Identification of ML251, a potent inhibitor of T. brucei and T. cruzi phosphofructokinase

Article abstract of DOI:10.1021/ml400259d

Human African Trypanosomiasis (HAT) is a severe, often fatal disease caused by the parasitic protist Trypanosoma brucei. The glycolytic pathway has been identified as the sole mechanism for ATP generation in the infective stage of these organisms, and several glycolytic enzymes, phosphofructokinase (PFK) in particular, have shown promise as potential drug targets. Herein, we describe the discovery of ML251, a novel nanomolar inhibitor of T. brucei PFK, and the structure-activity relationships within the series.

Full text of DOI:10.1021/ml400259d

Letter
pubs.acs.org/acsmedchemlett
Identification of ML251, a Potent Inhibitor of T. brucei and T. cruzi
Phosphofructokinase
†
,∥
†,∥
†
‡
‡
́
Kyle R. Brimacombe, Martin J. Walsh, Li Liu, Montserrat G. Vasquez-Valdivieso, Hugh P. Morgan,
‡
‡
§
†
†
Iain McNae, Linda A. Fothergill-Gilmore, Paul A. M. Michels, Douglas S. Auld, Anton Simeonov,
‡
†
,†
Malcolm D. Walkinshaw, Min Shen, and Matthew B. Boxer*
†National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville,
Maryland 20850, United States
‡
Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building,
The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
§
Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite
Hippocrate 74, B-1200 Brussels, Belgium
́
catholique de Louvain, Avenue
*
S Supporting Information
ABSTRACT: Human African Trypanosomiasis (HAT) is a
severe, often fatal disease caused by the parasitic protist
Trypanosoma brucei. The glycolytic pathway has been identified
as the sole mechanism for ATP generation in the infective stage of
these organisms, and several glycolytic enzymes, phosphofructo-
kinase (PFK) in particular, have shown promise as potential drug
targets. Herein, we describe the discovery of ML251, a novel
nanomolar inhibitor of T. brucei PFK, and the structure−activity
relationships within the series.
KEYWORDS: Trypanosoma brucei, Trypanosoma cruzi, phosphofructokinase, inhibitors, glycolysis, high-throughput screening
1
1
uman African Trypanosomiasis (HAT), also known as
sleeping sickness, is a disease caused by the parasitic
the first committed step of glycolysis. In addition, the
necessity of glycolysis for parasite growth has been confirmed
by siRNA-mediated knockdown; a 50% decrease in glycolytic
flux was found to be sufficient to significantly decrease parasite
viability, further suggesting that inhibition of glycolysis may be
H
protist, Trypanosoma brucei, and is endemic to sub-Saharan
Africa. More than 60 million people are estimated to be at risk
of HAT infection, due to the widespread infestation of the
parasite’s tsetse fly vector, as well as the density of humans and
cattle in the region, which serve as reservoirs for the causative
9
a viable strategy for HAT therapy. Currently, no inhibitors of
Tb PFK have been described that demonstrate submicromolar
potency or selectivity for the enzyme. Reported inhibitors
include suramin, a classic antitrypanosomal drug in use since
920 with noted promiscuous activity, inhibiting 80 assays
among 370 tested in PubChem (IC ≤ 1 μM in 26 assays), as
well as a furanose based analogue reported by Nowicki et al.
1
,2
parasites. HAT is almost always fatal if left untreated, and
classic therapies used to treat it have historically suffered from a
range of deleterious side effects, due in part to their
nonselective mechanisms of action, and most approved
therapies require either intravenous or intramuscular admin-
1
50
3
,4
istration, further complicating treatment. Second-stage HAT,
characterized by the infiltration of parasites into the central
nervous system, remains a particular challenge because of the
need for therapeutic molecules to cross the blood−brain
11,12
with an IC50 of 23 μM (Figure 1).
We report here the discovery and structure−activity
relationship (SAR) of novel and potent inhibitors of T. brucei
and T. cruzi PFK. A library of 330,683 compounds from the
Molecular Libraries Small Molecule Repository (MLSMR,
http://mli.nih.gov/mli/compound-repository/mlsmr-
compounds/) was screened at 6 concentrations (spanning a
5
barrier. Furthermore, growing resistance to these drugs has
6
highlighted a need for the identification of novel therapies.
The glycolytic enzyme phosphofructokinase (PFK) has
recently been recognized as a potential drug target in T. brucei,
as the infectious bloodstream stage of the parasite is solely
dependent on the metabolism of glucose for ATP gener-
Received: July 8, 2013
Accepted: October 30, 2013
Published: October 30, 2013
7
−10
ation.
PFK catalyzes an essentially irreversible reaction
under physiological conditions in parasites and thus represents
©
2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Table 1. SAR Assessing the Core of PFK Inhibitors
Figure 1. (a) Chemical structures of Tb PFK inhibitors suramin, a
glycoside analogue, and our qHTS hit (1). Compound 1 is an obvious
derivative of the antibiotic sulfamethoxazole, which shows no
inhibitory activity against PFK. (b) Synthetic route to 1, which was
the general synthetic route to most analogues in Tables 1 and 2. (c)
Synthetic route to 31, which was also used for analogues in Table 3
concentration range from 57.5 μM to 3.7 nM) against
13
recombinant Tb PFK for inhibitory activity. PFK activity
was assessed by coupling PFK-mediated ADP production to a
modified luciferase-based detection assay (ADP-Glo), providing
a luminescent end point readout. A stepwise description of the
1
536-well assay is shown in Supplementary Table 1. Complete
screening and follow-up data have been made available in
PubChem (PubChem BioAssay summary identifier 488768;
http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=
488768). Subsequent hit confirmation with an orthogonal ATP
depletion-based assay (description in Supplementary Table 2;
data not shown) led to the identification of the para-
amidosulfonamide scaffold as one of the hit series, typified by
the screening hit (1) with an IC of 1.1 μM (Figure 1). While
a
IC50 values represent the average of at least 3 separate experiments
reported as the half maximal (50%) inhibitory concentration as
determined in the ADP-Glo assay. All IC50s <57 μM have a maximum
inhibition of >80% at 57 μM.
5
0
1
contains the same core as the antibiotic sulfamethoxazole, the
latter shows no inhibitory activity against T. brucei PFK.
The resynthesis of 1 is shown in Figure 1b and followed a
simple sequence of sulfonamide formation, acid catalyzed acetyl
hydrolysis, and finally amide formation. Gratifyingly, the
resynthesized version had an IC50 of 410 nM (Table 1).
Though the original screening campaign was designed to
identify inhibitors of PFK in T. brucei, the related organism,
Trypanosoma cruzi, responsible for Chagas disease, possesses a
similar reliance on ATP generation through glycolysis; the T.
brucei and T. cruzi PFK isoforms display 77% overall sequence
identity, with greater than 90% sequence identity within the
Figure 1b was used to initiate synthesis, with all analogues
being tested against both T. brucei and T. cruzi PFK isoforms.
The SAR tracked very well with few exceptions between the
species, though the discussion of SAR here will focus solely on
T. brucei activity. The first changes attempted were to
understand if we could modify the para-amidosulfonamide
core (Table 1). The 1,4-disubstitution arrangement on the
phenyl ring proved to be optimal as the meta-substituted
analogue (2) suffered from greatly diminished activity.
Attempts to change to saturated (3), alkyl chain (4), and
heteroaryl (5) cores all resulted in less active analogues. This
1
4
active site. As such, the general synthetic strategy shown in
1
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ACS Medicinal Chemistry Letters
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initial round of SAR concluded with efforts to understand the
importance of the sulfonamide and amide linkers. Very tight
SAR was observed with respect to both of these functionalities,
as indicated in analogues 6−12.
Table 2. SAR Assessing the Benzyl Group of PFK Inhibitors
As a number of hits from the quantitative high-throughput
screen (qHTS) campaign, as well as the furanose analogue
reported by Nowicki, contained the 3,4-dichlorobenzyl motif
present in 1, we wanted to test the role of these substituents for
inhibitory activity (Table 2). While both single- and double-
point deletions of the chloro groups led to less active
compounds (13−15), we realized that chloro substitution at
the 3- or 4-position contributed significantly to the potency as
the phenyl analogue was 5- and 20-fold less potent than the 4-
and 3-chloro analogues, respectively, for T. brucei. Using this
information, we next synthesized a few analogues with p-halo or
-
1
methyl substituents and found that the p-halo analogues (17−
9) had similar activity among themselves and slightly
diminished activity compared to the dichloro analogue. The
p-methyl analogue (20) showed a ∼8-fold loss in potency
compared to 18 highlighting the importance of these halogens
at the 4-position. Further manipulations of these 3,4-
substituents led to significantly lower or a complete loss of
activity (21−24). Finally, removal, extension, and substitution
of the benzylic methylene group all resulted in reduced
potencies (25−27).
Realizing that the 3,4-dihalo substitution pattern was key for
activity, this motif was carried on to an additional round of
analogues aimed at investigating the SAR around the isoxazole
moiety using the chemistry shown in Figure 1c (Table 3).
Interestingly, sulfonic acid analogue 28 had submicromolar
activity while the more hydrophobic phenyl analogue (29) lost
considerable activity. The 5-des-methyl analogue 30 (ML251)
showed a slight improvement in potency compared to the hit 1
and led us to hypothesize that analogues with hydrophilic and/
or hydrogen bond donors/acceptors at this position may lead
to improved activity. Therefore, we synthesized analogues 31−
42 containing various heterocycles and substituted phenyl
groups. While pyrazole analogues 31 and 32 had appreciable
activity, a significant jump in potency against PFKs from both
species was observed for the carboxylic acids 33 and 35.
Interestingly, the parent ester (34) had no inhibitory activity.
Pyrimidine, phenol, and aniline containing analogues 36−38 all
showed diminished activity, leading us to investigate small
heterocyclic analogues. Switching from the isoxazole to a
thiazole resulted in an improvement in activity with analogues
3
9 and 40 having 79 and 24 nM potency, respectively, for T.
brucei and 41 and 120 nM for T. cruzi, respectively. The
thiadiazole analogue (41) saw only a very slight decrease in
activity against both T. brucei and T. cruzi compared to 39.
Interestingly, the thiadiazole analogue with the 4-chloro-3-
fluoro substitution pattern (42) gave the most potent analogue
against T. brucei at (IC = 15 nM), while 39 remained the most
5
0
potent inhibitor of T. cruzi (IC50 = 41 nM).
To identify the mechanism of action of this para-
amidosulfonamide chemotype, competition studies were
performed by titrating individual substrates and the potent
thiadiazole analogue, 42, using a coupled biochemical PFK
assay. Lineweaver−Burk transformations of the initial velocities
suggest that 42 engages in competition for binding with
fructose 6-phosphate (F6P) (Figure 2a), while demonstrating
mixed inhibition with respect to ATP (Figure 2b). Determi-
nation of inhibition constants for 42 against PFK for F6P
a
IC50 values represent the average of at least 3 separate experiments
reported as the half maximal (50%) inhibitory concentration as
determined in the ADP-Glo assay. All IC s <57 μM have a maximum
inhibition of >80% at 57 μM.
50
(under saturating F6P levels) further decreases the affinity of
showed a K of 52 nM (Supplemental Figure 1a), while ATP
42 by 4.5-fold (K = 240 nM, Supplemental Figure 1b). Indeed,
i
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Table 3. SAR of the Heterocycle
Figure 2. Assessment of mechanism of action of para-amidosulfona-
mide analogue 42. Lineweaver−Burk transformations of initial velocity
are shown for (a) F6P and (b) ATP, demonstrating competitive and
mixed modes of inhibition, respectively. The second (untitrated)
substrates were held constant at saturating concentrations in both
studies (0.5 mM ATP and 2 mM F6P, respectively). Concentration of
4
2 is shown at 0 (○), 1.5 (□), 12.5 (◆), 50 (▲), and 100 (■) nM.
Secondary plots to derive inhibition constants for 42 are shown for (a)
F6P (K ) and (b) ATP (K ). (c) Selectivity of ML251 (circles) and 42
i
i′
(triangles) against T. brucei (black) and T. cruzi (white) PFK isoforms.
(d) Toxicity of 1, ML251, and 42 against in vitro T. brucei cultures
(solid bars) and MRC-5 human lung cell line (dashed bars).
Typified by 30 and 42, comparable inhibitory activity was
seen in both T. brucei and T. cruzi PFK isoforms (Figure 2c),
and this trend was anticipated as these isoforms display
11
significant sequence identity (vide supra). Furthermore, many
analogues displayed increased potencies against the T. cruzi
isoform compared to T. brucei, suggesting that this series may
find further utility outside of T. brucei alone. Encouragingly, 42
was tested at 1 μM against the rabbit isoform of PFK and
showed no significant inhibition (Supplemental Figure 2).
These data suggest that a significant window of selectivity exists
for this chemotype, and importantly, a mammalian isoform of
the enzyme does not appear sensitive to inhibition. This is of
particular importance for potential HAT therapies, as cross-
species promiscuity and polypharmacology continue to plague
many frontline treatments currently used to treat trypanoso-
miasis (e.g., suramin).
Further validation of this chemotype was performed by
evaluating toxicity in T. brucei (strain Lister 427) bloodstream-
form cultures in vitro. This subspecies, while not human-
infective, is highly related to the HAT causative subspecies T. b.
gambiense and T. b. rhodesiense and is therefore commonly used
as a model in laboratory settings. Activity of a subset of para-
amidosulfonamide analogues was determined (Figure 2d), and
a
IC50 values represent the average of at least 3 separate experiments
reported as the half maximal (50%) inhibitory concentration as
determined in the ADP-Glo assay. All IC s <57 μM have a maximum
inhibition of >80% at 57 μM.
50
modest dose-dependent toxicity was seen with both 1 (ED =
50
15.18 μg/mL/26.8 μM) and 30 (ED50 = 7.24 μg/mL/16.3
μM). The most potent analogue in the PFK enzymatic assay,
42, did not show any appreciable parasite toxicity, though it is
unclear whether the lack of activity is due to poor compound
permeability or decreased potency/binding against the target in
the parasite. These compounds were additionally tested for
toxicity against the MRC-5 human lung cell line, a commonly
used surrogate for human host toxicity. No appreciable toxicity
was observed at any concentration of compound (ED50 > 20
μg/mL/46 μM), suggesting that these compounds do not exert
any off-target or PFK-related effects in a human cell
environment. It should be noted that the overall reduction in
previous studies have shown that T. brucei PFK undergoes a
conformation transition upon ATP binding, inducing a
significant change in the active site, which may contribute to
the reduced affinity of these inhibitors in the presence of
ATP. In this view, our study is consistent with the inhibitors
binding to the free enzyme and inhibiting activity through
direct competition with F6P. The commonality of the 3,4-
dichlorobenzyl motif in our inhibitors with the furanose
analogues reported by Nowicki also supports interaction with
the sugar pocket and our enzyme.
1
0
1
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ACS Medicinal Chemistry Letters
Letter
potency relative to the primary assay was anticipated, as
multiple studies have shown translation from enzymatic to
cultured parasite assays is often accompanied by a significant
Funding
This research was supported in part by Award No.
R03MH092153-01 from the Molecular Libraries Initiative of
the NIH Roadmap for Medical Research (grant
U54MH084681).
1
5,16
loss of activity.
We also assessed these select analogues via in vitro ADME
assays to evaluate aqueous solubility, stability in mouse, rat and
human liver microsomes, and plasma stability (Table 4). Aside
from a liability in rat microsomes for analogue 1 (t1/2 = 9.7
min), none of the analogues showed major issues with all
having very good aqueous solubility.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Sam Michael and Michael Balcom for assistance with
robotic HTS and William Leister, Chris LeClair, Danielle van
Leer, James Bougie, Heather Baker, Paul Shinn, and Tom
Daniel for help with analytical chemistry and compound
management. DF1020DE3 was received as a generous gift from
Simon H. Chang (Louisiana State University, Baton Rouge,
LA). We are grateful to Edinburgh Protein Production Facility
(EPPF) and to staff at the synchrotron facility at Diamond,
U.K. The crystal structure of tetrameric Tb PFK (PDB# 3F5M)
in the graphical Table of Contents was generated using
QuteMol open source molecular visualization software (http://
qutemol.sourceforge.net).
Table 4. Select in Vitro ADME Properties for 1, ML251, and
4
2
plasma stability
(% remaining
after 2 h)
aqueous kinetic
solubility
liver microsomal stability
(t1/2 in min.)
compd
μg/mL
mouse
rat
human mouse human
1
23.3
408
9.7
N/D
330
N/D
>95
>95
N/D
>95
>95
ML251
>81.0
>81.0
231
>30
>30
42
N/D
N/D
ABBREVIATIONS
■
The para-amidosulfonamide chemotype, exemplified by 30
PFK, phosphofructokinase; HAT, Human African Trypanoso-
miasis; T. brucei, Trypanosoma brucei:; T. cruzi, Trypanosoma
cruzi; MLSMR, Molecular Libraries Small Molecule Repository;
SAR, structureactivity relationship; qHTS, quantitative high-
throughput screen; F6P, fructose-6-phosphate; ADME, absorp-
tion, distribution, metabolism and excretion
and 42, represent the first small molecules to possess
submicromolar inhibitory activity against T. brucei and T.
cruzi PFK. Furthermore, 1 and 30 had micromolar activity in
cultured parasite growth assays. This, coupled with our
mechanistic and selectivity data, provides the first evidence
that specific inhibition of T. brucei PFK by a small molecule
may be realized. Lastly, members of this series have
demonstrated encouraging properties in a panel of in vitro
ADME assessment assays. As such, they represent useful tools
for advancing our understanding the role that PFK and
glycolysis play in trypanosome infection.
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AUTHOR INFORMATION
Corresponding Author
(M.B.B) E-mail: boxerm@mail.nih.gov.
■
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Author Contributions
These authors (K.R.B. and M.J.W.) contributed equally to this
work.
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