Chroman-4-one derivatives targeting pteridine reductase 1 and showing anti-parasitic activity

Article abstract of DOI:10.3390/molecules22030426

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivat

Full text of DOI:10.3390/molecules22030426

molecules
Article
Chroman-4-One Derivatives Targeting Pteridine
Reductase 1 and Showing Anti-Parasitic Activity
Flavio Di Pisa 1, Giacomo Landi 1, Lucia Dello Iacono 1, Cecilia Pozzi 1, Chiara Borsari 2,
Stefania Ferrari 2, Matteo Santucci 2, Nuno Santarem 3, Anabela Cordeiro-da-Silva 3,
Carolina B. Moraes 4, Laura M. Alcantara 4, Vanessa Fontana 4, Lucio H. Freitas-Junior 4,5
,
Sheraz Gul 6, Maria Kuzikov 6, Birte Behrens 6, Ina Pöhner 7, Rebecca C. Wade 7,8,9
,
Maria Paola Costi 2,* and Stefano Mangani 1,10,
*
1
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy;
dipisa2@unisi.it (F.D.P.); landi31@unisi.it (G.L.); delloiacono3@unisi.it (L.D.I.); pozzi4@unisi.it (C.P.)
Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy;
chiara.borsari@unimore.it (C.B.); stefania.ferrari@unimore.it (S.F.); matteo.santucci@unimore.it (M.S.)
Institute for Molecular and Cell Biology, 4150-180 Porto, Portugal and Instituto de Investigação e Inovação
em Saúde, Universidade do Porto and Institute for Molecular and Cell Biology, 4150-180 Porto, Portugal;
santarem@ibmc.up.pt (N.S.); cordeiro@ibmc.up.pt (A.C.-d.-S.)
Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e
Materiais (CNPEM), Campinas SP13083-100, Brazil; carolinaborsoi@gmail.com (C.B.M.);
laura.alcantara@lnbio.cnpem.br (L.M.A.); fontana_vanessa@yahoo.com.br (V.F.);
lucio.freitas@butantan.gov.br (L.H.F.-J.)
2
3
4
5
6
GARDE, Instituto Butantan, São Paulo SP05503-900, Brazil
Fraunhofer Institute for Molecular Biology and Applied Ecology Screening Port,
D-22525 Hamburg, Germany; Sheraz.Gul@ime.fraunhofer.de (S.G.);
Maria.Kuzikov@ime.fraunhofer.de (M.K.); Birte.Behrens@ime.fraunhofer.de (B.B.)
Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies,
69118 Heidelberg, Germany; ina.poehner@h-its.org (I.P.); rebecca.wade@h-its.org (R.C.W.)
Center for Molecular Biology (ZMBH), DKFZ-ZMBH Alliance, Heidelberg University,
69120 Heidelberg, Germany
Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, 69120 Heidelberg, Germany
Magnetic Resonance Center CERM, University of Florence, 50019 Sesto Fiorentino (FI), Italy
Correspondence: mariapaola.costi@unimore.it (M.P.C.); stefano.mangani@unisi.it (S.M.);
Tel.: +39-0592-058579 (M.P.C.); +39-0577-234-255 or +39-0577-234-252 (S.M.)
7
8
9
10
*
Academic Editor: Thomas J. Schmidt
Received: 7 February 2017; Accepted: 3 March 2017; Published: 8 March 2017
Abstract: Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1
(PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one
analogues (1–3) of previously published chromen-4-one derivatives were synthesized and biologically
evaluated against parasitic enzymes (Trypanosoma brucei PTR1–TbPTR1 and Leishmania major–LmPTR1)
and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex
with compound
were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained
by comparison of observed and predicted binding modes of the compounds. Compound showed
1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3)
1
activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a
low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design
aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.
Keywords: pteridine reductase 1; Trypanosoma brucei; Leishmania spp.; chroman-4-one; chromen-4-one;
crystallographic studies
Molecules 2017, 22, 426; doi:10.3390/molecules22030426
www.mdpi.com/journal/molecules

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1. Introduction
In modern drug discovery, crystallography is an essential tool for rapid scaffold optimization
through structure-based drug design. It provides a basis for the application of computational
methods to design ligands for the targeted proteins [1]. Thus, the determination of novel crystal
complexes of parasitic proteins and ligands represents a starting point for a medicinal chemistry
approach to the discovery of novel antiparasitic compounds. Trypanosomatid parasites are the
etiologic agents of serious human and animal vector-borne infections, such as Human African
Trypanosomiasis (HAT, also known as sleeping sickness) and Leishmaniasis. The bloodstream form of
the protozoan parasite Trypanosoma brucei (T. brucei) causes HAT [
macrophages and causes different clinical forms ranging from cutaneous lesions to potentially
fatal visceral infections [ ]. Since vaccines to prevent HAT and Leishmaniasis are currently not
2], while Leishmania spp. infects
3
available, the control of these diseases is essentially based on chemotherapy. Almost all the
drugs used to combat parasitic infections were discovered decades ago and nowadays drug
resistance is a major threat. Moreover, the drugs currently available present several problems
such as high toxicity, limited efficacy, parenteral administration regimens and long periods of
treatment [
and an ongoing challenge. Drug discovery for neglected tropical diseases relies both on phenotypic
screening and target-based approaches [ ]. Dihydrofolate reductase (DHFR) is a well-established
target for the treatment of bacterial infections and some parasitic diseases, such as malaria [ ].
4,5]. Thus, the discovery of novel, safe and effective drugs is an unmet medical need
6,7
8
The classical inhibitors of DHFR have reduced activity against Leishmania and Trypanosoma due
to the upregulation of a gene encoding the dihydronicotinamide adenine dinucleotide phosphate
(NADPH)-dependent pteridine reductase 1 (PTR1). PTR1 is present in Leishmania spp. and
Trypanosoma brucei parasites, but not in the human cells. It is able to reduce both unconjugated
and conjugated pterins and provides a metabolic bypass to alleviate DHFR inhibition [
considered a promising target for the development of novel antitrypanosomal and antileishmanial
candidates and has recently been genetically validated as a drug target in T. brucei 15]. In the literature,
different scaffolds, such as pteridine [16], pyrrolopyrimidine [17 18] and benzimidazole [19 20],
9–14]. PTR1 is
[
,
,
have been reported to bind in the biopterin binding site and to inhibit PTR1 activity. In our previous
work, we have shown that chromen/chroman-4-ones were promising scaffolds for the development
of PTR1 inhibitors and antiparasitic agents. Four crystal structures of Trypanosoma brucei pteridine
reductase 1 (TbPTR1) in complex with three flavonols and one flavanone had been solved, providing
essential tools for the development of improved inhibitors [14]. Our previous attempts to obtain X-ray
crystal structures of Leishmania major pteridine reductase 1 (LmPTR1) in complex with inhibitors failed
and no crystal structure complexes of LmPTR1-flavonoids have been reported in the literature to date.
In the present work, we synthesized three flavanones (
published flavonols (1A 3A) [14] (Figure 1) in order to compare their binding mode in the PTR1 active
site and their biological activity. The crystal structure of TbPTR1 in a ternary complex with compound
1–3) that are analogues to our previously
–
1
was solved and the first crystal structures of LmPTR1-flavanone (compounds 1 and 3) ternary
complexes were obtained. Computational studies were also performed to explain the differences
between the binding modes of the chromen-4-one and the chroman-4-one moiety. We evaluated the
compounds for inhibitory activity against PTR1 and for antiparasitic activity against the parasites
T. brucei and Leishmania infantum (L. infantum). Finally, a wide panel of in vitro toxicological studies to
assess the safety of the proposed scaffold were undertaken.

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Figure 1. Chemical structures of the previously published flavonols [14] (1A–3A) and of the flavanone
analogues studied in the present work (1–3).
2. Results and Discussion
2.1. Synthesis and Inhibitory Activity towards PTR1
Previously [14], we had synthesized a library of 16 flavonols and compound 1A turned out to be
the most potent TbPTR1 inhibitor. In the present work, we designed and synthesized compounds
analogues of 1A 3A respectively, in order to compare the binding modes and the affinities of flavanones
with those of flavonols. Compounds were synthesized by condensation between substituted
1–3,
–
1–3
acetophenone and benzaldehydes in the presence of thionyl chloride in ethanol, as previously reported
(Scheme 1) [21]. The compounds were characterized by 1H-NMR, 13C-NMR and mass analysis and the
data are reported in the Supporting Information (p. S2).
Scheme 1. Synthesis of compounds 1–3. Reaction conditions: (a) SOCl2, EtOH, r.t.
The three flavanones were investigated for their activity against TbPTR1 and LmPTR1. The data
were compared with those of compounds 1A–3A. The results are reported in Table 1.
Table 1. IC50 values of compounds
(TbPTR1) and Leishmania major pteridine reductase 1 (LmPTR1). Standard deviations agreed
to 10%. NI: No Inhibition. The percentage of inhibition at 50 M is reported in Table S1 of the
1–3 and 1A–3A against Trypanosoma brucei pteridine reductase 1
±
µ
Supporting Information. The control compound was pyrimethamine, a PTR1 inhibitor (100% inhibition
at 50 µM against both PTR1 enzymes). NI = No Inhibition (percentage of inhibition lower than 15%).
Compound
IC50 TbPTR1 (µM)
IC50 LmPTR1 (µM)
1
2
3
31.0
133.0
82.0
57.0
35.0
36.0
1A
2A
3A
4.3
12.5
NI at 50 µM
35.0
NI at 50 µM
38.0

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Among the three synthesized flavanones, compound
1
was the most potent derivative against
TbPTR1, with a percentage of inhibition of 81% and an IC50 value of 31
µM. All three flavanones show
a percentage of inhibition of LmPTR1 greater than 50%, with compounds
(IC50 35 and 36 µM, respectively).
2
and being the most active
3
2.2. Crystallographic Structures of LmPTR1 and TbPTR1 in Complex with Flavanones
The compounds were subjected to crystallization trials to gain further information for
structure-based drug design. Ternary complex structures of LmPTR1-NADP+ with compounds 1 and 3
and TbPTR1-NADP+ with compound
were obtained at resolutions ranging from 1.70 Å to 2.35 Å.
Data collection and processing statistics are reported in Tables S2 and S3 of the Supporting Information.
PTR1 is a functional homotetramer with four catalytic sites. Each subunit has a single
structured domain with the typical topology of short-chain dehydrogenases/reductases (SDR) and
is characterized by a seven-stranded parallel -sheet sandwiched by three -helices on each side,
that represents the classical dinucleotide-binding motif known as the Rossmann fold. The active
site (about 30 Å 22 Å 15 Å) is mainly formed by a single chain, and is blocked at one end by
the C-terminal residues of an adjacent subunit. The catalytic centre is formed by residues from the
C-termini of 4 and 5, by the two loops connecting 5- 5 and 6- 6, and the nicotinamide of the
cofactor, which creates the floor of the active site [10 22] (Figure 2). The highly conserved Ser-Tyr-Lys
,
1
α/β
β
α
×
×
β
α
β
α
β α
,
catalytic triad, typical of the SDR family, is replaced by Asp-Tyr-Lys in both TbPTR1 and LmPTR1
(Asp181, Tyr194 and Lys198 in LmPTR1; Asp161, Tyr174 and Lys178 in TbPTR1). The biopterin binding
pocket is further completed by the aromatic side chain of Phe113 in LmPTR1 and Phe97 in TbPTR1,
forming an overhang under which the substrate binds through a characteristic
involving the nicotinamide moiety of the cofactor [23].
π-sandwich interaction
Figure 2. Ribbon diagram of the LmPTR1 subunit A showing the tertiary and secondary structures as
well as the location of the cofactor (green ball and sticks) and an inhibitor (Compound ; yellow ball
and stick) in the active site. The subunit D (pale lilac cartoon) contributing to the active site of the A
subunit with Arg287 (cyan sticks) is also shown. The helices of subunit A are colored red, the -strands
1
β
blue, and the secondary structure elements are numbered following the sequence. TbPTR1 has the
same fold and topology as LmPTR1.
The asymmetric units of all three structures presented here contain one PTR1 homotetramer
with identical subunits within experimental error (pairwise root mean square deviation, rmsd, values,

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calculated on C
α
atoms, ranging from 0.26 Å to 0.38 Å for LmPTR1-NADP+-compound
1
, 0.21 Å to
0.27 Å for LmPTR1-NADP+-compound 3 and 0.14 to 0.34 Å for TbPTR1-NADP+-compound 1).
Structural alignments of the Cα atoms of each ternary complex of LmPTR1 and TbPTR1 and
the respective PTR1-NADP+ binary complexes (Protein Data Bank (PDB) code 2BFO for LmPTR1
and unreleased structures from our laboratory for TbPTR1) resulted in rmsd values ranging from
0.18 Å to 0.39 Å, indicating that inhibitor binding does not cause large conformational changes of
the enzyme. Even the superposition of the LmPTR1 and TbPTR1 ternary complexes with the ternary
structures present in the PDB reveals no major structural changes. The high degree of structural
similarity also extends to many side chains, the conformation of the cofactor and the positions of
several water molecules. This is a clear indicator of the high level of structural conservation of the
protein, irrespective of the binding of substrate, product or inhibitors.
TbPTR1-NADP+-1 complex. The structure was determined at 1.70 Å resolution. Ligand placement
and key interactions within the active site cavity are reported in Figure 3A. The chroman-4-one moiety
of compound
ether O1 points towards the side chains of Asp161 and Tyr174. The hydroxyl group in position 6
establishes a H-bond with the oxygen atom O2A of NADP+
-phosphate and a weaker one (3.45 Å)
1 is involved in a π
-sandwich between the nicotinamide of NADP+ and Phe97, and the
β
with the Ser95 side chain. The carbonyl group in position 4 is H-bonded to the amino group of Arg14
and linked to the oxygen atom (O2A) of the cofactor through a water molecule. The phenol linked to
position 2 of the chroman-4-one is located in a prevalently hydrophobic pocket lined by Met163, Val206,
Leu209, Met213, Trp221 and Leu263, and is able to make a T-shaped stacking interaction with the side
chain of Trp221 and a water-mediated interaction with the side chain of Asp161. Even though the
soaking procedure was performed using a racemic mixture only the R-enantiomer has been observed
in the active site cavity (no evidences for the S-enantiomer in the electron density map as shown in
Figure 3A).
Figure 3. Cont.

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Figure 3. Binding of the NADP+ cofactor (ball and sticks, green carbon atoms) and compounds
(ball and sticks, yellow carbon atoms) in the active site of: ( ) TbPTR1 (grey ribbons); and ( ) LmPTR1
(pink ribbons). Binding of the NADP+ cofactor (ball and sticks, green carbon atoms) and compound
(ball and sticks, purple carbon atoms) in the active site of: ( ) LmPTR1 subunit C, and ( ) LmPTR1
subunit A. The 2Fo Fc electron density maps corresponding to the inhibitors (dark-blue wire) and
NADP+ (light-blue wire), contoured at the 1
level, are shown. H-bonds involving the cofactor and
1
A
B
3
C
D
−
σ
inhibitor are shown as dashed lines. It is worth noting the different orientation and environment of the
compound 3 catechol ring between subunits C and A of LmPTR1.
LmPTR1-NADP+-1 and LmPTR1-NADP+-3 complexes. The structures have been determined
at 2.35 Å and 2.10 Å resolution, respectively. The orientations of compounds
1 (Figure 3B) and 3
(Figure 3C,D) in the catalytic pocket of LmPTR1 resemble that of compound in the TbPTR1 active
1
site (Figure 3A). Moreover, as reported above for the complex with TbPTR1, only the R-enantiomer
of the compounds has been observed within the catalytic cavity of LmPTR1. The chroman-4-one
scaffold establishes a
interactions with the surrounding residues are retained across the two species: the hydroxyl group in
position 6 of both compounds is located within H-bonding distance of the -phosphate oxygen atom
O2A of NADP+, and Ser111 side chain. Compound
is also able to establish a polar contact with the
π
-sandwich between the nicotinamide of NADP+ and Phe113 and most of the
β
1
Ser111 side chain (Figure 3B). In both compounds, the carbonyl group in position 4 is H-bonded to
the NH2 group of Arg17. As in TbPTR1, the phenol moieties are located in a prevalently hydrophobic
pocket lined by Leu188, Leu226, Leu229, His241 and Arg287 (for clarity, leucine residues are not
labelled in Figure 3B–D). The phenol ring of compound
the Arg287 side chain, which points into the catalytic pocket from an adjacent subunit of the tetramer,
and a water-mediated interaction with Asp181. In the complex LmPTR1- , the involvement of the
1 is able to establish a strong H-bond with
3
m-hydroxyl group of the catechol ring in the interaction with the NH2 group of Arg287 is visible only
in chains C and D (Figure 3C), whereas in chains A and B (Figure 3D), due to a flip of the catechol ring
of about 120◦, the p-hydroxyl group interacts with the side chain of Tyr283.

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Irrespective of the compound in the catalytic pocket, different conformations of His241 in LmPTR1,
at the corresponding position of Trp221 of TbPTR1, were observed in the tetramer, indicating a high
flexibility of this residue which delimits the catalytic pocket.
2.3. Docking Studies and Crystal Structure Comparison
The binding modes of the flavanones with those of their corresponding flavonols in TbPTR1 and
LmPTR1 were compared by performing docking studies with a ligand-based constraint in order to favor
binding modes closely related to the crystallographic reference modes as previously described [14].
The crystal structures of TbPTR1 and LmPTR1 show almost identical binding modes for
compound
1
(Figure 3A,B). Interestingly, compound
1
is about twice as potent against TbPTR1 as
against LmPTR1 (IC50 TbPTR1 = 31
µM, LmPTR1 = 57
µM). The LmPTR1 binding pocket is smaller
than the TbPTR1 pocket, since one residue from the neighboring subunit, which is pointing into the
active site, namely Arg287 of LmPTR1, is replaced by the smaller His267 in TbPTR1. In LmPTR1, ring B
is in close proximity to Arg287 and adopts a slightly different orientation allowing a hydrogen bond
between 30-OH and Arg287 to be formed. Notably, in TbPTR1, no direct interaction with His267 is
0
possible and the 3 -OH can only interact with a fully conserved water molecule, and ring B is stabilized
by a T-shaped stacking interaction with Trp221. The corresponding His241 of LmPTR1 does not allow
a similar stabilizing interaction. We conclude that the gating role of Trp221 in TbPTR1, leading to a more
closed pocket, is favorable for activity. The LmPTR1 pocket remains more open and more accessible
for bulk water; the placement of a hydrophobic ligand moiety in a solvent-exposed environment
could affect the binding of compound
modes of compound and flavonol 1A are almost identical. Nevertheless, compound 1A is about
eight-fold more active against TbPTR1 than compound (IC50 TbPTR1 4.3 and 31.0 M, respectively).
As shown in Figure 4, both the chromanone of compound and the chromenone of compound 1A
occupy the stacking position between Phe97 and the cofactor nicotinamide. Compound 1A can
form extended aromatic interactions with the -system of the cofactor; on the contrary, only the
aromatic ring A of compound can interact in a similar manner, while ring C contributes solely to
1 to LmPTR1, leading to the lower affinity. The binding
1
1
µ
1
π
1
hydrophobic stabilization. Moreover, ring C is located in proximity of the rather polar carboxyamide
group of NADP+, which may further explain why the binding of a chromanone (compound 1) is less
favorable than that of a chromenone (compound 1A). This difference in activity is also observed in
LmPTR1 (IC50 LmPTR1 compound 1A: 12.5 µM and compound 1: 57.0 µM).
Figure 4. Comparison of the binding modes of compound
1 (ball-and-stick, yellow carbons) and
compound 1A (lilac carbons) in TbPTR1 (gray cartoon). The NADP+ cofactor (in ball-and-stick,
green carbon atoms) and Phe97 are included for reference. Possible
by cyan dotted lines.
π-stacking interactions are indicated

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Compound 3A is less active than 1A (IC50 TbPTR1 = 38.0 and 4.3
µM and IC50 LmPTR1 = 35.0
and 12.5
µM, respectively). According to the crystal structures, compounds 1A and 3A show
identical binding modes in TbPTR1; however, the 40-OH is solvent-exposed and it does not establish
hydrogen bonds. The same pattern is observed for the inhibitory activity of the corresponding
flavanones towards TbPTR1 (compounds
1 and 3, IC50 TbPTR1 = 31 µM and 82 µM) leading to
the suggestion that the flavanone compounds may share a similar binding mode. This 0idea is
supported by our docking studies, indicating only minor reorientation of the non-interacting 4 -OH in
some docking solutions (data not shown). However, due to the loss of aromatic stacking capability
with the chroman-4-one ring (Figure 4), compound 3A is again more active than compound
(IC50 TbPTR1 = 38 µM and 82 M, respectively). On the other hand, the crystal structure of LmPTR1
with compound reveals a binding mode different from that of compound . In the latter, ring B
3
µ
3
1
is straightened up and hydrogen bonds are established either with Arg287 from the neighboring
subunit or with Tyr283 and water. The difference in activity towards LmPTR1 between flavonol 3A
and flavanone
binding modes are observed in the two different receptors. For example, His241 lies at about 4 Å from
ring B of (see Figure 3D) and is part of an alpha-helix adjacent to the substrate loop. Thus, different
3 is negligible (IC50 LmPTR1 = 35 µM and 36 µM,) and only subtle differences in the
3
substituents in the meta position of ring B may promote additional interactions of this helix with the
ligand and stabilize the substrate loop in a more closed conformation, prohibiting the solvent exposure
of the chroman-4-one/chromen-4-one.
The rather elongated, largely planar structure of compound 2A did not fit in the LmPTR1 active
site with a classical binding mode. The difference in activity between the inactive compound 2A and
the corresponding flavanone
50 M) may be explained by a different mode of binding of the kinked chroman-4-one as shown in
Figure 5. In fact, compound
adopts the typical binding mode in the LmPTR1 pocket with the 40-OH
2 (IC50 LmPTR1 compound 2 = 35 µM; compound 2A: no inhibition at
µ
2
in hydrogen bonding distance to His241 and Arg287, while compound 2A contacts the fully conserved
water molecule bridging to Asp181 with the 6-OH of the chromen-4-one. As a consequence, ring B of
compound 2A is located close to the cofactor and rather solvent-exposed. Also in TbPTR1, flavanone
shows slight activity whereas compound 2A is completely inactive (IC50 TbPTR1 compound = 133 M;
compound 2A: no inhibition at 50 M). The 4 OH does not have suitable hydrogen bonding partners
2
2
µ
µ
'
to allow major stabilization of the binding mode in TbPTR1, thus even the kinked conformation of
the chroman-4-one core is not able to introduce new direct contacts, which is in agreement with the
overall low activity.
Figure 5. (A) Docking poses of compound 2 (ball-and-stick, yellow carbons) and 2A (blue carbons)
in LmPTR1 (pink cartoon with Arg287 from the neighboring subunit shown in magenta sticks;
NADP+ cofactor in ball-and-stick with green carbons). Interactions are indicated as dashed lines
colored according to the interacting compounds’ carbon color. Interacting residues are shown in stick
◦
representation; (
B
) As (
A
) but rotated by approx. 90 . The yellow arrows indicate the kink in the ligand
that is introduced by the chromanone ring system.

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2.4. Biological Profile of Compounds 1–3
In order to evaluate whether the chroman-4-one is a safe scaffold for a drug discovery program,
the in vitro toxicological properties for the three compounds were determined. The inhibitory activity
towards the hERG potassium channel, Aurora B kinase and five cytochrome P450s (CYP1A2, CYP2C9,
CYP2C19, CYP2D6 and CYP3A4) were assessed as well as the mitochondrial toxicity and cytotoxicity
(measured as A549/W1-38 cell growth). The compounds showed a safe profile: they are neither
mitotoxic nor cytotoxic and they do not inhibit hERG and Aurora B kinase. Only compound 1 inhibited
a single cytochrome P450 isoform (CYP2C19) at more than 70%. The data are reported in Table S4 of
the Supporting Information.
Compounds
L. infantum amastigotes at 50
response curve studies for compounds
were the most active molecules against T. brucei with EC50 values of 12.6
Compound showed an EC50 value against T. brucei of 34.8 1.1
for cytotoxicity on THP-1 macrophage-like cells to determine the NOAEL (no observed adverse
effect level). All the compounds presented a NOAEL higher than 100 M. The data are shown in
Table S5 of the Supporting Information. The selectivity index (SI), given by the ratio between the CC50
toward THP-1 and the EC50 toward T. brucei, was higher than 7 for compounds and . Compounds
and also weakly inhibited L. infantum cell growth (% inhibition at 50 M: 31% and 29%, respectively),
1
–
3
were tested against the bloodstream form of T. brucei at 10
µM and against
µM, since it is usually more difficult to find antileishmanial hits. Dose
1–
3
against T. brucei were performed. Compounds
1.7 and 13.0
1
1.8
and
2
±
±
µM.
3
±
µM. The compounds were assessed
µ
1
2
1
2
µ
while compound 3 was inactive against L. infantum.
Among the chroman-4-one inhibitors, compound 1 was the most potent derivative against both
T. brucei and L. infantum. The data for compound
parasitic activity. At the enzyme level, there is a 2.5-fold activity difference between compounds
with compound being the most potent inhibitor of TbPTR1. At the biological level, the difference in
potency between compounds 1 and 3 is 2.8-fold, suggesting that PTR1 might be one of the molecular
targets. Compound does not follow this apparent trend since it is not a good inhibitor of TbPTR1,
but it shows antiparasitic activity comparable to that of compound . Therefore, its potency against
3
support the involvement of PTR1 inhibition in the
1
and 3,
1
2
1
the T. brucei parasite might result from effects on other protein targets.
3. Materials and Methods
3.1. General Information
All commercial chemicals and solvents were reagent grade and were used without
further purification. Reaction progress was monitored by thin layer chromatography (TLC) on
pre-coated silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany) and visualization was
1
accomplished with UV light (254 nm). H- and 13C-NMR spectra were recorded on a Bruker FT-NMR
AVANCE 400 (CIGS, Centro Interdipartimentale Gradi Strumenti, Modena, Italy). Chemical shifts are
reported as
(DMSO) at
δ
values (ppm) referenced to residual solvent (CHCl3 at
δ 7.26 ppm, dimethyl sulfoxide
δ
2.50 ppm, MeOD at 3.31 ppm); J values were given in Hz. When peak multiplicities are
δ
given, the following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broadened signal. Silica gel Merck (60–230 mesh) was used for column chromatography.
The reaction progress was monitored by TLC (Merck F-254 silica gel). Mass spectra were obtained on
a 6520 Accurate-Mass Q-TOF LC/MS and 6310A Ion Trap LC-MS(n) (CIGS, Centro Interdipartimentale
Gradi Strumenti, Modena, Italy). The detailed nuclear magnetic resonance (NMR) and mass data of
the synthesized compounds are reported in the Supporting Information (p. S2).
3.2. General Procedure for the Synthesis of Compounds 1–3
To a stirred mixture of 20,50-dihydroxyacetophenone (0.30 g, 1.97 mmol) and the appropriate
aldehydes (1 eq.) in absolute ethanol (2 mL), thionyl chloride (120
µL) was added dropwise over 5 min.
The reaction was stirred at room temperature for 6 h. Ethanol and excess of thionyl chloride were

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removed under reduced pressure on a rotary evaporator. Column chromatography was carried out to
purify the desired product (eluent system: cyclohexane/ethyl acetate 9.8/0.2).
3.3. Computational Methodology
Compounds were generated with the LigPrep routine of Schrödinger Maestro [24] as previously
described [14]. Important structural water sites in the crystal structures were identified with a WatCH
clustering approach [25] for both LmPTR1 and TbPTR1 as previously published [14]. Docking grid
preparation and docking studies followed the previously published methodology for docking with
Glide [14,26–28]. Two crystal structures are reported in the present work, namely PDB code 5K6A for
docking to TbPTR1 and 5L4N for LmPTR1.
The crystal structures of TbPTR1 have oxo-cysteine at residue 168. To assess the possible impact
of the oxo-modification on the ligand binding pose, the complex of TbPTR1 was prepared with
compound 1 twice: once including the modification and once with a mutation to a normal cysteine
using the program PYMOL [29]. However, we found no major impact of this modification on our
docking results.
3.4. Protein Purification and Kinetic Profile Characterization
TbPTR1 and LmPTR1 were purified as reported in literature [14]. The enzyme kinetic profile
of each selected protein was characterized by a spectrophotometric assay. The experimental assay
consists of enzyme kinetic reaction monitoring at 340 nm for a total time of 180 s. The enzyme kinetic
activity was measured at steady-state concentration of 7,8-dihydro-L-biopterin (H2B) and NADPH
substrates equal to 50 and 120
µM and varying the enzyme concentration in the 0.001–0.006 µM and
0.005–0.010–0.015–0.020–0.025–0.030
µ
M range for Lm- and TbPTR1, respectively. The resulting kcat
values were equal to 0.68 (s−1) and 7.2 (s−1) for Tb- and LmPTR1, respectively. For both TbPTR1
and LmPTR1, the Km value evaluation assay for H2B- and NADPH-substrates was performed in the
0.3-0.6–0.9–1.2–2.4–4.8
µM and 3.0–6.0–12.0–40.0–90.0–190.0
µM concentrations range, respectively.
The obtained H2B- and NADPH-Km data were equal to 5.5
µ
M, 6.8 M and to 10.5 M, 16.0 M for
µ
µ
µ
Lm- and TbPTR1 respectively, and thus consistent with the data previously published [14].
3.5. TbPTR1 and LmPTR1 Enzyme Assay
Compounds 1A–3A were evaluated as reported in our previous paper [14]. The novel synthesized
compounds were tested towards TbPTR1 and LmPTR1 using the in vitro TbPTR1/LmPTR1 enzyme
1–3
reporter assay. The experimental procedure consists of monitoring the non-enzymatic reduction
reaction of cytochrome-c at 550 nm, related to the reduction reaction of H2B to tetrahydrobiopterin
(H4B) by using NADPH as the cofactor. TbPTR1 and LmPTR1 enzyme activity was assayed in a buffer
containing 20 mM sodium citrate (pH 6.0) in a final experimental volume equal to 600
reaction mixture contained different substrates added in the following order: TbPTR1/LmPTR1
(0.018 M/0.002 M) in 20 mM Tris-HCl, glycerol 10% (v/v), pH 7.5; H2B at a final concentration
equal to 0.3 M; cytochrome-c (80 M); Milli-Q water to a final volume of 600 L and NADPH to
M. Before the addition of NADPH, the reaction mixture was incubated for
µL. The final
µ
µ
µ
µ
µ
a final concentration of 500
10 min at 30 C and 80 RPM. To allow the starting of the reaction, NADPH was added and the enzyme
reaction was detected for a total time of 10 min at 30 C, 550 nm. The inhibition profile characterization
µ
◦
◦
of compounds
CA, USA). Each compound was assayed in duplicate against Tb/LmPTR1 target enzymes in the
0–10–25–50–80–150 M concentrations range. At the different concentration values, the compounds
1–3 was performed with a DU-640 spectrophotometer (Beckman Coulter, Brea,
µ
were added to the reaction mixture (enzyme,◦H2B, cytochrome-c in 20 mM sodium citrate buffer and
Milli-Q water) and incubated for 10 min at 30 C, 80 RPM. The reaction was initiated by the addition of
NADPH (500 µ
M) and monitored for 10 min at 30 ◦C, 550 nm. The absorbance values were processed
by software analysis and transformed into the corresponding slopes. The percentages of inhibition
and the IC50 values were determined.

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3.6. Crystallization
TbPTR1. Crystals of TbPTR1 were obtained by the vapor diffusion sitting drop technique at room
temperature [30]. Drops were prepared by mixing equal volumes of protein (TbPTR1 6–10 mg
mL−1 in
20 mM Tris-HCl pH 7.5 and 10 mM DTT) and precipitant (1.5–2.5 M sodium acetate and 0.1 M sodium
·
citrate pH 5.0) solutions and equilibrated over 600
grew in several days.
µL of reservoir. Well-ordered monoclinic crystals
The ternary TbPTR1-cofactor-compound
1 complex was prepared by diffusion soaking of a 2 mM
solution of the inhibitor (dissolved in a 1:1 mixture of 1,4-dioxane and water) into pre-formed crystals
of the native enzyme for 4 h. Crystals were then transferred in a cryo-protectant, prepared by adding
30% glycerol to the precipitant solution, and flash frozen in liquid nitrogen.
LmPTR1. Crystals of the binary complexes of LmPTR1 and NADPH were obtained as previously
described [23], with minor modifications. The protein (12.5 mg
sodium acetate pH 5.3 containing 1 mM NADPH and 20 mM DTT, incubated at 4 C for 1.5 h and later
concentrated up to the starting volume with centrifugal concentrators (cutoff 10 kDa). The resulting
·
mL−1) was 10-fold diluted in 20 mM
◦
sample was used to obtain crystals with the hanging-drop technique [30]. A drop containing 2
µL
complex solution plus 2 L of a reservoir solution consisting of 12% PEG 4600, 100 mM sodium
µ
acetate buffer pH 5.5 and 120–160 mM calcium acetate was set up against reservoir solution at 293 K.
The crystals grew as clumps of thin fragile rods.
The inhibitor-bound complexes of LmPTR1 with compounds
1 and 3 were prepared by diffusion
soaking of a 2 mM solution of the inhibitors (dissolved in a 1:1 mixture of 1,4-dioxane and water) into
pre-formed crystals of the native enzyme for 4.5 h.
Fragments of the crystal rods were cleaved for analysis, cryopreserved by transferring into
a solution consisting of 70% reservoir solution and 30% glycerol and flash-cooled in liquid nitrogen.
3.7. Data Collection, Structure Solution and Refinement
X-ray diffraction data of TbPTR1 ternary complex with NADP+ and compound
1 were collected
at Elettra Synchrotron (Trieste, Italy) on beamline XRD-1, equipped with a Pilatus 2 M detector,
◦
using a wavelength of 1.0000 Å, a ∆ϕ of 1 and an exposure time of 15 s per image. Diffraction data of
the ternay complex of LmPTR1-NADP+-1 were collected at Diamond Light Source (DLS, Didcot, UK)
on beamline I04, equipped with a Pilatus 6M-F detector, using a wavelength of 0.9795 Å, a ∆ϕ of
0.2◦ and an exposure time of 0.2 s per image. Diffraction data of the complex LmPTR1-NADP+-
3
were collected at Elettra Synchrotron (Trieste, Italy) on beamline XRD-1, equipped with a Pilatus 2M
detector, using a wavelength of 1.0000 Å, a ∆ϕ of 1◦ and an exposure time of 35 s per image.
Data was integrated with MOSFLM 7.0.4 [31] or XDS [32] and scaled with the program SCALA [33
]
from the CCP4 suite (Collaborative Computational Project, 1994). Structures of complexes were solved
by molecular replacement with the software MOLREP [34] using the coordinates of a whole tetramer
of TbPTR1 (PDB code 2X9G) and LmPTR1 (PDB code 2BFA) as searching models. In all models,
non-protein atoms and water molecules were omitted. Structures were refined using REFMAC5 [35].
The refinement protocol consisted of a sequence of iterative manual rebuilding of the models and
maximum likelihood refinement. Visual inspection, manual rebuilding of the models and modeling of
the missing atoms into the electron density between refinement cycles were performed with Coot [36].
Water molecules were added using default parameters as implemented in the software ARP/wARP
suite [37] and checked by visual inspection [38].
The final models were inspected manually and checked with both Coot and Prockeck [39]. Data
collections and refinement statistics are reported in Tables S2 and S3.
Figures of the electron density were generated using CCP4mg [40].
PDB codes: 5K6A (TbPTR1 in complex with compound
compound 1), 5L42 (LmPTR1 in complex with compound 3).
1), 5L4N (LmPTR1 in complex with

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3.8. In Vitro Evaluation of Activity against L. infantum Intramacrophage Amastigotes
The efficacy of compounds against L. infantum (MHOM/BR/1972/BH46) intracellular amastigotes
was measured at a single concentration of 10 M. THP-1 cells were plated onto 384-well plates in
µ
Roswell Park Memorial Institute (RPMI) 1640 medium, Hyclone, GE Healthcare Life Sciences (Issaquah,
WA, USA) containing 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, Saint Louis,
MS, USA), and incubated for 48 h. Then, 6 day-old promastigotes were added, at a ratio of 50 parasites
per each THP-1 cell seeded earlier. After 24 h of infection, negative controls (0.5% DMSO), positive
controls (10 µM amphotericin B) or compounds were added to the plate. Assay plates were incubated
for 48 h, and then fixed with 4% paraformaldehyde and stained with Draq 5. The Operetta high-content
automated imaging system was used to acquire images and the Harmony Software was optimized
to quantify the host cell number, the infection ratio and the number of parasites per infected cell.
The ratio between infected cells and total number of cells is then calculated, and defined as the
Infection Ratio (IR). The raw data for IR values was normalized to negative—DMSO (mock)-treated
infected cells—and positive (non-infected cells) controls to determine the normalized antiparasitic
activity [41].
3.9. In Vitro Evaluation of Activity against T. brucei
The efficacy of compounds against T. brucei bloodstream forms was evaluated using a modified
resazurin-based assay previously described in literature [42].
3.10. Cytotoxicity Assessment against THP-1 Macrophages
The effect of compounds 1–3 on THP-1-derived macrophages was assessed by the colorimetric
(MTT) assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide).
3.11. hERG Assay
The assay made use of Invitrogen’s Predictor
a membrane fraction containing hERG channel (Predictor
fluorescent hERG channel ligand, or “tracer” (Predictor
™
hERG Fluorescence Polarisation Assay. This uses
hERG Membrane) and a high-affinity red
hERG Tracer Red), whose displacement by
™
™
test compounds can be determined in a homogenous, Fluorescence Polarisation (FP)-based format [43].
3.12. Cytochrome P450 1A2, 2C9, 2C19, 2D6 and 3A4 Assays
These assays made use of the PromegaP450-Glo™ (Madison, AL, USA) assay platform.
Each CYP450 assay includes microsomal preparations of cytochromes from baculovirus-infected
insect cells. Action of the CYP450 enzymes on each substrate ultimately resulted in the generation of
light and a decrease in this was indicative of inhibition of the enzymes [44].
3.13. Cytotoxicity Assay against A549 and W1-38 Cells
The assays were performed using the CellTiter-Glo assay from Promega (Madison, AL, USA).
The assay detects cellular adenosine triphosphate (ATP) content with the amount of ATP being
directly proportional to the number of cells present. The A549 cells were obtained from DSMZ
(German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and W1-38 cells
were obtained from ATCC (ATCC® CCL-75
(DMEM) from Capricorn Scientific GmbH, Ebsdorfergrund, Germany with fetal bovine serum (FBS)
™) and were grown in Dulbecco's Modified Eagle Medium
(10% v/v) from Capricorn Scientific GmbH, Ebsdorfergrund, Germany, streptomycin (100 µg/mL) and
penicillin G (100 U/mL) [45].

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3.14. Aurora B Kinase Assay
This assay made use of the ADP-Glo Kinase Enzyme System from Promega (Madison, AL, USA),
which is a bioluminescent assay that employs firefly luciferase in a coupled-enzyme assay format to
enable detection of adenosine diphosphate (ADP) levels from ATPase assays [46].
3.15. Assessment of Mitochondrial Toxicity
This made use of MitoTracker® Red chloromethyl-X-rosamine (CMXRos) uptake and High
Content Imaging to monitor compound mediated mitochondrial toxicity in the 786-O (renal carcinoma)
cell line. Cells were maintained using Roswell Park Memorial Institute-1640 (RPMI-1640) medium from
Capricorn Scientific GmbH, Ebsdorfergrund, Germany containing 2 mM glutamine, FBS (10% v/v),
streptomycin (100 µg/mL) and penicillin G (100 U/mL) [47].
4. Conclusions
PTR1 is a promising target for the development of antitrypanosomal and antileishmanial agents.
The identification of novel scaffolds for the design and synthesis of PTR1 inhibitors is an ongoing challenge.
Crystallographic structures are an essential tool for structure-based drug discovery programs, since they
allow rapid scaffold optimization and hit-to-lead progression. Herein, we solved the crystal structure of
the TbPTR1-
1
complex. Moreover, these experiments provided the first X-ray crystallographic structures
and ). The chroman-4-one scaffold
of LmPTR1 in complex with chroman-4-one derivatives (compounds
1
3
binds to TbPTR1 and LmPTR1 with an orientation similar to that of the biopterin substrate, suggesting
that it is a promising scaffold for the development of PTR1 inhibitors. The compounds were assessed
for early toxicity properties and were found to have a safe profile. The three synthesized compounds
were evaluated against T. brucei and L. infantum. Compound
enzymes (TbPTR1/LmPTR1) and the parasites (T. brucei and L. infantum) together with a selectivity index
greater than 7 and a low toxicity. Thus, compound is a promising hit. The use of the flavanone scaffold
1 showed activity both against the targeted
1
guided by the crystal structures could lead to the design of more potent and selective PTR1 inhibitors.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This project has received funding from the European Union’s Seventh Framework
Programme for research, technological development and demonstration under grant agreement No. 603240
(NMTrypI—New Medicines for Trypanosomatidic Infections) http://www.nmtrypi.eu/. I.P. and R.C.W. gratefully
acknowledge the support of the Klaus Tschira Foundation. We acknowledge Wolfgang Mueller and Ulrike Wittig
for the platform SEEK (https://fp7h-synergy.h-its.org/) where rough structural and biological data are reported
for the reader’s interest. We also acknowledge Diamond Light Source and Elettra Synchrotron for access to
beamline I04 and XRD-1, respectively.
Author Contributions: C.B. synthesized the compounds and analyzed the data. I.P. and R.C.W. analyzed the data
and performed docking studies. F.D.P., G.L., L.D.I. and C.P. expressed, purified and crystallized the proteins,
collected the crystallographic data, solved and refined the crystal structures. M.S. evaluated the activity of
the compounds towards the targeted enzymes. A.-d.C.S., N.S., C.B.M., L.M.A., V.F. and L.H.F.-J. assessed the
antiparasitic activity. S.G., M.K. and B.B. carried out the in vitro toxicological evaluation. C.B., S.M., F.D.P., C.P.,
I.P., R.C.W., M.P.C. and S.F. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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