Discovery of Membrane-Bound Pyrophosphatase Inhibitors Derived from an Isoxazole Fragment

Article abstract of DOI:10.1021/acsmedchemlett.9b00537

Membrane-bound pyrophosphatases (mPPases) regulate energy homeostasis in pathogenic protozoan parasites and lack human homologues, which makes them promising targets in e.g. malaria. Yet only few nonphosphorus inhibitors have been reported so far. Here, w

Full text of DOI:10.1021/acsmedchemlett.9b00537

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Discovery of Membrane-Bound Pyrophosphatase Inhibitors Derived
from an Isoxazole Fragment
Niklas G. Johansson,# Ainoleena Turku,# Keni Vidilaseris,# Loïc Dreano, Ayman Khattab,
́
Daniel Ayuso Perez, Aaron Wilkinson, Yuezhou Zhang, Matti Tamminen, Evgeni Grazhdankin,
Alexandros Kiriazis, Colin W. G. Fishwick, Seppo Meri, Jari Yli-Kauhaluoma, Adrian Goldman,*
̈
Gustav Boije af Gennas, and Henri Xhaard*
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ABSTRACT: Membrane-bound pyrophosphatases (mPPases) regulate energy homeostasis in
pathogenic protozoan parasites and lack human homologues, which makes them promising
targets in e.g. malaria. Yet only few nonphosphorus inhibitors have been reported so far. Here,
we explore an isoxazole fragment hit, leading to the discovery of small mPPase inhibitors with
6−10 μM IC50 values in the Thermotoga maritima test system. Promisingly, the compounds
retained activity against Plasmodium falciparum mPPase in membranes and inhibited parasite
growth.
KEYWORDS: Membrane-bound pyrophosphatase, protozoan diseases, drug design, isoxazoles
rotozoan diseases such as malaria and leishmaniasis affect
maritima13−15 and mung bean Vigna radiata14,16 mPPases have
Pmany vertebrate species and are a major cause of human been solved to date by us and other researchers. Some of these
mortality or morbidity worldwide.1,2 They are caused by
structures have been cocrystallized with the substrate analogue
imidodiphosphate (IDP), yet only very few nonphosphorus
protist parasites such as Plasmodium spp., Toxoplasma spp.,
Trypanosoma spp., and Leishmania spp. Common to these
parasites is the acidocalcisome, a vesicle that accumulates
phosphorus compounds (e.g. bisphosphonates and polyphos-
phates, with concentrations of the latter estimated in the 3−5
M range3,4), metal ions such as Mg2+ and Ca2+, as well as
positively charged compounds (e.g. diamines).5 These
organelles have been associated with several functions: short-
chain polyphosphate (poly-P) metabolism, storage of cations,
calcium homeostasis, intracellular pH homeostasis, and
osmoregulation. Acidocalcisomes and their major component
poly-P are essential for the response of trypanosomatids to
different types of stress,4 especially low-energy conditions.3,6−9
Parasitic protists express one or two types of membrane-
bound pyrophosphatases (mPPases), which are the focus of
this study.10,11 mPPases are large (70−81 kDa) homodimeric
integral membrane proteins, consisting of 15−17 trans-
membrane helices per monomer, which hydrolyze pyrophos-
phate into orthophosphate.11 They occur in bacteria, archaea,
plants, and protozoa.12 The process is coupled to active
transport (H+, Na+, or H+/Na+) across membranes and can be
regulated by monovalent cations (K+ dependence). Various X-
ray structures of the hyperthermophilic bacterium Thermotoga
compounds have been reported.15,17
Membrane-bound pyrophosphatases have been suggested to
be valuable targets for chemotherapeutic action.3,10,18,19
Indeed, siRNA-induced mPPase knockouts in Trypanosoma
brucei compromised the function of its acidocalcisomes,
causing severe reduction in poly-P quantity and acidocalcisome
acidity.8 In Toxoplasma gondii, mPPase knockouts increased
sensitivity to extracellular conditions and compromised
virulence in mice.7 Additionally, in parasitic protozoa, such
as P. falciparum, T. gondii, L. donovani, T. brucei, and T. cruzi,
pyrophosphate (and bisphosphonate) derivatives are able to
inhibit growth.10,20 However, this is thought to result from the
action of both soluble and membrane-bound pyrophospha-
tases,20,21 in particular by inhibition of the prenyl diphosphate
synthase (isoprenoid pathway).22 As a chemotherapeutic
target, mPPases offer significant advantages. First, they would
Received: November 20, 2019
Accepted: February 10, 2020
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allow targeting not only the digestive vacuole in the asexual
blood stage parasites but also the gametocyte stage.19,23,24
Second, since no human homologues exist, inhibitors are
highly likely to be specific, provided that phosphorus-
containing compounds are avoided due to their tendency to
recognize unrelated structural motifs.25,26
Here, we take advantage of our ability to assay the purified
T. maritima protein on 96-well plates to discover non-
phosphorus inhibitors27 useful as tool compounds for target
validation and X-ray crystallographic studies and with a
translation potential into therapeutic molecules. Of the 110
compounds presented here, 46 were obtained synthetically and
64 were purchased from commercial sources. The purity of the
commercial compounds was assessed independently (99
compounds in total met the >95% purity standard).
At the start of the study, no nonphosphorus ligands were
available. A pilot screen of 16 phosphate-mimicking fragments
(range 143−338 Da, average 236 Da) at 100 μM
concentration was first conducted on the Thermotoga maritima
mPPase (TmPPase) model system (see Supporting Informa-
tion, Table S1_1). Compounds in the library contained
common phosphate isosteres such as boronate, sulfone,
sulfonamide, or isoxazole moieties.26 As a result of the
experimental screen, four compounds (1−4) showed over
50% inhibition (Figure 1). Compounds 1−4 all include a
Figure 2. Selected heteroaryl analogues of 1 and the most potent hit
in the sulfonamide series.
showing that not all replacements of the aryl group in 1 were
tolerated.
A common feature of the other three initial hits (compounds
2−4, Figure 1) was that they contained a sulfone or
sulfonamide linker. To explore the outcome of introducing
sulfonamides to the 5-arylisoxazole-3-carboxylate core, we
acquired analogues of 1, where primarily the ortho-nitro group
had been replaced with a para-methoxy substituent (seven of
them had acceptable purity). In this series (Supporting
Information, Table S1_3) the most potent sulfonamide 7
(Figure 2) exhibited a 2-fold improvement in activity
compared to the parent compound 1. Otherwise this
exploration of aryl and sulfonamides did not provide
interesting inhibitors.
The first hit series showed a loss of activity for most
structural alterations of 1, except for compound 5. Building on
this initial knowledge, we started a synthetic expansion around
the isoxazole-3-carboxylic acid moiety of 1 and 5, introducing
various phenyl substituents to the 5-position of the common
core, as well as alterations to the carboxyl parts of the
molecule. In total, five commercially available analogues
complemented the 28 compounds synthesized at that stage
(see Supporting Information, Table S1_4).
As illustrated in Scheme 1, the isoxazole series was
constructed using mainly two different synthetic approaches.
The isoxazole ring can be formed from e.g. substituted
alkynes28 8 or ketones29,30 9. Aryl alkyne 8 and ethyl 2-
chloro-2-(hydroxyimino)acetate (forming nitrile oxide in situ)
were used in a one-step [3 + 2] cycloaddition reaction to give
ethyl 5-arylisoxazole-3-carboxylate 10. Alternatively, 10 was
obtained via a crossed condensation reaction between aryl
ketone 9 and diethyl oxalate in the presence of sodium
ethoxide, with a subsequent condensation reaction in the
presence of hydroxylamine to form the isoxazole ring. All ethyl
esters 10a−n were subsequently hydrolyzed to their
corresponding carboxylic acids 11a−n with lithium hydroxide.
Some of the carboxylic acids obtained (11i−l) were activated
as acyl chlorides before converting them to 2-bromophenyl 5-
arylisoxazole-3-carboxylates 12i−l.
We started by exploring variations of the phenyl moiety by
synthesizing 15 5-arylisoxazole-3-carboxylic acid derivatives.
Unfortunately, no active inhibitors were obtained (Table
S1_4). We first investigated 11a, with an unsubstituted phenyl
ring. Substitution of the phenyl ring with electron withdrawing
(as in 11b−d, 11h and 11m) or donating (as in 11e−g and
11i−l) groups did not lead to the desired activity. As part of
the aryl exploration, molecular modeling suggested two
positively charged interaction sites in the substrate site
(K199 and K664; R191 and K499). We attempted to bridge
Figure 1. Top four hits identified in the pilot screen. MW, molecular
weight; IC50, half maximal inhibitory concentration; CI95%, half
maximal inhibitory concentration expressed as a 95% confidence
interval (given in square brackets).
heterocyclic five-membered ring and an aryl group: 1 has a 5-
phenylisoxazole core (IC50 = 71 μM); 2 contains a sulfonyl-
substituted benzo[d]oxazol-2(3H)-one moiety (IC50 = 43
μM); 3 is a benzenesulfonylproline (IC50 = 110 μM); and 4 is
a sulfamoylbenzene-linked thiazolone (IC50 = 120 μM).
The isoxazole 1 was selected for further exploration. We first
supplemented the series with six commercial heteroaryl
analogues (see Supporting Information, Table S1_2) and
seven sulfonamides (see Supporting Information, Table S1_3).
The commercial availability of analogues was rather limited,
and the purity of 11 other sulfonamides was insufficient
(Supporting Information, Table S1_3). Screening led to the
discovery of 5 (Figure 2), a rather small compound (210 Da)
with an IC50 of 17 μM and a high ligand efficiency (LE 0.44).
Compound 5 was used from this point on as a positive control
in experimental tests, in addition to IDP. Unlike 5, the 1,5-
dimethyl-1H-pyrazol-4-yl analogue 6 lacked biological activity,
B
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a
Scheme 1. Main Synthesis Routes Used
a
Reagents and conditions: (a) Ethyl 2-chloro-2-(hydroxyimino)acetate, NaHCO3, MeCN, rt, 3−5 d, 41−99%; (b) NaOEt, diethyl oxalate, EtOH,
reflux, 2 h (for 10a) or NaOEt, diethyl oxalate, EtOH/Et2O, rt, overnight (for 10b−n); (c) H2NOH·HCl, EtOH, reflux, 3 h, 20−80% (after two
steps); (d) LiOH, EtOH/H2O, 1−48 h, 42−99%; (e) 2-Bromophenol, EDC·HCl, DMAP, DIPEA, DCM, rt, overnight (for 12h), 15%, or (i)
(COCl)2, DMF, DCM, rt, 0.5−1 h, (ii) 2-bromophenol, Et3N, 0 °C → rt, 1 h (for 12i−l), 28−82%.
these sites. However, neither the carboxylic acid 11n nor the
ester 10n were notably active.
Table 1. Selected 3,5-Di-tert-butylphenyl Analogues
Precursors of the already tested carboxylic acids (i.e., the
corresponding ethyl carboxylates) were conveniently available
and could be tested to expand the chemical series (Table
S1_4). Generally, this set of esters did not show any biological
activity, with the exception of 10l (IC50 = 72 μM), which was
more active than its carboxylic acid analogue 11l.
Initially the presence of an isoxazole core was central to the
compound design. Based on the activity of 10l, and due to the
difficulty in obtaining active compounds, we decided to
investigate a 3,5-di-tert-butylphenyl core with a set of 16
commercially available analogues (see Table 1; Supporting
Information, Table S1_5). Three of these derivatives (13−15)
had TmPPase IC50 values in the 39−47 μM range. These
compounds showed a 2-fold gain in activity compared to our
starting point 1.
We also investigated different ring replacements (see Figure
3; Supporting Information, Table S1_6). Replacing the
isoxazole ring of the inactive 11a with pyrazole (as in 21) or
thiazole (as in 23) led to active compounds, with at best an
IC50 of 29 μM for 23.
Based on the activity of 15 (as well as the 17 μM IC50 of
commercially available compound SI-54, purity 87%, see Table
S1_7), we decided to introduce 2-bromophenyl groups to our
most active compounds. Bromine atoms are useful in structural
studies because the anomalous signal can aid in identifying
both the presence of the molecule and its orientation in low-
resolution (3.5 Å or worse) diffraction data. These 2-
bromophenyl carboxylate modifications were successful for
compounds 12h−l, leading to the most active compounds
obtained so far (Figure 4A). The alkylated compounds 12i−l
gained up to a 77-fold activity increase (IC50 values in the 6.1−
10 μM range), making them the most potent inhibitors in this
study.
We also attempted to modify the thiazoles 5 (Figure 2) and
23 (Figure 3). Disappointingly, their corresponding 2-
bromophenyl carboxylates 25 and 26 showed a 27-fold and
1.6-fold loss in activity compared to the carboxylic acids,
respectively (Figure 4B).
To obtain higher ligand efficiency for compounds 12k and
12l, we tried to remove some redundant atoms. One of the
alkyl chains (as in 12i and 12j) could easily be removed
without any change in activity, whereas removing the bromine
atom (as in 27, Figure 4C) caused a 3.6-fold loss of inhibition.
We also explored changing the ester moiety of 12k into the
corresponding amide 28 (Figure 4C) to increase the chemical
stability and explore new interactions. However, the resulting
compound was inactive. Changing the isoxazole 12i to the
Figure 3. Selected isoxazole ring modifications.
C
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Figure 4. (A) Most potent hits of the 2-bromophenyl carboxylate series. (B) 2-Bromophenyl carboxylates of 5 and 23. (C) Selected modifications
of 12k. (D) Inhibition of P. falciparum vacuolar H+-translocating pyrophosphatase (PfPPase-VP1) by 12k (filled circles) and 12l (circles). (E)
Effect of 12k on P. falciparum growth. All data are shown as mean SD with three replicates.
corresponding pyrazole analogue (as in SI-53) did not seem to
be advantageous either.
μM) that inhibit the Thermotoga maritima mPPase and are
among the first nonphosphorus inhibitors reported for that
system. They were discovered through a chemical exploration
of 110 compounds from a fragment hit, achieving a 10-fold
activity gain. The most potent hits possess a bromine atom,
which should facilitate future structural studies. Those studies
are particularly relevant since, while we used the mPPase from
a hyperthermophilic bacterium as a test system, the substrate
binding sites are almost entirely conserved in the mPPases of
many pathogens.18,34 Preliminary tests against PfPPase-VP1 in
membranes and in a parasite growth assay showed that some
activity was retained, indicating their potential to be developed
for target validation and therapeutic applications in parasites.
We then addressed the potential of 12k and 12l for
translation to pathogenic proteins. These analogues differ by
replacement of two methyl groups by two tert-butyl groups. In
order to do so, we expressed the PfPPase-VP1 gene
(PF3D7_1456800) in baculovirus-infected insect cells and
performed the inhibition assay on the PfPPase-VP1 in the
membrane (the method and further analysis will be described
in more detail elsewhere). Compound 12k was able to inhibit
the PfPPase-VP1 activity with an IC50 = 55 μM, whereas
compound 12l did not show inhibition at the tested
concentrations (Figure 4D; Supporting Information). We
then tested 12k in a P. falciparum survival assay in
erythrocytes,31,32 and it inhibited the growth of P. falciparum
with an IC50 of 99 μM (Figure 4E; Supporting Information).
This latter finding does not prove a direct mechanism of
action, since inhibition may occur e.g. through the soluble
pyrophosphatase or some other mechanism. Nonetheless,
taken together, these data strongly suggest that the hits
discovered in this study are relevant to drug discovery against
parasitic mPPases. Interestingly, similar isoxazole compounds
have been found to have an IC50 of 3.3 μM in T. cruzi
parasites.33
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00537.
List of screened compounds (Table S1); aggregation
data for key compounds 5, 12k, and 12l (Figure S1);
IC50 plots from the inhibition assay; general exper-
imental methods, synthetic procedures or commercial
sources, characterization data, and KNIME workflow
(PDF)
Herein we report a set of compounds (12i, IC50 = 6.1 μM;
12j, IC50 = 6.7 μM; 12k, IC50 = 6.9 μM; and 12l, IC50 = 10
Molecular formula strings (XLSX)
D
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00537
AUTHOR INFORMATION
■
Corresponding Authors
Adrian Goldman − Department of Biosciences, Division of
Biochemistry, University of Helsinki, FI-00014 Helsinki,
Finland; School of Biomedical Sciences and Astbury Centre for
Structural Molecular Biology, University of Leeds, Leeds LS2
9JT, United Kingdom; Phone: +44 113 3438537;
Email: a.goldman@leeds.ac.uk
Henri Xhaard − Drug Research Program, Division of
Pharmaceutical Chemistry and Technology, Faculty of
Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland;
orcid.org/0000-0002-3000-7858; Phone: +358 2941
59190; Email: henri.xhaard@helsinki.fi
Author Contributions
#N.G.J., A.T., and K.V. contributed equally. The manuscript
was written through contributions of all authors. All authors
have agreed to the final version of the manuscript.
Funding
This work was supported by grants from Jane and Aatos Erkko
Foundation (to A.G., H.X., and J.Y.-K.; and to S.M. and A.K.),
the Biotechnology and Biological Sciences Research Council
(BBSRC, No. BB/M021610/1 to A.G.), the Academy of
Finland (No. 265481 to J.Y.-K., No. 308105 to K.V., No.
́
310297 to H.X., and No. 1323237 to S.M.), Sigrid Juselius
Foundation (to S.M), University of Leeds (to A.W.), and
University of Helsinki Research Funds (to G.B.a.G. and
N.G.J.).
Authors
Niklas G. Johansson − Drug Research Program, Division of
Pharmaceutical Chemistry and Technology, Faculty of
Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland;
orcid.org/0000-0002-8226-4813
Notes
The authors declare no competing financial interest.
Ainoleena Turku − Drug Research Program, Division of
Pharmaceutical Chemistry and Technology, Faculty of
Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland
Keni Vidilaseris − Department of Biosciences, Division of
Biochemistry, University of Helsinki, FI-00014 Helsinki,
Finland
Loïc Dreano − Drug Research Program, Division of
Pharmaceutical Chemistry and Technology, Faculty of
Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland
Ayman Khattab − Malaria Research Laboratory, Translational
Immunology Research Program, Department of Bacteriology
and Immunology, Haartman Institute, University of Helsinki,
FI-00014 Helsinki, Finland
ACKNOWLEDGMENTS
■
The authors wish to gratefully acknowledge CSC−IT Center
for Science, and the Drug Discovery and Chemical Biology
(DDCB) network for computational resources; Nina Sipari
and Jenna Lihavainen, from the Viikki Metabolomics Unit−
Helsinki Institute of Life Science (HiLIFE) for mass
spectrometry services; as well as Olga Lukkonen for assistance
with the experiments.
ABBREVIATIONS
■
DIPEA, N,N-diisopropylethylamine; poly-P, polyphosphate;
siRNA, small interfering ribonucleic acid; TmPPase, Thermo-
toga maritima mPPase; PfPPase-VP1, Plasmodium falciparum
vacuolar H+-translocating pyrophosphatase VP1
́
Daniel Ayuso Perez − Drug Research Program, Division of
Pharmaceutical Chemistry and Technology, Faculty of
Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland
Aaron Wilkinson − School of Chemistry, University of Leeds,
Leeds LS2 9JT, United Kingdom
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