Pyrazolyl-pyrimidones inhibit the function of human solute carrier protein SLC11A2 (hDMT1) by metal chelation

Article abstract of DOI:10.1039/d0md00085j

Solute carrier proteins (SLCs) control fluxes of ions and molecules across biological membranes and represent an emerging class of drug targets. SLC11A2 (hDMT1) mediates intestinal iron uptake and its inhibition might be used to treat iron overload diseases such as hereditary hemochromatosis. Here we report a micromolar (IC50 = 1.1 μM) pyrazolyl-pyrimidone inhibitor of radiolabeled iron uptake in hDMT1 overexpressing HEK293 cells acting by a non-competitive mechanism, which however does not affect the electrophysiological properties of the transporter. Isothermal titration calorimetry, competition with calcein, induced precipitation of radioactive iron and cross inhibition of the unrelated iron transporter SLC39A8 (hZIP8) indicate that inhibition is mediated by metal chelation. Mapping the chemical space of thousands of pyrazolo-pyrimidones and similar 2,2′-diazabiaryls in ChEMBL suggests that their reported activities might partly reflect metal chelation. Such metal chelating groups are not listed in pan-assay interference compounds (PAINS) but should be checked when addressing SLCs.

Full text of DOI:10.1039/d0md00085j

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Pyrazolyl-pyrimidones inhibit the function of
human solute carrier protein SLC11A2 (hDMT1) by
Cite this: DOI: 10.1039/d0md00085j
metal chelation†
bcd
e
Marion Poirier,‡a Jonai Pujol-Giménez,
‡
Cristina Manatschal,
Sven Bühlmann,a Ahmed Embaby,a Sacha Javor,
a
bcd
a
*
*
Matthias A. Hediger
and Jean-Louis Reymond
Solute carrier proteins (SLCs) control fluxes of ions and molecules across biological membranes and
represent an emerging class of drug targets. SLC11A2 (hDMT1) mediates intestinal iron uptake and its
inhibition might be used to treat iron overload diseases such as hereditary hemochromatosis. Here we
report a micromolar (IC50 = 1.1 μM) pyrazolyl-pyrimidone inhibitor of radiolabeled iron uptake in hDMT1
overexpressing HEK293 cells acting by a non-competitive mechanism, which however does not affect the
electrophysiological properties of the transporter. Isothermal titration calorimetry, competition with calcein,
induced precipitation of radioactive iron and cross inhibition of the unrelated iron transporter SLC39A8
(hZIP8) indicate that inhibition is mediated by metal chelation. Mapping the chemical space of thousands
of pyrazolo-pyrimidones and similar 2,2′-diazabiaryls in ChEMBL suggests that their reported activities
might partly reflect metal chelation. Such metal chelating groups are not listed in pan-assay interference
compounds (PAINS) but should be checked when addressing SLCs.
Received 11th March 2020,
Accepted 6th May 2020
DOI: 10.1039/d0md00085j
rsc.li/medchem
pathologies
such
as
hereditary
hemochromatosis,
Introduction
β-thalassemia, Parkinson's disease and Alzheimer's disease,
highlighting that its pharmacological inhibition may be
beneficial to treat human diseases.4–9 While metal chelators
have been classically used to treat metal intoxication10 and
neurodegenerative diseases,11,12 a recently reported highly
potent and specific inhibitor of ferroportin (SLC40), a different
iron transporter acting on the same pathway as hDMT1, has
been shown to have clinical efficacy against β-thalassemia.13
Two families of small molecule hDMT1 inhibitors have been
reported in the literature as the results of high-throughput
screening campaigns, namely bis-cationic isothioureas such as
dibenzofurans 1 and mesitylene 2,14 as well as pyrazolyl-pyridine
3 (Fig. 1).15 In our own investigations on hDMT1, we used the
inhibitors mentioned above as seeds for a ligand-based virtual
screening campaign guided by 3D-shape and pharmacophore
similarity16 and discovered bis-isothiourea 4 and pyrazolyl-
pyrimidone 5 as two additional hDMT1 inhibitors.17 Kinetic
Solute carrier proteins (SLCs) control fluxes of ions and other
molecules across biological membranes and represent an
emerging class of drug targets.1–3 Here, we investigate inhibitors
of the human H+-coupled transporter of ferrous iron (Fe2+),
SLC11A2 (hDMT1). This transporter is expressed in the
intestinal brush border membrane, where it acts as the key
mediator of dietary iron uptake. hDMT1 is also linked to
a Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3,
3012 Bern, Switzerland. E-mail: jean-louis.reymond@dcb.unibe.ch
b Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse
28, 3012 Bern, Switzerland
c Membrane Transport Discovery Lab, Department of Nephrology and Hypertension,
Inselspital, University of Bern Kinderklinik, Freiburgstrasse 15, 3010 Bern,
Switzerland. E-mail: matthias.hediger@ibmm.unibe.ch
d Department of Biomedical Research, University of Bern, Murtenstrasse 35, 3008
Bern, Switzerland
e Department of Biochemistry, University of Zürich, Winterthurerstrasse 190,
Zürich, Switzerland
studies, an X-ray structure of
a related brominated bis-
isothiourea inhibitor in complex with a bacterial analog of the
transporter and mutational studies recently showed that bis-
isothiourea-based compounds act as competitive inhibitors of
hDMT1.18 On the other hand, pyrazolyl-pyrimidone 5, whose
small size and better drug-like properties made it an attractive
candidate compound, acted as a non-competitive inhibitor. Here
we set out to investigate the pyrazolyl series (3 and 5) closer and
understand its mechanism of action.
† Electronic supplementary information (ESI) available: Methods for bioassays,
details of kinetic studies, SMARTS pattern analysis, chemical synthesis
procedures and compound characterization, X-ray deposition information, HPLC
purity table, NMR spectra of final compounds, and SMILES list of final
compounds with activities and of compounds extracted from ChEMBL. CCDC
1976759, 1976832 and 1976862 For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/d0md00085j
‡ These authors contributed equally to the work.
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We measured hDMT1 activity using a radioactive iron
uptake assay in HEK293 cells stably overexpressing hDMT1
as described previously.17 Dibenzofuran inhibitor 1 showed
only micromolar potency in this assay, which is 5-fold weaker
than the originally reported value based on a calcein assay,
while the mesitylene inhibitor 2 showed a comparable value
to the originally reported inhibition (Table 1). For the present
study, we selected dibenzofuran inhibitor 1 as positive
control. However, we were unable to determine an IC50 value
for the known pyrazolyl-pyridine 3 due to a lack of inhibition
at high compound concentration.
Activity screening of the twenty synthesized analogs of 5
for inhibition of hDMT1 revealed that seven pyrazoles (6, 7,
13, 16–19) showed similar or improved inhibition compared
to 5. On the other hand, pyrazoles 8–12, 14–15 and 20 as well
as pyrazolones 21–25 did not show significant inhibition of
iron uptake in our assay. Across all compounds tested,
pyrazole 13 stood out as one of the most potent compounds
with IC50 = 1.1 0.01 μM, representing a 10-fold improvement
over our initial inhibitor 5 while preserving the original ligand
efficiency. Note that the bis-cyclohexane analog 18 was
slightly more potent, however at the cost of higher
hydrophobicity.
Fig. 1 Structure and reported activity of DMT1 inhibitors. Data for
inhibition of Fe2+ uptake into transfected HEK cells measured by
calcein fluorescence assay (1–3) from ref. 14 and 15 or radioactive
55Fe2+ uptake assay (4 and 5) from ref. 17.
Results and discussion
1. Optimization of pyrazolyl-pyrimidone 5
2. Characterization of pyrazolyl-pyrimidone 13
Closer characterization of pyrazolyl-pyrimidone 13 in the
radioactive 55Fe2+ uptake assay showed that this compound
was a non-competitive inhibitor with similar inhibition
constants Ki = 2.0 μM ([Fe2+] → 0) and Kii = 1.7 μM (saturating
iron), reproducing the behavior of the previously reported
less potent pyrazolyl-pyrimidone 5. By comparison,
isothiourea 1 showed a competitive inhibition with inhibition
constant Ki = 1.4 μM ([Fe2+] → 0), Kii = 15.6 μM (saturating
iron) determined by Dixon plot analysis,21 similarly to the
previously reported less potent isothiourea 4 (Fig. S1†).
We prepared twenty new analogs (6–25) of pyrazolyl-pyrimidone
5 by condensing aminoguanidines with 1,3-diketones and keto-
esters to form pyrazoles 6–20 and pyrazolones 21–25 using
known chemistry (Scheme 1).14,15,19,20 For pyrazoles the
synthesis gave only one product from symmetrical diketones
(5–9, 16, 18–20). With non-symmetrical diketones synthesis also
mostly yielded a single product. X-ray crystal structures of 13
and 17 showed that the product from non-symmetrical
diketones was the isomer with the larger pyrazole substituent
pointing away from the aminopyridone. By analogy, the same
arrangement was assigned to the other pyrazoles. The synthesis
of pyrazolones 21–25 only gave a single product whose
structures were confirmed by an X-ray crystal structure of 24 as
hydrochloride salt (Fig. 2).
On the other hand, the uptake of radioactive iron in
Xenopus oocytes overexpressing hDMT1 was totally inhibited
by the bis-thiourea inhibitor 1, while pyrazolyl-pyrimidone 13
only
inhibited
50%
of
the
uptake
(Fig.
3a).
Electrophysiological recordings in this system furthermore
showed that only inhibitor 1 blocked hDMT1-induced current
with a potency comparable to the iron uptake experiment
(IC50 = 0.14 μM), but that pyrazolyl-pyrimidone 13 had no
measurable effect on the recorded currents (Fig. 3b/c).
Taken together, these experiments showed that 13 was
indeed
a more potent version of the non-competitive
inhibitor 5. However, in contrast to the well-behaved bis-
isothiourea 1, the inhibitory effect of 13 was only detected in
iron uptake experiments and was not observed in
electrophysiological recordings. A similar effect had been
reported for the literature inhibitor 3.15
3. Chemical evidence for metal complex formation
At this stage we suspected that pyrazolyl-pyrimidone 13 might
inhibit hDMT1 indirectly by chelating iron, although this
Scheme 1 Representative synthesis of pyrazolyl-pyrimidones at the
example of 13 and 19.
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Fig. 2 Chemical structures and X-ray crystallography of analogs of pyrazole 5. Carbon is in gray, nitrogen in blue, oxygen in red and chlorine in green.
Table
1 Activity of DMT1 inhibitors as measured by inhibition of
N:NIJ:C)–C:N substructure which is potentially capable of
forming a 5-membered ring chelate with metal ions via the
two terminal sp2 nitrogen atoms, although this substructure
is present in all the synthesized analogs including those that
did not show any inhibition. The formation of such
5-membered ring chelate has been recently established by
X-ray crystallography of a ruthenium complex containing
both a pyrazolyl-pyridine and a pyrazolyl-pyridone ligand and
investigated as C–H oxidation catalyst.22 Unfortunately, UV-
vis titration of 13 could not be used to provide any evidence
for metal complexation since the UV-vis spectrum did not
significantly change upon titration with Fe2+ and Cd2+.
Attempts to crystallize the iron complex of 13 as well as
investigation by 1H-NMR and mass spectrometry similarly
did not yield any indication of complex formation.
A first indication that pyrazolyl-pyrimidone 13 might
interact with divalent metal ions was obtained by isothermal
titration calorimetry (ITC) with Cd2+, which we used as a
redox stable analog of Fe2+ less prone to hydroxide formation
at pH = 7.4 and which is also transported by DMT1. At a pH
of 7.4, the ITC data indicated an apparent dissociation
constant of KD = 6.4 μM with n = 0.54, consistent with the
formation of a 2:1 ligand–metal complex (Fig. 4a). At pH 5.5
under which the hDMT1 transport experiment is carried out,
radioactive iron uptake in HEK293 cells
a
Compound
IC50 (μM)
Ligand efficiency
1
2
3
5
6
7
13
16
17
18
19
1.76 0.06
0.35 0.04
≫10 μMb
13.1 0.7
10.0 0.4
4.2 0.4
1.1 0.01
3.8 0.2
2.26 0.3
0.94 0.2
12.5 0.3
0.35
0.25
0.46
0.44
0.40
0.45
0.45
0.42
0.42
0.43
a
IC50 values were calculated using the radiolabeled iron uptake
assay in HEK293 cells (source: American Type Culture Collection,
catalog no. CRL-1573) stably overexpressing hDMT1. Absorbed
radioactive iron (1 μM) was measured after 15 min of incubation in
the presence of the indicated compound at different concentrations
b
at extracellular pH 5.5. Partial inhibition was detected at 10 μM,
however apparent uptake increased at higher concentrations.
Compounds 8–12, 14–15, 20–25 showed less than 20% inhibition at
10 μM in this assay.
possibility was judged unlikely for the related inhibitor 3 by
lack of conclusive evidence.15 These inhibitors contain an
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Fig. 3 Characterization of Fe2+ transport inhibition by 1 and 13 in X. laevis oocytes (collected from frogs under license number BE60/18 conceded
by the Veterinärdienst des Kantons BE, Sekretariat Tierversuche). (a) Upper panel: Average 55Fe2+-uptake (20 μM) by hDMT1-expressing and non-
injected oocytes in the absence and the presence of the indicated compounds (10 μM). Data was normalized to 55Fe2+-uptake (pmol min−1) in the
absence of compounds represented as mean SD (16–29 oocytes). Lower panel: Net uptake of 55Fe2+ (20 μM) by hDMT1 in the absence and the
presence of the indicated compounds (10 μM). For each batch of oocytes, uptake values were corrected for unspecific iron uptake in non-injected
oocytes. Data were normalized to the mean iron uptake by hDMT1 WT (pmol min−1) and represented as mean SD (14–34 oocytes). (b and c)
Electrophysiology. Oocytes were held at −50 mV (Vh) and a voltage-step protocol was applied (Vm = −150 mV to +50 mV) before and after the
addition of Fe2+ (20 μM) in the absence or the presence of the indicated compounds (10 μM). For each trace, recorded currents were corrected for
the background currents observed without substrate. (b) Representative traces recorded with hDMT1 injected oocytes (upper panel) and non-
injected oocytes (lower panel). (c) Upper panel: Average current traces of the response of hDMT1 injected oocytes to the indicated compounds.
Data was normalized to Fe2+ (20 μM) evoked currents at −150 mV and represented as mean SD (8 to 13 oocytes). Lower panel: Average dose–
response (0.01–10 μM) of the inhibition of the indicated compounds over the Fe2+ (20 μM) evoked currents at −70 mV. Data was normalized to
Fe2+ (20 μM) + compound (0.01 μM) evoked currents and represented as mean
SD (4 oocytes). Kinetic parameters were obtained by fitting
experimental results to a 4-parameter sigmoidal curve (black line). Compound 13 did not fit to the equation while for compound 1 the obtained
IC50 was 0.14 μM. All the experiments were performed with oocytes from at least 3 different oocyte batches. Statistical differences were assessed
using T-test or Mann–Whitney U test (Fe2+-uptake by hDMT1 WT vs. non-injected oocytes or vs. compound); p > 0.05 = ns; p < 0.001 = ***.
the ITC data could not be interpreted with certainty. Upon
titration of Cd2+ into a solution of 13, the exothermic signal
seems to saturate after the first injections, but later results in
a noisy signal, indicating precipitation (Fig. 4b). The less
potent pyrazolyl-pyrimidone DMT1 inhibitor 5 also interacted
with Cd2+ at pH 7.4, although binding was much weaker (KD
∼ 250 μM, n = 0.74).
precipitation with the literature inhibitor 3 corresponding to
the pH of the assay with hDMT1 (Fig. 4d and e). Such
precipitation might indicate chelation of iron to form an
insoluble complex. Taken together, these data pointed to
potential iron chelating abilities of our inhibitors, however
the evidence was inconclusive and did not match well with
the observed inhibitory potencies.
A calcein competition assay to detect Fe2+ chelation at pH
5.5 also indicated a significant level of iron chelation by 13,
although the effect was weaker than with the positive control
bipyridine or the literature inhibitor 3, and pyrazolyl-
pyrimidone 5 did not show any iron chelation in this assay
(Fig. 4c). Tracking the radioactivity of 55Fe2+ in solutions after
incubation with the various inhibitors indicated that
pyrazolyl-pyrimidone 13 as well as the literature inhibitor 3
induced significant precipitation of Fe2+ at pH 7.4, while bis-
isothiourea 1, bipyridine or pyrazolyl-pyrimidone 5 had no
effect. On the other hand, at pH 5.5, we only observed
4. Cross-inhibition of human SLC11A2 (hDMT1) and SLC39A8
(hZIP8)
Complex formation with Fe2+ should result in non-selective
inhibition of different metal transporters. To test this
hypothesis, we investigated whether human SLC39A8 (hZIP8),
a transporter of Zn2+ and Fe2+ unrelated to SLC11 family,
might also be inhibited by our hDMT1 inhibitors. Both SLCs
can be tested by inhibition of radioactive iron uptake into
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Fig. 4 Chemical evidence of divalent metal complexation by hDMT1 inhibitors. (a and b) Isothermal titration calorimetry of pyrazole 13 (0.4 mM)
with Cd2+ (5 mM) in aqueous buffer pH 7.4 and pH 5.5, 25 °C. (c) Calcein quenching assay in aqueous buffer pH 5.5 (2 μM Fe2+, 10 μM ligand, 1 μM
calcein). Fe2+ (2 μM), ascorbic acid (200 μM) and compounds (20 μM) were incubated for 5 min before the addition of calcein (1 μM) in Krebs buffer
at pH 5.5. Fluorescence was measured after 5 min incubation. (d and e) Removal of radioactive 55Fe2+ from buffers upon incubation with ligands and
centrifugation. 55Fe2+ (1 μM) precipitation upon interaction with the indicated compounds (10 μM). 55Fe2+ and the indicated compounds were
incubated for 15 min, then, an aliquot of the solution supernatant was taken for determination of the remaining 55Fe2+. Data was normalized to the
values obtained for the Fe2+ + DMSO (0.1%) solution and represented as mean SD (N = 15; 2 independent experiments). Statistical differences
were assessed using T-test or Mann–Whitney U test (Fe2++ DMSO vs. each compound); p > 0.05 = ns; p < 0.05 = *; p < 0.001 = ***.
HEK293 cells expressing the transporter, at pH 5.5 for
hDMT1 and at pH 7.4 for hZIP8.
comparable IC50 values on both transporters (13: IC50 ∼ 1.1
μM, bipyridine: IC50 ∼ 6.5 μM, Fig. 5b/c).
Indeed, activity screening showed that bipyridine, a well-
known iron chelator, non-specifically inhibited both
transporters. Similarly, the literature pyrazolyl-pyridine 3, our
optimized pyrazolyl-pyrimidone 13, and to a lesser extent
pyrazolyl-pyrimidone 5, all inhibited both transporters,
although the inhibition by 3 was not consistently observed at
higher concentrations, probably due to adsorption of the
complex to the cell material and assay plate, resulting in
apparent uptake. On the other hand, bis-isothiourea 1, which
inhibits hDMT1 by competing in the iron binding site, only
inhibited hDMT1 and had no measurable activity on hZIP8
(Fig. 5a). A precise determination of the inhibitory effects
with pyrazolyl-pyrimidone 13 and with the known iron
chelator bipyridine showed that these compounds had
5. Mechanistic model
We interpret the non-selective inhibition of bipyridine and 13
on both hDMT1 and hZIP8 as an indication that inhibition is
caused by chelation of iron without specific interactions with
the transporters. Metal chelation most likely involves a five-
membered ring chelate with the two cyclic nitrogen atoms
within the N:NIJ:C)–C:N substructures, similar to the well-
known chelation by bipyridine forming a tight 3:1 complex
with Fe2+. Acid–base titration of 3, 5 and 13 shows in each
case two ionizable groups (3: pKa1 = 3.3, pKa2 = 6.7; 5: pKa1 =
3.6, pKa2 = 6.5; 13: pKa1 = 3.3, pKa2 = 7.0, Fig. 6a). The
transition at neutral pH (pKa2) can be attributed to the
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Fig. 5 Cross reactivity of hDMT1 inhibitors over the functional activity of the divalent metal transporter hZIP8. (a) 55Fe2+ uptake (1 μM) by
HEK293T cells (source: American Type Culture Collection, catalog no. CRL-3216) transiently transfected with the empty vector (Ev), hDMT1 or
hZIP8. Uptake assay was performed at the optimal pH for the functional activity of each transporter (pH 5.5 and pH 7.4 respectively). The indicated
compounds (10 μM) were pre-incubated for 5 min, and then, for 15 min with both compound and 55Fe2+. Measured 55Fe2+ was corrected by
subtracting the background iron uptake measured in non-transfected cells. The data was normalized to the Fe2+ uptake measured in the presence
of DMSO (0.1%) and represented as mean SD (N = 5–16; obtained from 2 independent experiments). IC50 determination for pyrazolyl-pyrimidone
13 (b) and bipyridine (c) in HEK293T cells transiently transfected with hDMT1 (left panels) or hZIP8 (right panels). 55Fe2+ uptake (1 μM) was
measured in the presence of the indicated compound concentration ranges, and the measurements were corrected by subtracting the background
iron uptake measured in non-transfected cells. IC50 values were calculated by fitting the data to a sigmoidal 4-parameter equation. Representative
experiments are depicted, while IC50 values were calculated as mean SD of 3 different experiments.
pyrazole hydroxyl group for 3 and to the pyrimidone NH
group for 5 and 13, resulting in a transition from the neutral
form below pH 7 to a mono-anionic form above pH 7
(Fig. 6a). Both forms possess an N:NIJ:C)–C:N substructure
and should have metal chelating abilities, the neutral form
existing at pH 5.5 where hDMT1 transport is measured, and
the anionic form existing at pH 7.4 where hZIP8 transport is
measured (Fig. 6b).
with the Fe2+ precipitation experiments at pH 5.5, where we
only observe high precipitation of iron in the presence of
compound 3. The lack of inhibition by most of the
compounds tested might therefore either reflect partial iron
chelation or formation of an iron complex that partially
absorbs on the cells, resulting in apparent iron uptake and
no net inhibition.
However, while all pyrazolyl-pyrimidones and pyrazolyl-
pyridines tested here should have comparable metal
chelating abilities, only a few of them actually showed activity
in the iron uptake assays with hDMT1 and hZIP8. We
therefore postulate that inhibition only occurs either if metal
complexation is tight, as for the reference chelator
bipyridine, or if metal complexation results in the formation
of an insoluble complex, as observed using radioactive 55Fe2+
with 3 and 13. Note that, while iron uptake into hDMT1
expressing cells was partially reduced with the literature
inhibitor 3, we did not observe inhibition at higher
concentrations (Table 1), probably because complexed iron
was in this case absorbed on the cell material and assay
plate, and resulted in apparent iron uptake. This is in line
6. Pyrazolyl-pyrimidones and pyrazolyl-pyridines in chemical
space
To better understand our compound series, we surveyed the
ChEMBL database, which lists 1.82 million bioactive
molecules and their annotated targets,23 and found that
0.86% of these (15 585 molecules) contain a potentially metal
chelating 2,2′-diazabiaryl group, including 1717 pyrazolyl-
pyridines related to 3 and 169 pyrazolyl-pyrimidones related
to 5 and 13 and annotated with bioactivities (Table 2). Among
the 9.2 million screening compounds available in stock from
commercial providers as listed in the ZINC database,24 we
similarly found 0.87% (80 153 molecules) 2,2′-diazabiaryls,
however with relatively fewer pyrazolyl-pyridines (4673
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Fig. 6 (a) Acid–base titration of 3, 5, 13 and neutral and basic forms of 3, 5, 13. (20 μmol of compound in 10 mL (10% DMSO, ∼2 mM), 3 and 5 were
acidified with HCl (50 μmol)). (b) Acid-base equilibria for inhibitors 3, 5 and 13 highlighting potentially metal chelating nitrogen atoms in red. (c)
TMAP of our inhibitors combined with 169 pyrazolyl-pyrimidones and 1717 pyrazolyl-pyridines found in ChEMBL, color coded by source: cyan =
pyrazolyl-pyridines from ChEMBL, blue = pyrazolyl-pyrimidones from ChEMBL, red = this work. Insert: Close-up view around pyrazolyl-pyrimidones
color-coded by target class: red = transporter, cyan = enzyme, magenta = unclassified protein, grey = unclassified target. The interactive TMAP
including further color-codes (target classes, QED, etc.) is accessible at: http://tm.gdb.tools/dmt1_inhibitors/pyrimidones_pyridines/
molecules) but more abundant pyrazolyl-pyrimidones (1960
molecules), which suggests that such 2,2′-diazabiraryl
compounds are relatively easy to synthesize and drug-like.
The abundance of 2,2′-diazabiaryls was significantly lower in
theoretically enumerated chemical space databases such as
FDB17,25 GDBMedChem26 and GDBChEMBL,27 reflecting the
fact that these databases contain a much higher fraction of
non-aromatic molecules.
To obtain a closer insight into the selected 2,2′-diazabiaryls,
we created
tree-map (TMAP)30 representing pyrazolyl-
a
pyridines and pyrazolyl-pyrimidones from ChEMBL (Fig. 6c).
The TMAP can be interactively analyzed in-browser by
loading its layout using the program Faerun.31 In this map
each compound is represented by a point that can be color-
coded according to selected properties, and the molecular
structure is shown upon mouse-over by the program
Smilesdrawer.32 The compounds are connected in a tree
topology according to their structural similarities as
measured by the MHFP6 molecular fingerprint.33 The TMAP
We recently developed interactive chemical space
visualization tools that provide useful insights into
polypharmacology and structure–activity relationships.28,29
Table 2 2,2′-diazabiaryls in compound databasesa
Database
ChEMBL24
ZINCinStock
FDB17
GDBMedChem
GDBChEMBL
Number of molecules
2,2′-Diazabiaryls
% of database
Pyrazolyl-pyridines
% of 2,2′-diazabiaryls
Pyrazolyl-pyrimidones
% of 2,2′-diazabiaryls
1 820 035
15 585
0.86%
1717
11%
169
9 238 092
80 153
0.87%
4673
5.8%
1960
10 101 204
13 880
0.14%
110
0.8%
0
9 994 112
6241
0.06%
72
1.2%
26
9 978 095
2629
0.03%
65
2.5%
0
1.1%
2.4%
0.0%
0.4%
0.0%
a
Compounds were extracted using the corresponding SMARTS patterns. A TMAP of the 15 585 2,2′-diazabiaryls from ChEMBL24 is available at
http://tm.gdb.tools/dmt1_inhibitors/diazabiaryls/. See also Fig. 6c–f and Table S1† for SMARTS patterns.
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illustrates the diversity of pyrazolyl-pyrimidones and
pyrazolyl-pyridines in ChEMBL. The pyrazolyl-pyrimidones
investigated here belong to the more drug-like molecules in
(PAINS), however these should be checked whenever
screening SLC targets.
the series, as can be appreciated by color-coding for drug- Conflicts of interest
likeness using the quantitative estimate of drug-likeness
There are no conflicts to declare.
(QED).34
The activities reported for these molecules in ChEMBL
cover a diversity of target types, spanning from transporters
Acknowledgements
and ion channels to enzymes and uncharacterized targets.
This work was supported financially by the Swiss National
We found 45 2,2′-diazabiaryl including 18 pyrazolyl-
pyrimidones closely related to our compounds and reported
with transporter activity in ChEMBL (Table S2†). The
ChEMBL record indicates, among various targets, inhibition
of glucose and hexose transporters identified in antiparasitic
Science Foundation, grants no. 51NF40-185544 (JLR) and
310030_182272 (MH). We thank the group of Chemical
Crystallography of the University of Bern (PD Dr. P. Macchi)
for the X-ray structure solution and the Swiss National
Science Foundation (R'equip project 206021_128724) for co-
funding the single crystal X-ray diffractometer at the
department of Chemistry and Biochemistry of the University
of Bern.
screening
campaigns
against
Leishmania
mexicana,
tuberculosis, and Plasmodium falciparum.35 However, the
screens performed were not transport assays but simple
cytotoxicity tests. Considering that metal chelation is known
to be a mechanism of toxicity against these parasites,36,37 one
can speculate that the reported transporter inhibitory activity
of these pyrazolyl-pyrimidones might in fact reflect toxicity as
a result of metal chelation. Further activities reported for
pyrazolyl-pyrimidones and pyrazolyl-pyridines might similarly
be the consequence of metal chelation rather than the
attributed activity, including a recently reported activity of 3
and 5 against cancer cells.38 Note that potentially metal
chelating 2,2′-diazabiaryls such as pyrazolyl-pyrimidones are
not listed in PAINS (pan-assay interference compounds).39–41
Indeed a systematic checking of our compounds with the
PAINS filter from RDkit does not flag any molecule in the
series.
References
1 A. César-Razquin, B. Snijder, T. Frappier-Brinton, R. Isserlin,
G. Gyimesi, X. Bai, R. A. Reithmeier, D. Hepworth, M. A.
Hediger, A. M. Edwards and G. Superti-Furga, Cell,
2015, 162, 478–487.
2 L. Lin, S. W. Yee, R. B. Kim and K. M. Giacomini, Nat. Rev.
Drug Discovery, 2015, 14, 543–560.
3 T. Schumann, J. König, C. Henke, D. M. Willmes, S. R.
Bornstein, J. Jordan, M. F. Fromm and A. L. Birkenfeld,
Pharmacol. Rev., 2020, 72, 343–379.
4 N. F. Olivieri, N. Engl. J. Med., 1999, 341, 99–109.
5 R. Mariani, P. Trombini, M. Pozzi and A. Piperno, Mediterr. J.
Hematol. Infect. Dis., 2009, 1, e2009006.
6 A. Shawki, P. B. Knight, B. D. Maliken, E. J. Niespodzany and
B. Mackenzie, Curr. Top. Membr., 2012, 70, 169–214.
7 N. Montalbetti, A. Simonin, G. Kovacs and M. A. Hediger,
Mol. Aspects Med., 2013, 34, 270–287.
8 L.-N. Lu, Z.-M. Qian, K.-C. Wu, W.-H. Yung and Y. Ke, Mol.
Neurobiol., 2017, 54, 5213–5224.
9 X. Wang, M. Zhang, S. R. L. Flores, R. R. Woloshun, C. Yang,
L. Yin, P. Xiang, X. Xu, M. D. Garrick, S. Vidyasagar, D.
Merlin and J. F. Collins, Mol. Ther., 2019, 27, 493–506.
10 S. J. S. Flora and V. Pachauri, Int. J. Environ. Res. Public
Health, 2010, 7, 2745–2788.
11 T. A. Sales, I. G. Prandi, A. A. de Castro, D. H. S. Leal, E. F. F.
da Cunha, K. Kuca and T. C. Ramalho, Int. J. Mol. Sci.,
2019, 20, 1829.
12 Y. P. Singh, A. Pandey, S. Vishwakarma and G. Modi, Mol.
Diversity, 2019, 23, 509–526.
13 V. Manolova, N. Nyffenegger, A. Flace, P. Altermatt, A. Varol,
C. Doucerain, H. Sundstrom and F. Dürrenberger, J. Clin.
Invest., 2020, 130, 491–506.
14 Z. Zhang, V. Kodumuru, S. Sviridov, S. Liu, M. Chafeev, S.
Chowdhury, N. Chakka, J. Sun, S. J. Gauthier, M. Mattice,
L. G. Ratkay, R. Kwan, J. Thompson, A. B. Cutts, J. Fu, R.
Kamboj, Y. P. Goldberg and J. A. Cadieux, Bioorg. Med. Chem.
Lett., 2012, 22, 5108–5113.
Conclusion
To date the pharmacology of divalent metal transporters is
extremely limited. In view of the increasing importance of
SLCs as drug targets, we investigated the inhibition of
hDMT1 mediated iron transport by two previously reported
inhibitors, namely pyrazolyl-pyridine
pyrimidone 5. Optimization of uncovered analog 13
showing potent inhibition of hDMT1 (IC50 = 1.1 μM).
However, this inhibitor did not modulate hDMT1-induced
currents in Xenopus oocytes and exerted cross-inhibition of
the unrelated iron transporter hZIP8.
Our combined experiments suggest that this non-selective
inhibition reflects chelation of Fe2+ in the assay medium by
these molecules to form an insoluble precipitate. Although
metal chelation is often exploited as a mechanism of drug
action,10–12 metal chelation is in principle an assay
interference. This effect might occur across thousands of
bioactive molecules reported in ChEMBL that share the same
potentially chelating substructure and explain some of their
reported bioactivities. Pyrazolyl-pyrimidones and related
potentially metal chelating 2,2′-diazabiaryls are not
considered in the list of pan-assay interference compounds
3
and pyrazolyl-
5
RSC Med. Chem.
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View Article Online
RSC Medicinal Chemistry
Research Article
15 J. A. Cadieux, Z. Zhang, M. Mattice, A. Brownlie-Cutts, J. Fu,
L. G. Ratkay, R. Kwan, J. Thompson, J. Sanghara, J. Zhong
and Y. P. Goldberg, Bioorg. Med. Chem. Lett., 2012, 22, 90–95.
16 C. Simonin, M. Awale, M. Brand, R. van Deursen, J.
Schwartz, M. Fine, G. Kovacs, P. Hafliger, G. Gyimesi, A.
Sithampari, R. P. Charles, M. A. Hediger and J. L. Reymond,
Angew. Chem., Int. Ed., 2015, 54, 14748–14752.
17 N. Montalbetti, A. Simonin, C. Simonin, M. Awale, J.-L.
Reymond and M. A. Hediger, Biochem. Pharmacol., 2015, 96,
216–224.
28 M. Poirier, M. Awale, M. A. Roelli, G. T. Giuffredi, L.
Ruddigkeit, L. Evensen, A. Stooss, S. Calarco, J. B. Lorens,
R.-P. Charles and J.-L. Reymond, ChemMedChem, 2019, 14,
224–236.
29 C. Delalande, M. Awale, M. Rubin, D. Probst, L. C. Ozhathil,
J. Gertsch, H. Abriel and J.-L. Reymond, Eur. J. Med. Chem.,
2019, 166, 167–177.
30 D. Probst and J.-L. Reymond, Aust. J. Chem., 2020, 12, 12.
31 D. Probst and J.-L. Reymond, Bioinformatics, 2018, 34, 1433–1435.
32 D. Probst and J.-L. Reymond, J. Chem. Inf. Model., 2018, 58,
1–7.
33 D. Probst and J.-L. Reymond, Aust. J. Chem., 2018, 10, 66.
34 G. R. Bickerton, G. V. Paolini, J. Besnard, S. Muresan and
A. L. Hopkins, Nat. Chem., 2012, 4, 90–98.
18 C. Manatschal, J. Pujol-Giménez, M. Poirier, J.-L. Reymond,
M. A. Hediger and R. Dutzler, eLife, 2019, 8, e51913.
19 A. V. Erkin and V. I. Krutikov, Russ. J. Gen. Chem., 2009, 79,
1204–1209.
20 J. Orrego-Hernández, J. Cobo and J. Portilla, Eur. J. Org.
Chem., 2015, 2015, 5064–5069.
21 W. J. Dixon, Annu. Rev. Pharmacol. Toxicol., 1980, 20,
441–462.
35 F.-J. Gamo, L. M. Sanz, J. Vidal, C. de Cozar, E. Alvarez, J.-L.
Lavandera, D. E. Vanderwall, D. V. S. Green, V. Kumar, S.
Hasan, J. R. Brown, C. E. Peishoff, L. R. Cardon and J. F.
Garcia-Bustos, Nature, 2010, 465, 305–310.
22 C.-B. Bo, Q. Bu, X. Li, G. Ma, D. Wei, C. Guo, B. Dai and N.
Liu, J. Org. Chem., 2020, 85, 4324–4334.
36 C. Raventos-Suarez, S. Pollack and R. L. Nagel, Am. J. Trop.
Med. Hyg., 1982, 31, 919–922.
23 A. Gaulton, A. Hersey, M. Nowotka, A. P. Bento, J. Chambers,
D. Mendez, P. Mutowo, F. Atkinson, L. J. Bellis, E. Cibrián-
Uhalte, M. Davies, N. Dedman, A. Karlsson, M. P. Magariños,
J. P. Overington, G. Papadatos, I. Smit and A. R. Leach,
Nucleic Acids Res., 2017, 45, D945–D954.
24 T. Sterling and J. J. Irwin, J. Chem. Inf. Model., 2015, 55,
2324–2337.
25 R. Visini, M. Awale and J. L. Reymond, J. Chem. Inf. Model.,
2017, 57, 700–709.
37 R. J. Wall, S. Moniz, M. G. Thomas, S. Norval, E.-J. Ko, M.
Marco, T. J. Miles, I. H. Gilbert, D. Horn, A. H. Fairlamb and
S. Wyllie, Antimicrob. Agents Chemother., 2018, 62, e00235-18.
38 A. L. Turcu, A. Versini, N. Khene, C. Gaillet, T. Cañeque, S.
Müller and R. Rodriguez, Chem. – Eur. J., 2020, DOI: 10.1002/
chem.202000159.
39 J. B. Baell and G. A. Holloway, J. Med. Chem., 2010, 53,
2719–2740.
40 S. J. Capuzzi, E. N. Muratov and A. Tropsha, J. Chem. Inf.
Model., 2017, 57, 417–427.
26 M. Awale, F. Sirockin, N. Stiefl and J.-L. Reymond, Mol. Inf.,
2019, 38, 1900031.
27 S. Bühlmann and J.-L. Reymond, Front. Chem., 2020, 8, 46.
41 J. B. Baell and J. W. M. Nissink, ACS Chem. Biol., 2018, 13,
36–44.
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