Benzoxazepine-Derived Selective, Orally Bioavailable Inhibitor of Human Acidic Mammalian Chitinase

Article abstract of DOI:10.1021/acsmedchemlett.0c00092

Human acidic mammalian chitinase (hAMCase) is one of two true chitinases in humans, the function of which remains elusive. In addition to the defense against highly antigenic chitin and chitin-containing pathogens in the gastric and intestinal contents, AMCase has been implicated in asthma, allergic inflammation, and ocular pathologies. Potent and selective small-molecule inhibitors of this enzyme have not been identified to date. Here we describe structural modifications of compound OAT-177, a previously developed inhibitor of mouse AMCase, leading to OAT-1441, which displays high activity and selectivity toward hAMCase. Significantly reduced off-target activity toward the human ether-à-go-go-related gene (hERG) and a good pharmacokinetic profile make OAT-1441 a potential candidate for further preclinical development as well as a useful tool compound to study the physiological role of hAMCase.

Full text of DOI:10.1021/acsmedchemlett.0c00092

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Letter
Benzoxazepine-Derived, Selective, Orally Bioavailable
Inhibitor of Human Acidic Mammalian Chitinase
Gleb Andryianau, Micha# Kowalski, Michal C. Piotrowicz, Adam A Rajkiewicz, Barbara Dymek,
Piotr L. Sklepkiewicz, El#bieta Pluta, Filip Stefaniak, Wojciech Czestkowski, Sylwia Olejniczak,
Marzena Mazur, Piotr Niedziejko, Robert Koralewski, Krzysztof Matyszewski, Mariusz Gruza,
Agnieszka Zagozdzon, Magdalena Salamon, Aleksandra Rymaszewska, Miko#aj Welzer,
Karolina Dzwonek, Jakub Golab, Jacek Olczak, Agnieszka Bartoszewicz, and Adam Golebiowski
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00092 • Publication Date (Web): 24 Apr 2020
Downloaded from pubs.acs.org on April 24, 2020
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Benzoxazepine-Derived, Selective, Orally Bioavailable Inhibitor of
Human Acidic Mammalian Chitinase
Gleb Andryianau,◊,‡ Michal Kowalski,◊,‡ Michal C. Piotrowicz,◊,‡ Adam A. Rajkiewicz,◊,○ Barbara
Dymek,◊ Piotr L. Sklepkiewicz,◊ Elzbieta Pluta,◊ Filip Stefaniak,◊,□ Wojciech Czestkowski,◊ Sylwia
Olejniczak,◊ Marzena Mazur,◊ Piotr Niedziejko,◊ Robert Koralewski,◊ Krzysztof Matyszewski,◊ Mariusz
Gruza,◊ Agnieszka Zagozdzon,◊ Magdalena Salamon,◊ Aleksandra Rymaszewska,◊ Mikolaj Welzer,◊
Karolina Dzwonek,◊ Jakub Golab,◊,∆ Jacek Olczak,◊ Agnieszka Bartoszewicz,*,◊ Adam Golebiowski◊
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◊ OncoArendi Therapeutics S.A., Żwirki i Wigury 101, 02-089 Warsaw, Poland
○ Centre of New Technologies, University of Warsaw, Banacha 2C, 02-097 Warsaw, Poland
□ Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw,
Ks. Trojdena 4, 02-109 Warsaw, Poland
∆ Department of Immunology, Medical University of Warsaw, Nielubowicza 5, 02-097 Warsaw, Poland
KEYWORDS Acidic mammalian chitinase, AMCase, inhibitor, CHIT1, chitotriosidase, hERG
ABSTRACT: Human acidic mammalian chitinase (hAMCase) in one of two true chitinases in humans, the function of which remains
elusive. In addition to the defense against highly antigenic chitin and
chitin-containing pathogens in the gastric and intestinal contents,
AMCase has been implicated in asthma, allergic inflammation, and
ocular pathologies. Potent and selective small molecule inhibitors of this
enzyme have not been identified yet. In this work, we describe structural
modifications of compound OAT-177, a previously developed inhibitor
of mouse AMCase, leading to OAT-1441, which displays high activity
and selectivity toward hAMCase. Significantly reduced off-target
activity toward the human Ether-à-go-go-Related Gene (hERG),
combined with a good pharmacokinetic profile, make OAT-1441 a potential candidate for further preclinical development, as well
as a useful tool compound to study the physiological role of hAMCase.
Acidic mammalian chitinase (AMCase) belongs to the
family 18 of glycosyl hydrolases displaying a chitinase
activity.1 It contains both a chitin-binding domain and an
AMCase action under the chitin-containing conditions is
understood, the modulation of the immune response in the non-
chitin-dependent pathologies is extremely interesting and
remains to be elucidated.23-27
enzymatically active domain that catalyzes the hydrolysis of β-
1,2
(1→4) glycosidic bonds in chitin.
AMCase is widely
In recent years, a number of natural product-derived
chitinase inhibitors have been described, including cyclic
peptides argifin and argadin,28-29 pseudosaccharide allosamidin
and its derivatives.30-34 However, the applicability of these
compounds as potential drugs is limited because of their
structural complexity and high molecular weight.35-37 A search
for suitable AMCase-selective inhibitors is challenging also due
to the high structural homology of this enzyme to the second
chitinase present in humans, namely CHIT1. Examples of small
molecule chitinase inhibitors identified so far that have shown
in vivo efficacy in murine inflammatory models, e.g., Bisdionin
C,38 Bisdionin F39 and Wyeth140 display low potency and/or
poor AMCase/CHIT1 selectivity, which makes it difficult to
discriminate the biological function of these enzymes (Figure
conserved from prokaryotes to eukaryotes,3-4 despite the limited
,6
evidence of endogenous chitin in the majority of organisms.5
Many studies have confirmed that active chitinases occur in
vertebrates,7 in particular, AMCase and a related chitotriosidase
(CHIT1) are abundant in humans.8 While AMCase is mostly
present in human stomach and gut,9-10 activated macrophage
secreted CHIT1 is present in a number of organs and its levels
correlate with the progression of various diseases.11-17 The
former enzyme is thought to function as a digestive enzyme that
breaks down chitin and is also considered to be a part of the host
defense against highly antigenic chitin and chitin-containing
pathogens in the gastric and intestinal contents.18-20
AMCase has attracted considerable attention due to its
increased expression under pathological conditions activating
Th2 inflammatory response, both in chitin dependent and
independent manner, in antigen-induced mouse models of
allergic lung inflammation.21-22 While the mechanism of
1).41
A reverse CHIT1/AMCase selectivity is equally
challenging to achieve, and the first CHIT1 selective inhibitors
that show in vivo efficacy in bleomycin induced fibrosis model
in mice have been described only very recently (Figure 1). 42
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aReagents and conditions: (a) NaBH(OAc)3, DCE, rt; 99% yield
(b) RCHO, NaBH(OAc)3, DCE, rt; (c) HCl, EtOAc, rt; (d) S,S′-
dimethyl N-cyanodithioiminocarbonate, K2CO3, MeCN, reflux; (e)
N2H4·H2O, MeCN, reflux, overall 15-32% yield over steps b-e.
Characteristics of the selected, highly potent compounds are
summarized in Table 1. In particular, phenol-containing
analogs 1 and 2 were quickly identified as excellent inhibitors
of hAMCase, also displaying a better hAMCase/hCHIT1
selectivity compared to OAT-177 (up to 40-fold for 2). On the
downside, however, they exhibit a noticeable off-target
inhibition of the hERG channel (IC50 = 100-150 nM) and poor
microsomal stability (T1/2 < 1 h; see the SI for selected data)
rendering their pharmacokinetic evaluation pointless. Our
initial strategy aimed at addressing these two problems, most
likely originating from the presence of the phenol moiety, was
modulating the electron density of the central nitrogen atom by
the incorporation of various electron-withdrawing substituents
to the benzyl ring. In general, all new inhibitors 3-10 displayed
comparable activity and selectivity, which seem to be a little
dependent on the substitution pattern (Table 1). For all the
analogs, with the exception of 6, the undesired hERG inhibition
decreased 10 to 20-fold compared to the initial hit compounds
1 and 2. In particular, the addition of a heteroaromatic ring in
10 led to the highest decrease in this off-target inhibitory
activity (hERG IC50 = 6.4 M). Nonetheless, the hERG IC50
values were still too low to be acceptable. Moreover, the
microsomal stability and pharmacokinetics parameters,
measured for selected most promising compounds in this series,
were only modest (Table 1; AUC and Cmax, Table S1 and S2).
Significantly higher concentrations in plasma obtained for
inhibitor 8 (AUC = 13.7 mg·h/L) suggest a possible correlation
between the electronic properties of the aromatic ring and the
metabolic stability of the compound. Overall, the insufficient
improvement of off-target hERG inhibition of the simple
benzylic analogs of OAT-177, together with the poor
pharmacokinetic profile prompted us to introduce further
modifications to the structure.
Figure 1. Selectivity of small-molecule inhibitors toward mouse
and human AMCase reported in literature.
In our ongoing program targeting chitinase inhibition as a
potential therapeutic approach for a broad range of pulmonary,
inflammatory, and fibrotic diseases, we have focused on finding
specific and potent compounds toward both AMCase and
CHIT1. Recently, we have reported selective inhibitors of
mouse CHIT1 (mCHIT1) and mouse AMCase (mAMCase)
enzymes, OAT-206843 and OAT-17744 respectively (Figure 1).
Both compounds displayed excellent in vitro activity and PK
profile. Moreover, OAT-177 has been found to be effective in
animal inflammation-driven models of asthma.44 Herein, we
describe the development of an inhibitor specifically tailored to
selectively target human AMCase (hAMCase), having
additionally excellent characteristics in terms of high selectivity
vs. human CHIT1 (hCHIT), lower hERG inhibition, and
sufficient pharmacokinetic properties in mice and rat.
In addition to potential therapeutic applications, the highly
active and hAMCase-selective small-molecule inhibitor can be
used as a pharmacological tool to evaluate the role of AMCase
in the modulation of chitin dependent vs. independent immune
responses.
Upon testing the inhibitory potency of several close analogs
of OAT-177, we recognized that the introduction of a benzyl
substituent at the central nitrogen atom leads to a notable
improvement in both the affinity toward hAMCase and
hAMCase vs. hCHIT1 selectivity. Therefore, a series of
compounds were synthesized from 2-(4-chlorophenyl)ethan-1-
amine by two sequential reductive amination steps with N-Boc-
piperidinone and appropriate benzaldehyde, followed by the
removal of Boc protecting group and the installation of
aminotriazole ring at the secondary amine (Scheme 1).
Compounds 2, 4 and 5 were synthetized via a similar pathway,
see the Supporting Information for the details.
Guided by the good performance of compound 1 in terms of
the affinity toward hAMCase, we designed conformationally
constrained, bicyclic benzoxazepine inhibitors, incorporating
the oxygen-substituted benzyl moiety, but lacking the free
phenol functionality. The additional, predicted advantage of
creating a ring fusion is that due to the presence of an oxygen
atom in the β-position relative to the central nitrogen, the latter
will become less basic, which in consequence, should hinder the
undesired binding to the hERG channel. The synthetic route to
the advanced lead compounds 14, 16, and 18 is depicted in
Scheme 1. Synthetic pathway for analogs 1, 3, 6-10.a
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Scheme 2. In the first step, enantiopure (S)-1-((tert-
1
2
3
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5
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8
butyldimethylsilyl)oxy)-3-(4-chlorophenyl)propan-2-amine
was reductively alkylated with ortho-chloro- or ortho-fluoro-
substituted Ar-aldehyde. Following the removal of tert-
butyldimethyl silyl (TBDMS) protecting group, the
benzodiazepine ring was closed under basic conditions. The
resulting secondary amine was then transformed into the final
products in a similar way as used before (Scheme 1).
Structurally related inhibitors, 11-13, 15, 17, and 19-23 were
synthesized via an alternative route described in the SI.
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Scheme 2. Synthetic pathway for analogs 14, 16 and 18.a
aReagents and conditions: (a) NaBH(OAc)3, DCE, rt; (b) TBAF,
THF or AcOH/H2O/THF, 65 °C (c) t-BuOK, THF, reflux
(d) N-Boc-piperid-4-one, AcOH, NaBH(OAc)3, DCE, 75 °C to rt;
(e) TFA, DCM, rt; (f) S,S′-dimethyl N-cyanodithioiminocarbonate,
K2CO3, MeCN, reflux; (g) N2H4·H2O, MeCN, reflux, overall 8-
27% yield over steps a-g.
Table 1. SAR of benzylic analogs
R
hCHIT1
(IC50 nM)a
hAMCase
(IC50 nM) a
hAMCase/hCHIT1 hERG
selectivity
AUC0-inf
Cmax
Compd #
(mg*h/L)c
(IC50 nM)b
(mg/L)
OAT-177
1
229±30
49±1
15±2
15
25
8000
0.09d
NT
NT
NT
2±0.5
100
2
3
4
79±9
2±1
5±2
3±1
40
33
31
150
NT
NT
NT
1.4
NT
NT
NT
122±3
104±4
2000
5
6
7
176±6
90±1
6±2
28
23
40
3700
400
7.8
7.0
7.5
0.78
NT
4±1
200±10
5±0.5
4000
0.69
8
310±10
12±0.5
26
2700
13.7
1.6
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277±32
620±20
5±0.5
15±2
55
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3400
6400
2.4
0.6
10
0.43
0.13
5
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8
aAverage IC50 values of two independent experiments and standard deviations. For practical reasons, to determine the structure-activity
relationships for hundreds of compounds toward human chitinases, IC50 were recorded instead of Ki. In the enzyme assays, the substrate
concentrations for hAMCase and hCHIT assays tests were fixed correspondingly at 2KM and 0.67KM Michaelis–Menten constants
determined for each enzyme. The IC50 values of competitive inhibitors toward hCHIT1 are close to the corresponding Ki constants, while
the IC50 values of the same inhibitors toward hAMCase are 2-3x higher than the corresponding Ki constants. Due to the above, the selectivity
of the inhibitors, measured as the ratio of IC50 toward hAMCase vs. hCHIT1, is usually underestimated. See the Supporting Information for
further details.; bIC50 values in a hERG fluorescence polarization assay, see the Supporting information for details; cAUC0-inf calculated based
on data from pharmacokinetics study in rat (PO administration, dose 10mg/kg), see the Supporting Information for the plasma concentration
vs. time plot; dOAT-177 is metabolized rapidly in rats. This seems to be species-dependent, as concentrations and AUC0-inf values for OAT-
177 in mice were >100 higher.
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The results of in vitro studies on the benzoxazepine
inhibitors are summarized in Table 2. Most of these compounds
show a significant, more than 10-fold improvement in the
hAMCase/hCHIT selectivity compared to the first series of
inhibitors (Table 1). The reasons for such behavior will be
discussed in detail in the following sections. The analog
containing an unsubstituted phenyl ring (11 – OAT-1441)
displays the most balanced, and already fully satisfactory,
properties in terms of the potency toward hAMCase (IC50 = 7
nM) and low affinity for hERG (IC50 hERG = 22 M). The
addition of halogen substituents (12, 13, and 15) resulted in an
increased binding to hERG. The introduction of a cyano group
to the aromatic ring (14) did not cause a meaningful
improvement in the hERG IC50 value, while it decreased the
potency toward hAMCase. Significantly lower hERG activities
were observed for analogs 17 and 18, bearing a pyridine ring
fused with the oxazepine core, but similarly to 14, the activities
toward hAMCase were only moderate. Neither of the open-
chain analogs, 19 and 20, displayed interesting inhibitory
activity or selectivity. This demonstrates that the advantageous
properties of the benzoxazepine inhibitors are not only due to
their electronic characteristics, but they originate also from a
restricted conformational freedom. The replacement of the
aromatic ring with a reduced π-system, as in 21, maintained the
high inhibitory activity for hAMCase, but had a detrimental
effect on the selectivity. Finally, two benzodiazepine analogs
were prepared (22 and 23). Despite their excellent potency
towards hAMCase and the best selectivity among all inhibitors,
this structural motif was not pursued further due to the high
affinity toward hERG (IC50 < 600 nM). In the next step, the
inhibitory activity of OAT-1441 in hERG patch clamp45 and
DAT (dopamine active transporter) were assessed,46 as these
two off target activities were the most problematic in the
previous generation of inhibitors developed in our laboratory.47
OAT-1441 was found to inhibit hERG potassium channel and
DAT transporter very weakly, 23 M and 37% at 10 M,
respectively.
Table 2. SAR of compounds with benzoxazepine and benzodiazepine core.
hCHIT1
(IC50 nM)a
hAMCase
(IC50 nM)a
hERG
(IC50 nM)b
hAMCase/hCHIT1
selectivity
Compd #
11(OAT-1441)
915±85
7±2
7±2
8±2
49±1
177
133
303
149
22000 (23000)c
6700
12
13
930±70
2300±150
7300±900
6700
14
15
23000
>20000
68±2
>294
6000
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18000±1000
4400±100
2400±100
18000±150
16000±500
350±90
290±40
55±3
62
80
80
73
31
29
17000
50000
>50000
2900
NT
1
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8
30±3
245±15
580±40
12±1
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NT
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23
275±25
420±40
1.4±0.3
0.7±1
196
600
3200
800
asee the footnote a, Table 1; asee the footnote b, Table 1; c hERG IC50 (hERG-CHO, automated patch-clamp)
To provide the rationale to the observed activity and
selectivity trends we performed a detailed analysis of the
structures of compounds 3 and OAT-1441 in complex with
hAMCase and hCHIT1 (Figure 2). For hCHIT1, the
experimentally solved structure of 3 in a complex with hCHIT1
has been determined previously (PDB ID: 5NRF) and it was
used for the prediction of a binding mode of OAT-1441 with
this enzyme using molecular docking. In the case of hAMCase,
we used molecular docking to the solved structure of the protein
complex with a structurally similar ligand (PDB ID: 3RM4)
(see the Supporting Information for details). In the hAMCase,
the chitin binding cleft is formed by the solvent-exposed
aromatic residues, including Trp71, Tyr34, Trp31, Trp360,
Trp99, Trp21848 and Phe58. These residues are highly
conserved across species. X-ray data for CHIT1 and molecular
modeling for AMCase show that the described compounds bind
to the pocket formed by three of them - Trp99, Trp360, and a
slightly more distant Trp218 (see Figure S1). For both enzymes,
the binding pocket is highly conserved. The only differences
between the active site residues occur at positions 269 (Arg in
hCHIT1 vs. His in hAMCase) and 300 (Met in hCHIT1 vs. Ile
in hAMCase). As it has been shown previously,44 the
interactions of aliphatic substituents present at the central
nitrogen atom of the inhibitors with these amino acids are
crucial for the high inhibitory activity toward hAMCase and the
selectivity against hCHIT1. Additionally, the orientation of
Trp99 in these two proteins may be different. Namely, in the
majority of the solved hCHIT1 structures, the indole ring is
directed slightly toward the binding pocket. Conversely, in most
structures of hAMCase, it is pointing slightly outside of the
binding pocket. It has been postulated earlier,44 that one of the
driving forces for the Trp99 flip may be the interaction of this
amino acid with an alkyl group of a ligand. In the current
modeling effort, for each enzyme we assumed the two opposite
orientations of the Trp99, i.e., the ones most frequently
observed, respectively.44
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Figure 2. Structures of hCHIT1 (A) co-crystallized with 3 (PDB: 5NRF) and (B) with docked OAT-1441. Structures of hAMCase with
docked (C) 3 and (D) OAT-1441. Both protein structures are shown in the same orientation.
Analysis of the structural data and docking results reveals
that in the case of compounds 3 and OAT-1441 interactions
with Arg269 (or lack thereof) may contribute to the observed
selectivity. As it was noted above, in the hCHIT1, the long
positively charged side chain of Arg269 interacts with the edge
of the hydrophobic benzene ring of 3, resulting in a negative
effect on compound binding. 44,47 In the case of OAT-1441, this
effect is even stronger, as the compound has a rigid
benzoxazepine ring, which cannot adapt itself to this
hydrophobic environment. The distance from Arg269 to C5 of
the benzoxazepine ring is 3.6 Å and is equal to the distance
observed for 3. It has been also recognized previously that the
formation of a hydrogen bond between the tertiary nitrogen
atom of a ligand and Asp213 is critical for potency. Compound
3 forms a hydrogen bond with this amino acid (at a distance of
2.8 Å), while according to the performed simulations - the
central nitrogen atom of OAT-1441 is more distant from
Asp213 and forms only a weak or no hydrogen bond. The
benzyl group of compound 3 may undergo rotation and adopt
itself to create a T-shaped -stacking interaction with Trp99
(ring centroids distance equal to 5.0 Å). Such interaction is less
probable for OAT-1441, as the planes of the ligand and the
indole of Trp99 are neither parallel nor perpendicular to each
other. Moreover, the rings are more distant (centroids distance
equal to 5.4 Å) and the constrained system of the ligand cannot
accommodate to form a stronger bond. Taken together, these
observations explain the lower potency of OAT-1441 toward
hCHIT1 compared to 3.
On the other hand, both compounds 3 and OAT-1441 form
a similar interaction network inside the binding pocket of
hAMCase. In this enzyme, according to our simulations,
His269 is too distant to the inhibitors and does not weaken the
binding, in a similar way as Arg269 in hCHIT1. The benzyl
group of compound 3 may form a -stacking interaction with
Trp99 in a sandwich configuration (ring centroids distance
equal to 3.2 Å). OAT-1441 may also form this kind of
interaction, but in a T-shaped configuration (ring centroids
distance equal to 3.9 Å). This results in a comparable inhibitory
activity of these two compounds toward hAMCase.
We postulate a similar binding pattern in the active site of
hAMCase for the other benzoxazepines (12-15) and
benzodiazepines (22-23), as well the three pirydooxazepines
(16-18). In the case of the last three inhibitors, their distinctly
lower potency, likely originates from an unfavorable interaction
of the lone electron pair of the pyridine nitrogen with the
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electron-rich aromatic rings of Trp99. This effect is the most
Table 3. Pharmacokinetic parameters of OAT-1441 in mice
/ rats
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7
8
pronounced for the inhibitor 16, whose lone electron pair is
oriented directly towards the ring center, and lower for
compound 17, whose lone electron pair is heading slightly off
the aromatic system of Trp99 (see Figure S1).
Route
IV
PO
Dose (mg/kg)
AUC0-inf(mg·h/L)
C0 or Cmax(mg/L)
Tmax(h)
3
10
A single dose pharmacokinetic studies with OAT-1441
were carried out after both intravenous (IV) and oral (PO)
administration to mice and PO administration to rats. As shown
in Table 3, OAT-1441 displays a significantly improved
pharmacokinetic profile compared to compounds 4-10 (AUC
values, Table 1; Figure S1). After IV administration to mice, the
2.9 / -
9.5 / -
- / -
3.9 / 22.5
3.1 / 3.4
0.5 / 0.5
- / -
CL (L/h/kg)
Vss (L/kg)
1.0 / -
0.35 / -
1.4 / -
0.34 / -
- / -
9
10
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12
13
14
15
16
17
18
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41
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57
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59
60
- / -
compound exhibits
a moderate plasma clearance (1.0
L·h−1·kg−1), a poor volume of distribution (0.35 L·kg−1) and T1/2
of 1.1 hours. After PO administration to mice compound OAT-
1441 was rapidly absorbed with Tmax at 0.5 hours and showed
good bioavailability of 41%. Pharmacokinetic parameters are
improved in rats, which is demonstrated by a much higher AUC
value, longer T1/2 and MRT. Overall, the pharmacokinetic
profile of compound OAT-1441 is sufficient to study its
pharmacological potential.
T½ (h)
1.1 / 4.2
0.84 / 4.2
41 / -
MRT (h)
Bioavailability (F%)
In conclusion, we have identified the first highly potent and
selective human acidic mammalian chitinase inhibitor OAT-
1441. The excellent selectivity of the compound vs. a closely
related human chitotriosidase was rationalized using molecular
docking investigations. Moreover, OAT-1441 has a good
pharmacokinetic profile and a significantly reduced off-target
inhibition of hERG channel. These overall features make it a
potential candidate for further preclinical development as well
as a useful tool compound to study the physiological role of
hAMCase.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website. Experimental data, 1H and 13C NMR spectra
for compounds 1-23, PK data and the details of molecular
modelling.
AUTHOR INFORMATION
Corresponding Author
* a.bartoszewicz@oncoarendi.com
Author Contributions
‡These authors contributed equally.
Notes
OAT-1441 displayed lower potency and selectivity toward
mAMCase (IC50 = 95 nM) and mCHIT (IC50 = 180 nM) than for
the human isoforms. This might be due to only a moderate
homology of hAMCase vs. mAMCase (80%) and hCHIT vs.
mCHIT (75%). In order to distinguish the functional differences
between AMCase and CHIT1, the drug candidate would need to be
tested in a humanized model.
The authors declare the following competing financial interest:
some of the authors are current employees of OncoArendi
Therapeutics S.A. and own company stocks.
ACKNOWLEDGMENT
Studies were supported by two projects: (1) “Selective inhibitors of
acidic mammalian chitinase as potential therapy for asthma”, co-
financed by the National Centre for Research and Development in
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 9
the framework of Innovative Economy Operational Program; (2)
“Preclinical research and clinical trials of first-in-class
development candidate in therapy of asthma and inflammatory
bowel disease”, co-financed by the National Centre for Research
and Development in the framework of European Funds Smart
Growth.
(14) Harlander, M.; Salobir, B.; Zupančič, M.; Dolenšek, M.;
1
2
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Kere, J.; Söderhäll, C.; Dahlén, B.; Boot, R.G.; Dahlén, S. E.
Increased YKL-40 and Chitotriosidase in Asthma and Chronic
Obstructive Pulmonary Disease. Am. J. Respir. Crit. Care Med.
2016, 193, 131–142.
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Malaguarnera, L. Evaluation of CHI3L-1 and CHIT-1 expression
in differentiated and polarized macrophages. Inflammation 2013,
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Gaspard, G.; Frère, J. M.; Galleni, M.; Filée, P. Comparative
functional analysis of the human macrophage chitotriosidase.
Protein Sci. 2011, 20, 1451–1463.
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F. Chitinase mRNA Levels by Quantitative PCR Using the Single
Standard DNA: Acidic Mammalian Chitinase Is a Major Transcript
in the Mouse Stomach. PLoS One 2012, 7, e50381.
(19) Kashimura, A.; Okawa, K.; Ishikawa, K.; Kida, Y.;
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ABBREVIATIONS
hAMCase/mAMCase, human/mouse acidic mammalian chitinase;
AUC0−inf, area under the curve; F, bioavailability; CHIT1,
chitotriosidase; CHO, Chinese hamster ovarian cell; CL, clearance;
Cmax, peak plasma concentration of a drug after administration;
DAT, dopamine active transporter; Ki, inhibitory constant; IC50,
half maximal inhibitory concentration; hERG, human Ether-à-go-
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go-Related Gene; PK, pharmacokinetics; tmax, time to reach Cmax
;
Vss, the steady-state volume of distribution.
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