = 19 µM). The SAR determined for mammalian
APN was very similar for its M1 orthologues from microorganisms, EcPepN and P. falciparum PfAM1,
with an improved affinity when the aminobenzosuberone scaffold was substituted in C-1 or C-4
position with a hydrophobic functional group (for instance 21i exhibited Ki values of 50 nM and 30 nM
against EcPepN and PfAM1, respectively). The influence of bromine in C-1 position remained similar
for the enzymes of both microorganisms with a 10 fold increase in potency when compared to the
unsubstituted derivative 21a. The main differences relied on the lack of synergistic effect (100-fold more
potent for APN) when both positions were occupied by these functions (Ph and/or Br). Compounds
21c and 21i showed similar potencies on PfAM1 (Ki values of 50 nM and 30 nM, respectively) and 21i
was only 3 times more potent than 21c on EcPepN.
As regard to human enzymes, HsPSA is well-inhibited with Ki values of 55 and 21 nM for 21c
and 21i, respectively. Within the oxytocinase subfamily, compounds were less active with inhibitory
potencies in the micromolar range, except for 21h on HsIRAP (Ki = 34 nM). Generally, the inhibitors
were less potent on HsERAP2 and HsERAP1 even if a similar trend emerged in the SAR studies.
Actually, substitution at C-4 position seemed to be more efficient than substitution in the C-1 position,
21c was usually more potent than 21f and 1,4-disubstituted derivatives 21h and 21i displayed the most
interesting binding affinity with a precise substitution pattern to achieve an excellent potency.

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Table 4. Inhibition of selected aminopeptidases by racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochloride salts.
M17 & M28
Aminopeptidases
‘Bimetallic’
M1 Aminopeptidases
‘Monometallic’
R1
R4
REcPepN m
rHsERAP1 n
*
rHsERAP2 o
*
rHsPSA p
rHsIRAP q
*
rHsLTA4H s [58]
rPfAM17 t
AAP u [58]
Papn k [58]
1
rPfAM1 l
15 ± 0.07
0.04 ± 0.009
0.05 ± 0.005 [69]
>50
21a
21b
H
H
H
Br
Ph
Fc
H
50 ± 3.0
1.07 ± 0.11
0.16 ± 0.012
0.06 ± 0.008
-
-
49 ± 3.8
18 ± 1.3
-
-
-
>100
>100
19
-
>100
0.04
120 ± 11
63 ± 3
-
1.63 ± 0.08
0.05 ± 0.004
1.0 ± 0.08
2.4 ± 0.3
-
>100
>100
28
21c
H
0.007
0.004 [60]
0.02
>100 [69]
21d
H
-
-
-
-
21e
Br
Ph
1 ± 0.04
2 ± 0.07
15 ± 1
0.005 ± 0.001
0.03 ± 0.005
4 ± 0.7
4.59 ± 0.17
56 ± 7
137 ± 5
-
104 ± 7
>200
1.07 ± 0.063
2.6 ± 0.2
2.2 ± 0.13
-
>100
>100
>100
>100
68% #
54% #
>100
>100
>100
>100
>100
39
21f
H
0.25
-
-
>100
21g
Benzo [1,2]
0.04
-
-
-
-
-
-
-
21h
Br
Br
Ph
Br
Ph
Br
0.006
0.00006
0.07
-
5.5 ± 1.1
1.6 ± 0.1
-
2.8 ± 0.3
0.39 ± 0.013
-
-
0.034 ± 0.001
0.12 ± 0.015
21i
0.05 ± 0.003
0.021 ± 0.003
21j
-
-
-
>100
Ki values (
µ
M) determined from Dixon plots or * IC50 values (
—human endoplasmic reticulum aminopeptidase 2 (rHsERAP2),
—human leukotriene A4 hydrolase (rHsLTA4H), —P. falciparum rPfAM17 and
r stands for recombinant and Fc for Ferrocenyl. # % of inhibition determined at 100 µM. Inhibition values for pAPN, HsLTA4H and AAP were already reported in literature [58,60].
µ
M) of compounds 21a
–
j
with,
k
—pAPN from porcine kidney,
l
—P. falciparum rPfAM1.
m
—E. coli rEcPepN,
n
—human
endoplasmic reticulum aminopeptidase 1 (rHsERAP1),
—human Leucyl-cystinyl aminopeptidase (rHsIRAP),
o
s
p
—human puromycine sensitive aminopeptidase (rHsPSA),
—aminopeptidase from A. proteolytica (AAP).
q
t
u

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3. Discussion
3.1. Structure-Based Analysis of the Structure-Activity Relationships
In the present work, we focused on subtle amino acid sequence differences in the active site
that may explain the various inhibition values experimentally determined for the selected M1
aminopeptidases. In addition, conformational dynamics have been considered for some members
of this family among the structures captured during various crystallization processes [20,21,24,70].
Data remain scarce due to the small number of solved 3D structures and no general statements could
be outlined, nevertheless our SAR studies enabled to precise the structural environment required for
potent inhibition by the aminobenzosuberone derivatives of a given enzyme within the M1 family.
Our discussion will essentially focus on “closed” conformation of different aminopeptidases.
The determined structures from various species indicate that the active site of a M1
aminopeptidase is buried inside the protein and its zinc binding site may be located in a spacious
cavity with wide openings, or located in a small and gated compartment as it is the case for
bacterial/parasite orthologues [71]: this was in agreement with the active site’s cavity volume
of different M1 aminopeptidases analysed by KVFinder (Table S2 and Figure S2) [72]. Local or
intradomain/interdomain movements can cause enlargement or contraction of the active site as
encountered for EcPepN or mammalian APN [20,25,71,73].
However, the architecture and amino acid composition of the active site are highly conserved
within the whole family [1], which permits to predict an identical binding mode of the
aminobenzosuberone ligand in the various M1 aminopeptidases similar to the previous model reported
for mammalian APN [60] or EcPepN-aminobenzosuberone co-crystallized structures (Figure 2) [74].
(a)
Tyr381
Glu298
O
Glu320
(HExxHx18E)
E
(catalytic, H xxH)
O
H
O
Zn2+
O
O
'
S1
Glu264
(GAMEN site)
O
Val294
His297
H
H
H
+O
A
O
O
H
H
N
4
Tyr376
Met260
Glu121
O
B
9
O
1
S1
(b)
Figure 2. Interactions of aminobenzosuberone derivative 21c with EcPepN. (a) Surface representation
of compound 21c in the active site of EcPepN (PDB 5MFS). The binding pocket is shown in light-blue
surface representation and the zinc ion is represented by a grey sphere (prepared using PyMOL
Molecular Graphics System). (b) Schematic representation of compound 21c binding mode in the active
site of EcPepN. Hydrogen bond and metal interactions are shown with dotted purple and red lines,
respectively. Green lines represent the hydrophobic residues located in the active site [74].

Molecules 2018, 23, 2607
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3.2. S1 Subsite
The S1 subsite is always described as a cylinder formed by hydrophobic residues and capped
by two polar amino acids [75 76]. In Table 5, key residues of this constitutive recognition site are
,
summarized. Interestingly, a highly variable residue (highlighted in “red”), both in steric size and
polarity is located at the entrance of this pocket and may contribute to the diversity of S1 specificities
(Table S3 and Figure S3) [75].
The aforementioned variable residue may be flexible upon ligand binding or might be sterically
hindered, restricting thus the S1 subsite entrance (Figure 3 and for more details, Figure S3). For instance,
in EcPepN it corresponds to Met260 (PDB 2DQM or 5MFS) with a temperature factor value of 28–37 Å2,
in agreement with a local significant flexibility of its side chain, which acts as a cushion to accommodate
various side chains in P1 position of a substrate/ligand. This local movement of Met residue can
cause the contraction of the active site cavity. KVFinder analysis revealed that the cavity volume
of EcPepN-bestatin 1 complex (PDB 2DQM) was higher (638.5 Å3), compared to 531.25 Å3 for the
EcPepN-aminobenzuberone 21c complex (PDB 5MFS) (Table S2). In this latter complex, this Met260
residue was found at two closed positions and moved slightly inward the S1 subsite, interrupting and
shortening its typical cylindrical shape. The distance between the sulphur atom of Met260 and the
centroid of the aromatic ring of the aminobenzosuberone scaffold is 5.3–5.7Å, which is consistent with
sulphur-carbon van der Waals contacts and the sulphur lone pairs might interact with the edge of the
aromatic core, providing additional stability (Figure S4) [77]. On the other hand, Val459 in PfAM1
(PDB 3EBH) and Pro333 in HsERAP2 (PDB 4JBS) might be considered as straight and bulky, shortening
the S1 pocket. In HsLTA4H, Tyr267 (PDB 1HS6) in this position induces the formation of a narrow
S1 tunnel. Moreover, domain III is missing in this latter enzyme and the presence of Tyr267, Lys565
and particularly Tyr378, which extends into the active site cavity pointing inward toward the Zn ion,
reduced substantially the active site cavity volume (Figure S5). As a result of this spatial arrangement,
steric clashes with the aromatic core of aminobenzosuberone might explain the very weak inhibitory
activities of this scaffold on HsLTA4H.
The aminobenzosuberone derivatives are markedly more potent on M1 aminopeptidases
exhibiting a shallow S1 cavity with small to moderate size (EcPepN, HsAPN, PfAM1 and a borderline
case for HsIRAP). At the top of the elongated S1 subsite cylinder are located two or three cap residues
(presented in bold type in Table 5 and depicted in stick in Figure 3a and Figure S3) from the C-terminal
domain of the proteins. These residues are known to influence S1 subsite specificity [78,79]. Indeed,
depending on the nature of the residues and through the conformational flexibility of their side chains,
they can mitigate any potential unfavourable steric clashes and favour van der Waals, H-bond or
electrostatic interactions with the substrates/inhibitors. Moreover, the closing of the S1 subsite induces
the expulsion of water molecules from the active site into the bulk (entropically favourable) and may
strengthen both van der Waals and electrostatic interactions. The analysis of superimposed structures
showed that our aminobenzosuberone inhibitors were not expected to penetrate into the S1 cavity as
deeply as other inhibitors (like bestatin) (Figure S4a and Figure S6), indicating they should therefore
interact more efficiently with shallow S1 subsite.
However, depending on the nature of the variable amino acid residue, the aminobenzosuberone
core might slightly move upward to minimize any potential steric hindrance with this special
‘gatekeeper’ residue. Substituents on position-1 most likely induced steric conflicts with residues at
the entrance of S1 cavities. More suitable substitutions in position-9 should offer new scopes for novel
series of more potent and selective aminobenzosuberone derivatives (Figure S6).

Molecules 2018, 23, 2607
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Table 5. Amino acid composition of the S1 subsite of selected M1 APs.
M1 AP
PDB
Amino Acid Composition of the S1 Subsite
4FYT
4FYR
Q211
Q211
Q181
D198
Q293
Q134
Q119
Q119
Q317
Q213
Q213
E183
E200
E295
Q136
E121
E121
E319
N350
N350
Q315
A332
E426
A351
A351
S316
P333
A427
Y267
G352
G352
G317
G334
G428
G268
A353 M354
A353 M354
A318 M319
A335 M336
A429 M430
G269 M270
A262 M263
A262 M263
A461 M462
E355
E355
E320
E337
E431
E271
E264
E264
E463
H392
H392
H357
H374
H468
H299
H301
H301
H500
E411
E411
E376
E393
E487
E318
E320
E320
E519
F472
F472
F433
F450
F544
Y378
Y376
Y376
Y575
Y477
Y477
Y438
Y455
Y549
Y383
Y381
Y381
Y580
F896
S897
S869
R895
Y961
HsAPN
A214
P184
P201
P296
A137
A122
A122
A320
D216
A186
Q203
S469
R430
Q447
E541
D375
S895
E865
Y892
P957
HsERAP1 2YD0 H160
F314
F331
F425
S868
HsERAP2
HsIRAP
4JBS
E177
5MJ6 Y272
HsLTA4H 1HS6
5MFS
M260 G261
N259 M260 G261
N458 V459 G460
R825
R825
EcPepN
2DQM
N373
E572
Q821
PfAM1
3EBH T305
T321
F457
M1034
In red, the variable residue determined for the corresponding pdb entries. In blue, residue situated at the top of S1 subsite and involved in the capping of the pocket.

Molecules 2018, 23, 2607
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(a)
(b)
Figure 3. Diversity in the S1 subsite of different M1 aminopeptidases (
a) Superimposition of the
protein backbone of S1 subsite of different M1 APs: HsAPN (cyan, PDB 4FYT), HsERAP1 (orange,
PDB 2YD0), HsERAP2 (yellow, PDB 4JBS), HsIRAP (white, PDB 5MJ6), EcPepN (green, PDB 5MFS),
PfAM1 (magenta, PDB 3EBH). (b) Surface representation of the S1 subsite of EcPepN (PDB 5MFS).
The catalytic zinc ion is shown as grey sphere. Residues involved in S1 plasticity are shown in stick.
Images generated with KVFinder plugin in PyMOL.

Molecules 2018, 23, 2607
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3.3. S10 Subsite
In contrast to S1, the S10 subsite is an open cavity of 9 to 10 residues for all enzymes. For instance,
in EcPepN, Gly261, Ala262, Tyr275, Arg293, Val294, His297, Glu298, Val324, Asp327, Tyr381 compose
this subsite. The careful analysis of the various 3D structures showed that only 2 to 3 residues
(highlighted in blue and red in Table 6) seem to be involved in S10 plasticity, thereby controlling
the cavity width and depth (Figures 4a and S7). Two amino acids, which are Tyr275 (located on the
sidewall opposite to the catalytic zinc ion) and Arg293 (partially capping the S10 cavity) for EcPepN,
vary in size and polarity within this enzyme family, as summarized in Table 6 and Figure 4a.
0
Table 6. S1 subsite composition.
0
M1 AP
HsAPN
Amino Acid Composition of the S1 Subsite
G352
G317
G334
G428
G268
G261
G460
A353
A318
A335
A429
G269
A262
A461
R381
T384
V385
T350
V367
I461
H388
H353
H370
H464
H295
H297
H496
E389
E354
E371
E465
E296
E298
E497
S415
K380
K397
T491
V322
V324
V523
E418
E383
E400
E494
E325
D327
E526
Y477
Y438
Y455
Y549
Y383
Y381
Y580
HsERAP1
HsERAP2
HsIRAP
HsLTA4H
EcPepN
PfAM1
G346 M349
W363 R366
S348
T442
L457
K460
N291
R293
T492
V292
V294
V493
R563 K565
Y275
R489
Highlighted residues are involved in S10 site. In blue, residues which reduce the cavity width; in red, residues
which modulate the cavity depth and in bold, flexible amino acid side chains pointing either inward or outward the
active site.
(a)
Figure 4. Cont.

Molecules 2018, 23, 2607
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(b)
Figure 4. Diversity in the S10 subsite of different M1 aminopeptidases. (
a
) Superimposition of the
protein backbone of S10 subsite of different M1 APs: HsAPN (cyan, PDB 4FYT), HsERAP1 (orange,
PDB 2YD0), HsERAP2 (yellow, PDB 4JBS), HsIRAP (white, PDB 5MJ6), EcPepN (green, PDB 5MFS),
PfAM1 (magenta, PDB 3EBH). (
b
) Surface representation of the S10 subsite of EcPepN (green, PDB
0
5MFS). The catalytic zinc ion is shown as grey sphere. Residues involved in S1 plasticity are shown in
stick. Images generated with KVFinder plugin in PyMOL.
For example, PfAM1 Arg489 is found more buried in the cavity than HsAPN Arg381, reducing
thus slightly the width of S10 subsite, whereas the EcPepN Tyr275 and HsAPN Arg381 are positioned
at a similar location, modifying thereby the local polarity. Also, the EcPepN Arg293 side chain
is located further inside the cavity reducing the cavity depth, whereas the less bulky and polar
PfAM1 Thr492 and HsAPN Thr384 occupy a similar position. Finally, local flexibility was observed
within captured structures. Indeed, the side chain of residue HsAPN Arg381, HsERAP2 Arg366 and
EcPepN Arg293 can adopt different positions upon ligand binding. A similar observation might
also be noticed for EcPepN Asp327, this carboxylate side chain is either orientated in- or outside
S10 cavity, with the inside orientation potentially involved in further interactions as exemplified by
N. meningitides alanyl aminopeptidase inhibition study [80]. In its close vicinity, a Lys residue is found
in HsERAP1 and HsERAP2, Lys380 and Lys397, respectively, with their side chains extending in the
cavity (residue in black bold in Figure S6). Developing interactions with this particular residue via
para- or meta-substituted phenyl ring at position 4 of the aminobenzosuberone core should improve
the selectivity and affinity for both members of this oxytocinase family.
All this information clarifies the inhibition observed for HsAPN and its microbial orthologues,
0
EcPepN and PfAM1. Despite PfAM1 S1 cavity volume being slightly larger than HsAPN and EcPepN
0
(Table S4), the S1 subsite cavity width of PfAM1 is shorter, leading to a lack of binding affinity for the
bulky ferrocenyl substituent of 21d which cannot fit appropriately the PfAM1 S1 subsite (Ki > 50
0
µM).
With regard to the SAR experimentally determined, it highlights the importance of the substitution
in position-4 of the aminobenzosuberone core, which, according to our model, almost fully occupies
this subsite in the evaluated enzymes. Most aminopeptidases can accommodate bromine or a phenyl
group with an improved affinity of the inhibitor, when compared to the unsubstituted derivative.
Nevertheless, a phenyl group is mostly preferred probably due to a possible π-stacking with EcPepN
His297, leading to the low Ki value of 21c when compared to 21a or 21b.
3.4. Intra/Inter-Domain Movements
An important feature to be considered for M1 aminopeptidases molecular recognition is their
inter- or intra-domain movements [20 25]. Various conformations have been captured during
–

Molecules 2018, 23, 2607
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crystallization processes and open, intermediate and closed conformations have been determined for
various given enzymes, suggesting that these dynamics are important for ligand binding and peptide
hydrolysis [21,22,70,81]. A large inter-domains motion has been markedly observed for HsERAP1,
associated with local changes for the highly conserved Tyr residue (Tyr438 for HsERAP1) in the
active site (corresponding to the position Tyr381 of EcPepN in Figure S1). In an “open” form of the
protein, this residue undergoes a 6 Å shift away from the zinc ion, whereas in a closed form, this Tyr
residue is proposed to both assist in the activation of the peptide bond and in the stabilization of
the tetrahedral intermediate during the proteolytic catalytic cycle [21,22]. It has been shown, by site
directed mutagenesis, that mutation of this Tyr residue into Phe completely abolished the enzyme
activity [82,83].
As a result of this dynamic, the open form of the enzyme is likely catalytically inactive and a
subsequent allosteric activation upon substrate binding might trigger a catalytically active closed form.
This dynamic transition between those different states might help the substrates/products to bind
to or to be released from the active site. In solution, a number of conformers might exist, from open
to closed states, along with different position of the Tyr residue. We have previously suggested [60
]
that this Tyr residue probably activates the carbonyl function of the aminobenzosuberone towards
a nucleophilic attack of the catalytic water to form a hydrated form of this ketone function and
generates in situ a potent chelator of the zinc ion and a perfect mimic of the transition state. So,
our low-molecular weight reversible inhibitors would be likely to bind to a closed form of HsERAP1
for optimum interactions. Interestingly, HsIRAP was reported to undergo a similar conformational
change with, this time, complete different orientation of the GAMEN motif upon inhibitor binding,
leading to a closed conformation of HsIRAP (PDB 5MJ6) and highlighting some significant structural
plasticity of the GAMEN loop [24]. Since those enzymes in solution should exist in a conformational
equilibrium between different forms (even so small angle X-ray scattering experiments have suggested
that the HsERAP1 average structure in solution adopts an open conformation) [70
could account for the moderate inhibition activity values observed with the HsERAP sub-family
(IC50 = 1.6 to 150 M) and, to a lesser extent, HsIRAP (IC50 = 0.034 to 2.6 M), indicating that the
,81], these processes
µ
µ
aminobenzosuberone scaffolds are not able to induce domain movements to a more closed form of
the enzyme.
Intra-domain mobility is also to be considered. For example, besides a more compact folding,
a particularity of the human and porcine APN structures determined up to now is the plasticity of
the S1 subsite through conformational changes. It looks like a small/shallow hydrophobic pocket,
compared to the deeper S1 subsites observed in other crystallized M1 aminopeptidases [20,25]. Indeed,
HsAPN Phe896 which serves to cap the S1 pocket belongs to a flexible loop (as indicated by an average
temperature factor of 25–35 Å2) of 8 residues (Y891GGGSFSF898) in domain IV and might act as a
gatekeeper restricting access and closing the S1 subsite. Upon ligand binding, this S1 pocket might
undergo a major conformational change via movement of the flexible 891–898 loop, revealing thus a
deeper cylindrical hydrophobic S1 subsite [25]. The cavity volumes of the active site of HsAPN varied
from 572.12 Å3 to 811.87 Å3 (Figure S8 and Table S2). Our series of compounds are very potent on this
human enzyme, probably through interactions with the “Phe-In” conformation of this loop via the
bromine substituent in position-1 of the aminobenzosuberone core and the aromatic ring of Phe896 (8i
,
Ki = 60 pM) [60 74]. These interactions might induce or stabilize the closure of domain IV, especially
,
that of mammalian APN and hence a closed APN form, which is an important feature to block porcine
coronavirus cell entry via its porcine APN receptor [23].
These different conformational dynamic processes seem to be an important aspect of catalytic
regulation for some M1 enzymes, although it has yet to be more documented with a larger number
of structures. These inter- or intra-domain movements are probably only one remarkable feature of
aminopeptidase M1 dynamics, according to their biological functions and their cellular localization.

Molecules 2018, 23, 2607
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3.5. ADME-Tox Properties
To gain insight into the physicochemical and ADME-Tox properties of these aminobenzosuberone
derivatives, several parameters were experimentally determined or computed (Table S5) [84].
The experimental logD7.4 values of the whole aminobenzosuberone series complied with Lipinski’s
“rule of five” [85,
86] and the calculated TPSA value is comprised between 44.71 and 68.10 Å2
for the ketone and hydrate forms, respectively. These compounds are predicted to penetrate the
blood-brain barrier, although some possible P-glycoprotein (P-gp) efflux issues should be taken
into consideration (predicted probability to be P-gp substrate is 0.5). They could be considered
as valuable chemical tools to target M1 aminopeptidases implicated in CNS diseases, mostly
involved in the brain renin-angiotensin system (HsIRAP, HsAPN, HsAPA) [87
pain management (HsAPN) [57 90]. However, the aminobenzosuberone scaffold requires careful
optimization and increasing selectivity toward targeted M1 aminopeptidase versus HsPSA, which
plays major neuroprotective roles [91 96]. Analysis of the calculated probabilities of drug-drug
–89] and in the
,
–
interaction with different CYP450 isozymes shows that this series of molecules should inhibit CYP1A2
and to a lesser extent CYP2D6. In addition, the probability of toxicity is high and is mostly associated
with cardiotoxicity with the inhibition of hERG channel and also hepatotoxicity. The uncertainties in
the predicted outcomes should be taken into account and these potential toxicity liabilities should
be investigated further. Thus, considering ADMET properties and inhibitory activities, the most
promising compound for subsequent studies is the 1,4-dibromo derivative 21h, which is also the
most synthetically feasible molecule. However, according to the predictive method, decreasing the
lipholicity (clogP ou logD7.4) by introducing heteroatoms into the phenyl ring of compound 21c might
also be a direction of modification since it should improve the ADME properties and have beneficial
effects on toxicity.
4. Materials and Methods
4.1. General Information
Reactants were purchased from usual provider. Usual solvents were freshly distilled, dry MeOH
distilled over Mg/MgI2, dry DME over Na and benzophenone, dry Et2O was distilled and stored over
Na, DCM was distilled over P2O5 and stored over anhydrous K2CO3.
Flash chromatography: silica gel (Merck 60, 230–400 mesh). TLC: Al-roll silica gel (Merck 60,
1
F254). Mp: Kofler hot bench, corrected. H- and 13C-NMR (400 MHz and 100.6 MHz resp.) spectra:
Bruker Avance 400; δ in ppm and J in Hertz.
All these racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochloride
salts were already fully described in literature [58,60] and were resynthesized to determine their
selectivity profiles. Their inhibitory activities against pAPN, hLTA4H and AAP were already
reported [58,60].
4.2. General Procedure for Rubottom Oxidation
To an ice-cold mixture of water and acetone (70:35 mL) were added NaHCO3 (6.18 g, 20 eq.) and
Oxone® (11.3 g, 5 eq.). The suspension was stirred at 0 ◦C for 30 min and then a solution of silyl enol
ether 15 (1.3 g, 1 eq.) in DCM (70 mL) was dropwise added. The mixture was warmed to r.t. and
stirred for 3 h (TLC monitoring). Layers were separated and aqueous layer was extracted with DCM.
Combined organic layers were washed with brine, dried on MgSO4, filtered and concentrated to give
silyl-oxy ketone 16, which was used without further purification.
4.3. General Procedure for Oxime Reduction
To a solution of hydroxy-oxime 17 (220 mg, 1 eq.) in methanol (13 mL) was added CoCl2
(388 mg, 2 eq.). The mixture was cooled to
30 ◦C and NaBH4 (462 mg, 15 eq.) was carefully added.
The reaction was slowly warmed to r.t. and stirred for 2 h (TLC monitoring). The mixture was diluted
·
6H2O
−

Molecules 2018, 23, 2607
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with water and extracted by AcOEt. Organic layers were washed with brine, dried on MgSO4, filtered
and concentrated to give amino-alcohol 18, which was N-protected without further purification.
4.4. Production and Purification of Recombinant Aminopeptidases
4.4.1. PfAM1 and PfAM17
Genes encoding residues 192–1085 of native Plasmodium falciparum alanyl aminopeptidase
PfAM1 (PlasmoDB PF3D7_1311800) and residues 84–605 of native Plasmodium falciparum leucyl
aminopeptidase PfAM17 (PlasmoDB PF3D7_1446200) were synthetized by Genecust (Luxembourg),
with the help of algorithms developed for optimizing, DNA sequences to improve proteins expression
in Escherichia coli [97]. iSynthetic genes were cloned into the T7 expression pET45b(+) vector (Novagen),
which appended a N-terminal hexahistidine tag (KnpI and SalI sites for PfA-M1–BamHI and SalI sites
for PfA-M17).
Escherichia coli Rosetta 2 (DE3) bacteria (Novagen) were transformed with these recombinant
plasmids, after their validation by Sanger sequencing (Beckman Coulter Genomics). Bacterial cultures
were grown in auto-induced LB medium (Merck) s◦upplemented with carbenicillin (50
µg/mL) and
chloramphenicol (34 µg/mL), during 24 h at 25 C, prior to bacterial extract preparations with
BugBusterTM (Novagen, Darmstadt, Germany). The clarified lysates were loaded onto Ni2-charged
HisTrap column (GE Healthcare) equilibrated in 20 mM imidazole phosphate buffer and washed in
the same buffer. Bound recombinant proteins were then eluted in 80 mM imidazole for PfAM1 and
200 mM imidazole for PfAM17, in phosphate buffer. Eluted fractions were finally purified by size
exclusion chromatography on a Superdex 200 10 300 (equilibrated with Tris HCl 50 mM, NaCl 200 mM,
ZnCl2 10 µM, pH 7.4 for PfAM1 and with Hepes 50 mM, NaCl 300 mM, ZnCl2 10 µM, 5% glycerol,
pH 8.5 for PfAM17) using an Äkta purifier chromatography system (GE Healthcare Life Science,
Little Chalfont, England).
4.4.2. HsERAP1, HsERAP2 and HsIRAP
The expression and purification of recombinant human endoplasmic reticulum aminopeptidase
1 (HsERAP1), endoplasmic reticulum aminopeptidase 2 (HsERAP2) and insulin-regulated
aminopeptidase (HsIRAP) have been described before [79,98,99]. Briefly, recombinant baculovirus
containing the gene for each enzyme was produced in sf9 cells according to the manufacturer’s
instructions (Bac-to-Bac baculovirus expression system, Invitrogen). Recombinant proteins were
expressed in Hi5 cells after infection with the appropriate recombinant baculovirus and purified by
NiNTA affinity chromatography as previously described (PMID: 21314638). Proteins were aliquoted
◦
and stored at
until needed.
−
80 C in a buffer containing 10 mM Hepes pH 7.0, 100 mM NaCl, 10% glycerol,
4.4.3. EcPepN and HsPSA
Both the enzymes were purified as reported earlier [73,100].
4.5. Enzymatic Assays and Kinetic Analysis
4.5.1. PfAM1 and PfAM17
Tests were carried out on HP/Agilent UV-Visible diode array spectrophotometer 8453
(HP/Agilent, Santa Clara, CA, USA), at 30 ◦C in Tris HCl 50 mM pH 7.4 for rPfAM1 and pH 8 for
rPfAM17, with a final DMSO concentration of 1%. Enzyme activities of rPfAM1 (10 nM) and rPfAM17
(60 nM) were determined by continuously measuring the release of para-nitroaniline at 405 nm with
alanine para-nitroanilide as substrate for rPfAM1 (Km = 1.5 mM) and leucine para-nitroanilide
for rPfAM17 (Km = 0.5 mM). After 10 min of incubation, CI50 measurements were conducted

Molecules 2018, 23, 2607
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during 30 min for rPfAM1 and 3 h for rPfAM17. Initial velocities were measured with increasing
concentrations of inhibitors and Ki values were determined by Dixon plots in triplicate.
4.5.2. LTA4H
5 µg of rLTA4H (Sigma) were used to measure the activity of this protein through the release
of alanine para-nitroanilide at 405 nm (Km = 2 mM). Experiments were performed on HP/Agilent
◦
UV-Visible diode array spectrophotometer 8453 at 30 C, during 30 min, in Tris HCl 10 mM, KCl 0.1 mM,
pH 7.5 to obtain IC50 values.
4.5.3. EcPepN and HsPSA
Enzyme activity assays were carried out in 10 mM Tris-HCl buffer at pH 7.5 with 150 mM NaCl,
◦
enzyme, 100
Concentrations ranging from 1 nM to 100
each of the enzymes (75 M of EcPepN, 120
µ
M substrate (Leu-pNA) in a 100
µ
L reaction at 37 C. The absorbance was read at 405 nm.
µ
M of inhibitors (dissolved in DMSO) were incubated with
◦
µ
µ
M of HsPSA) for 30 min at 37 C in a 96 well plate.
After addition of the substrate, immediate increase in absorption was measured. All inhibition assays
were performed in triplicates and initial velocities were measured. Percentage inhibition and log
values of inhibitor concentrations were plotted using SigmaPlot to determine IC50 values. Ki values
were determined by the Dixon method. The assay consists of reaction buffer, enzyme and varied
concentrations of Leu-pNA (25, 50, 100, 200 and 400
µM). Based on the IC50 values, at each substrate
concentration, inhibitor concentration was varied in the range of 50 nM to 500 nM or 0.5
µ
M to 25 M.
µ
Data were plotted as 1/rate versus inhibitor concentration for each substrate concentration and a linear
fit was calculated by non-linear regression using SigmaPlot12.5. All the reactions were performed in
triplicates, SD values were reported [73].
4.5.4. HsERAP1, HsERAP2 and HsIRAP
The enzymatic activity of HsERAP1, HsERAP2 and HsIRAP was determined by following the
time-dependent increase in fluorescence at 460 nm (excitation: 380 nm) of the fluorigenic substrates
L-Leucine-7-amido-4-methyl coumarin (L-AMC; Sigma, Darmstatd, Germany) for HsERAP1 and
HsIRAP and L-arginyl-7-amido-4-methyl coumarin (R-AMC; Sigma) for HsERAP2. Measurements
were performed on a 96-well plate (150
microplate fluorescence reader. 30 nM of HsERAP1, 6 nM of HsERAP2 or 6 nM of HsIRAP was added
in each well, along with 50 M of substrate and varied concentrations of compound (within the range
of 1 nM to 200 M). The reaction was followed for 10 min at room temperature and the remaining
µL total volume in each well) on a TECAN infinite M200
µ
µ
enzymatic activity was calculated by measuring the slope of the time course. For calculation of the
in vitro IC50 values, experimental data were fit to the following equation using the GraphPad Prism
software package:
Y = Bottom + (Top − Bottom)/(1 + 10ˆ((LogIC50 − X) * HillSlope))
(1)
where Y is the enzymatic activity and X the inhibitor concentration.
4.6. Measurement of Octanol/Water Partition Coefficient (logD7.4
The lipophilicity (logD7.4 values) of the amino-benzosuberones was determined by reversed-phase
HPLC on a C-18 column (Eclipse XDB-C18, 5 m, 4.6 150 mm, from Agilent, France) according to the
method previously described by Minick and Pomper [101 102]. Measurement of the chromatographic
)
µ
×
,
capacity factors (k0) for each compound was done at various concentrations in the range of 90–60%
methanol (containing 0.25% octanol) and an aqueous phase consisting of 0.15% n-decylamine in 0.02 M
MOPS (3-(N-morpholino)propanesulfonic acid) buffer pH 7.4 (prepared in 1-octanol-satured water).
The flow was 0.9 mL/min. The aminobenzosuberone derivatives were dissolved at 0.4
µM in methanol.
The standards were also dissolved in methanol. Column void volume was estimated from the retention

Molecules 2018, 23, 2607
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time of uracil, which was included as a non-retained internal reference with each injection. The capacity
factors (k0) are extrapolated to 100% of the aqueous component given the value of k0w. The logD7.4 is
then obtained by the formula:
logD7.4 = 0.13418 + 0.98452k0w
(2)
Positive control with
β-oestradiol, experimental logD7.4 3.043
±
0.093 (n = 9), literature value 3.261.
4.7. In Silico Prediction of ADMET Properties
The study of ADMET properties was carried out on the website http://admet.scbdd.com [84].
5. Conclusions
M1 family aminopeptidases have broad and overlapping substrate specificity; hence
small-molecule inhibitors may not always be specific, especially regarding their selectivity toward
bimetallic enzymes. The aminobenzosuberone scaffold demonstrated exclusive selectivity for
the monometallic M1 aminopeptidase family with particular potent inhibitory activities against
mammalian APN and its microbial orthologues EcPepN and PfAM1 (Ki values in the nanomolar
range). It retains interesting potency on HsPSA and also on HsIRAP (Ki values in the nano- to
sub-micromolar range) but it is seemingly less active on both oxytocinase enzymes HsERAP1/2
(IC50 values in the micromolar range) and totally inactive against HsLTA4H.
This promising scaffold represents an attractive new class of potent antimalarial compounds
targeting PfAM1 [69], a crucial enzyme involved in at least the last committed step of haemoglobin
degradation by Plasmodium falciparum [17,31,35]. In addition, growing evidences highlight the crucial
role played by mammalian aminopeptidases in a great variety of cancer types, especially HsAPN and
HsERAP1/2 via their proteolytic activity or their ability to modulate protein-protein interactions [103].
The aminobenzosuberone core is an attractive starting point to design triple inhibitor of all these
three enzymes, acting by interfering with endothelial cell morphogenesis and cell motility [61] and by
modulating antigen processing to trigger cancer immunotherapy [14–16].
The next challenge is to rationally design selective inhibitors for individual M1 aminopeptidase,
to study their biological roles and precise their functions, or to avoid any potential adverse effects
following in vivo treatment with aminobenzosuberone derivatives by targeting other members of this
diverse family. To achieve this goal, the design process efforts should take into account the plasticity of
the active site and the conformational dynamics of these M1 aminopeptidases. Interesting hints suggest
deeper interactions into the S1 subsite through a substitution on position-9 of our scaffold should
offer new opportunities to improve both activity and selectivity. Another approach for achieving
selectivity is to look for the cellular/subcellular localization and/or tissue distribution of the targeted
aminopeptidase, to design targeted-prodrug to improve site-specific drug delivery. These different
strategies are currently under investigation and will be reported in due course.
Supplementary Materials: The following are available online. Figure S1: Conserved active site residues involved
in substrate recognition and catalysis, Table S1: Human proteinase selectivity profiles of 21a–c and 21i, Table S2:
Cavity volume of the active site of “closed” conformation of M1 APs, Figure S2: Surface representation of the
active site of studied M1 Aminopeptidases, Figure S3: Surface representation of the S1 subsite of different M1
aminopeptidases, Table S3: Cavity volume of the S1 subsite of “closed” conformation of M1 APs, Figure S4:
Local movement of Met260 in the active site of EcPepN, Figure S5: Superimposition of the protein backbone of
the aminobenzosuberone 21c in EcPepN complex (green, PDB 5MFS) with HsLTA4H (slate blue, PDB 1HQ6),
Figure S6: Superimposition of the protein backbone of the aminobenzosuberone 21c in EcPepN complex (green,
0
PDB 5MFS) with different M1 aminopeptidases, Figure S7: Surface representation of the S1 subsite of different
0
M1 aminopeptidases, Table S4: Cavity volume of the S1 subsite of “closed” conformation of M1 APs, Figure S8:
Intra-domain movement in the active site of HsAPN, Table S5: Determination and prediction of various ADME-Tox
properties of substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochloride salts.
Author Contributions: C.T. and S.A. conceived and supervised the study and A.A. and E.S. helped in designing
the experiments. E.S. and S.A. performed the synthesis and characterisation of aminobenzosuberone derivatives;
M.S., A.K.M., A.S. and C.S. produced proteins and performed enzymatic tests under the guidance of C.T., I.F.,

Molecules 2018, 23, 2607
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A.A. and E.S.; G.R. and S.A. wrote the original draft; C.T. and S.A. wrote the whole article, made discussion and
conclusions and S.A. prepared the figures/tables/schemes. All authors reviewed the manuscript.
Funding: This research was funded by Agence Nationale de la Recherche, grant number ANR-12-BS07-0020-01.
Funding was also received by the European Union (European Social Fund) and Greek national funds through
the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework:
Research Funding Program of the General Secretariat for Research & Technology and the Harry J. Lloyd Charitable
Trust (to E.S.). The use of infrastructure associated with the National Research Infra- structure in Structural
Biology, INSTRUCT-EL for recombinant protein production is also acknowledged. A.K.M. thanks Council of
Scientific and Industrial Research (CSIR), New Delhi, India, for research fellowship. A.A. thanks Department of
Science and Technology (DST), New Delhi, India with the grant number EMR/2015/000461.
Acknowledgments: The support of the Université de Haute-Alsace and the Ecole Nationale Supérieure de Chimie
de Mulhouse is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 21a–j are available from the authors.
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