Deorphaning pyrrolopyrazines as potent multi-target antimalarial agents

Article abstract of DOI:10.1002/anie.201311162

The discovery of pyrrolopyrazines as potent antimalarial agents is presented, with the most effective compounds exhibiting EC₅₀ values in the low nanomolar range against asexual blood stages of Plasmodium falciparum in human red blood cells, and Plasmodium berghei liver schizonts, with negligible HepG2 cytotoxicity. Their potential mode of action is uncovered by predicting macromolecular targets through avant-garde computer modeling. The consensus prediction method suggested a functional resemblance between ligand binding sites in non-homologous target proteins, linking the observed parasite elimination to IspD, an enzyme from the non-mevalonate pathway of isoprenoid biosynthesis, and multi-kinase inhibition. Further computational analysis suggested essential P. falciparum kinases as likely targets of our lead compound. The results obtained validate our methodology for ligand- and structure-based target prediction, expand the bioinformatics toolbox for proteome mining, and provide unique access to deciphering polypharmacological effects of bioactive chemical agents. Pyrrolopyrazines show strong activity against Plasmodium falciparum parasites and Plasmodium berghei liver schizonts. In-depth bioinformatical analysis and target panel screening, suggesting IspD and multi-kinase inhibition, revealed their likely mode of action. The composite computational approach provides a unique access to deciphering polypharmacological effects of new bioactive chemical agents.

Full text of DOI:10.1002/anie.201311162

Angewandte
Chemie
DOI: 10.1002/anie.201311162
Medicinal Chemistry
Deorphaning Pyrrolopyrazines as Potent Multi-Target Antimalarial
Agents**
Daniel Reker, Michael Seet, Max Pillong, Christian P. Koch, Petra Schneider,
Matthias C. Witschel, Matthias Rottmann, Cꢀline Freymond, Reto Brun, Bernd Schweizer,
Boris Illarionov, Adelbert Bacher, Markus Fischer, FranÅois Diederich, and Gisbert Schneider*
Abstract: The discovery of pyrrolopyrazines as potent anti-
malarial agents is presented, with the most effective compounds
exhibiting EC50 values in the low nanomolar range against
asexual blood stages of Plasmodium falciparum in human red
blood cells, and Plasmodium berghei liver schizonts, with
negligible HepG2 cytotoxicity. Their potential mode of action
is uncovered by predicting macromolecular targets through
avant-garde computer modeling. The consensus prediction
method suggested a functional resemblance between ligand
binding sites in non-homologous target proteins, linking the
observed parasite elimination to IspD, an enzyme from the
non-mevalonate pathway of isoprenoid biosynthesis, and
multi-kinase inhibition. Further computational analysis sug-
gested essential P. falciparum kinases as likely targets of our
lead compound. The results obtained validate our method-
ology for ligand- and structure-based target prediction, expand
the bioinformatics toolbox for proteome mining, and provide
unique access to deciphering polypharmacological effects of
bioactive chemical agents.
Initially, we discovered pyrroloquinoxaline 1 (Figure 1) in
a high-throughput screening (HTS) campaign performed at
BASF, which aimed at finding low-molecular-weight com-
pounds that inhibit the herbicidal target 4-diphosphocytidyl-
2C-methyl-d-erythritol synthase (IspD) from the plant Ara-
M
alaria poses a severe health threat, and the rising
emergence of multi drug-resistant pathogens underlines the
urgent necessity of drug development with innovative modes
of action.[1–3] Herein, we disclose the identification of unique
antimalarial agents with nanomolar growth inhibition effect
on P. falciparum. We demonstrate how computational meth-
ods can be efficiently used to reveal their potential mecha-
nism of action. Pursuing a rigorous sequence- and structure-
based bioinformatics procedure, the approach starts from
identifying human targets and then makes the transition to
potential P. falciparum targets by orthologous identification
and experimental validation.[4–7]
Figure 1. Structures of reference compound 1 (IC50 A. thaliana
IspD=1.6 mm) and lead compound (Æ)-18, and concentration-depen-
dent growth inhibition of drug-sensitive P. falciparum NF54 parasites
by compound (Æ)-18. Untreated parasite growth served as control
(=100%). n=4 (two independent experiments with two technical
replicates); meanÆSEM.
[*] D. Reker,[+] Dr. M. Seet,[+] M. Pillong, Dr. C. P. Koch, Dr. P. Schneider,
Dr. B. Schweizer, Prof.Dr. F. Diederich, Prof. Dr. G. Schneider
Departement Chemie und Angewandte Biowissenschaften
ETH Zꢀrich, Vladimir-Prelog-Weg 3-4, 8093 Zꢀrich (Switzerland)
E-mail: gisbert.schneider@pharma.ethz.ch
Dr. P. Schneider, Prof. Dr. G. Schneider
inSili.com GmbH
Segantinisteig 3, 8049 Zꢀrich (Switzerland)
[+] These authors contributed equally to this work.
[**] We thank Dr. Case McNamara for support and discussion, and Dr.
Michael D. Wçrle for help with the X-ray crystal structures. This work
was supported by a grant from the ETH research council and F.
Hoffmann-La Roche, Ltd, by BASF SE, Ludwigshafen, and the OPO
Foundation Zꢀrich.
Dr. M. C. Witschel
BASF SE, Boschstrasse 14, 67056 Ludwigshafen (Germany)
Dr. M. Rottmann, C. Freymond, Prof. Dr. R. Brun
Schweizerisches Tropen- und Public-Health Institut
Socinstrasse 57, 4051 Basel (Switzerland)
and
Supporting information for this article (including experimental
procedures, chemical synthesis, cytotoxicity data, spectral data,
assay conditions, X-ray data, and detailed computational methods
and results) is available on the WWW under http://dx.doi.org/10.
1002/anie.201311162.
Universitꢁt Basel, Petersplatz 1, 4003 Basel (Switzerland)
Dr. B. Illarionov, Prof. Dr. A. Bacher, Prof. Dr. M. Fischer
Universitꢁt Hamburg, Hamburg School of Food Science
Institut fꢀr Lebensmittelchemie
Grindelallee 117, 20146 Hamburg (Germany)
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bidopsis thaliana.[8,9] A large set of derivatives was
synthesized and tested, but none of the follow-up
compounds exhibited lower than micromolar IC50
values against A. thaliana IspD. We decided to
transfer the observed moderate activity of the HTS
hits from the plant IspD enzyme to a potential
antimalarial application, because IspD is essential for
the survival of Plasmodium spp.[10] Plants and Api-
complexa such as malaria parasites possess the ability
to produce isoprenoids via the non-mevalonate path-
way as an alternative to the classical hydroxymethyl-
glutaryl-CoA reductase route. In fact, IspD as an
integral enzyme of the non-mevalonate route is an
attractive drug target for antimalaria research
because there is no human IspD homologue.
As no direct inhibition assay with purified
plasmodial IspD was available, we tested compound
1 in a phenotypic assay against asexual blood stages
of the drug-sensitive P. falciparum NF54 strain. We
observed almost complete inhibition of parasite
growth (> 99% inhibition) at a compound concen-
tration of 0.5 mgmLÀ1, which corresponds to an EC50
value of approximately 200 nm. Further chemical
optimization of the initial hit revealed improved
growth inhibition when the quinoxaline core was
replaced by a dicyanopyrazine. In total, we synthe-
sized and tested a set of 32 derivatives. The results for
compounds 2–(Æ)-33 show a remarkable antimalarial
potential of this substance class (Table 1). Synthesis
Table 1: Structure, antiplasmodial activity (NF54 strains) and cytotoxicity (L6 cells)
of pyrrolopyrazines 2–(Æ)-33.[a]
EC50 [mm] EC50 [mm]
R
Y
(NF54)
0.25
0.28
(L6)
85
3
SI LLE
348 3.5
11 5.3
2
3
NH
NH
4
NH
>100
>100
n.a. n.a.
13 3.6
5
6
NH
NH
NMe
NH
NH
NH
0.29
0.16
4.80
0.25
1.27
0.26
4
174 1093 3.6
7
188
148
187
141
39 1.7
591 3.7
148 3.1
625 3.1
8
9
10
11
12
NH
NH
0.41
0.28
80
198 3.0
519 2.7
of all derivatives followed
a two-step route
(Scheme 1). Commercially available pyrazine deriv-
ative 34 was substituted by acetonitrile derivatives
35–37 to give 38–40 with subsequent formation of the
pyrrole ring upon treatment with a primary amine
under basic conditions.[11,12]
143
13
14
NH
NH
0.19
0.22
52
40
273 2.9
182 2.8
In the cell-based assay, almost all pyrrolopyra-
zines tested in our study turned out to be strongly
active against P. falciparum, with EC50 values in the
nanomolar range. To assess the cytotoxicity of the
compounds, they were tested on rat skeletal myo-
blasts (L6 cells) as a mammalian model system. The
selectivity index (SI) was expressed as the ratio of
EC50 on L6 cells versus EC50 against Plasmodium. As
a general trend, we observed that the aromatic side
chains ensure low cytotoxicity with acceptable activ-
ity against Plasmodium, while derivatives with basic
side chains seem to be the most active ones.
Pyrrolopyrazines (Æ)-18 and (Æ)-20 in particular
exhibited strong antiplasmodial activity, with EC50
values in the double-digit nanomolar range (Table 1).
The selected lead compound (Æ)-18 (P. falciparum
NF54 EC50 = 53 nm; DpEC50 = 0.02 log units;
Figure 1) features a side chain resembling the one
from chloroquine, with adequate selectivity (SI = 40),
and remarkable lipophilic ligand efficiency[13,14]
(LLE = 4.0). We also tested this lead against the
chloroquine-resistant P. falciparum K1 strain, yield-
ing an EC50 value of 40 nm (DpEC50 = 0.75 log units).
Of note, this substance also exhibited strong inhib-
15
16
NH
NH
0.14
0.10
6
1
42 5.6
14 5.3
17
(Æ)-18
(Æ)-19
(Æ)-20
NH
NH
NMe
S
0.09
0.05
0.34
0.04
1
2
11 4.7
40 4.0
37 2.8
210 3.2
12
9
21
22
NH
S
0.30
0.59
>100 >820 5.0
18
30 3.8
23
24
NH
NH
0.12
0.74
4
34 6.0
81
109 3.1
25
26
NH
NH
0.36
0.28
42
55
116 3.3
194 3.3
2
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Table 1: (Continued)
ries.[23] Background inter-target compound similar-
ities were used to rationalize similarities between
(Æ)-18 and the co-clustered drug molecules by
ranking predictions according to their false-positive
error probability.[20] Retrospective evaluation dem-
onstrated that such a scoring scheme is remarkably
well-suited for making true-positive predictions,
yielding an average receiver-operator characteristic
(ROC) area-under-curve (AUC)[24] value of 0.78 Æ
0.14 in 10-fold cross validation on the level of 212
target families, with AUC values of up to 1.0 for
EC50 [mm] EC50 [mm]
R
Y
(NF54)
(L6)
SI LLE
27
28
29
NH
0.53
158
301 3.7
NH
NH
0.32
0.18
129
13
401 3.1
72 2.6
individual
targets
(Supporting
Information,
Table S13). Calculating rank scores for background
data allowed us to compute conservative p-values.[25]
Table 2 presents the suggested target list for com-
pound (Æ)-18 with p < 0.1. The predictions clearly
implied kinases as potential targets. This result is in
line with a study published by Volovenko and co-
workers,[26] who hypothesized, on the basis of an
observed general antiproliferative potential of pyr-
rolopyrazines, a binding mode of these compounds
to the hinge region of several kinases.
30
31
32
NH
NH
NH
1.98
0.32
5.23
100
53
51 2.9
158 3.6
22
4 2.6
(Æ)-33
NH
0.44
16
36 3.8
In an attempt to biochemically probe for the
computationally predicted kinase inhibition, we
subjected compound (Æ)-18 and selected analogues
[a] SI: selectivity index, LLE: lipophilic ligand efficiency.
Table 2: Top-ranking (p value <10%) predicted targets and target
classes for compound (Æ)-18.
Rank
Predicted target
p value [%]
1
2
3
4
5
6
thrombin receptor
serine-threonine kinase
adenosine kinase
tyrosine kinase
growth-hormone-releasing peptide receptor
DNA topoisomerase
7.2
8.7
9.1
9.6
9.7
9.9
Scheme 1. General synthetic strategy. a) DMF, 408C, 3–4 h; b) RNH2,
NEt3, DMF, 50–708C, 2–23 h; c) Me2SO4, K2CO3, DMF, 408C, 10 h.
6, 8, and 16 to activity testing in a human kinase panel (Cerep
ExpresS diversity kinase profile; Table 3). Compound (Æ)-18
inhibited 21 out of 48 tested kinases with > 50% effect at
a testing concentration of 10 mm. The basic derivative 16
showed a slightly weaker overall inhibitory effect (17/48), and
pyrrolopyrazines 6 and 8, featuring an aromatic side chain and
weaker antiplasmodial activity, exhibited no observable
kinase inhibition. For the four kinases that were inhibited
with ꢀ 99% efficiency by compound (Æ)-18, IC50 values were
determined. Low single-digit micromolar values corroborate
the target prediction, specifically for insulin receptor protein-
tyrosine kinase (IRK): 2.5 mm, serine-threonine-protein
kinase RAF-1: 2.3 mm, tyrosine protein kinases Src: 2.3 mm,
and tropomyosin receptor kinase TrkA: 1.0 mm (SEM < 1 log
unit for all IC50 values; Figure 2A). These results designate
members of the pyrrolopyrazine class as potent kinase
inhibitors and uphold our working hypothesis that the
observed high antiplasmodial activity of compound (Æ)-18
might be caused at least in part by multi-target effects.
itory activity against the liver schizont stage of P. berghei
(EC50 = 0.3 Æ 0.1 mm) and did not show cytotoxicity against
HepG2 cell cultures over a period of 48 h.
The discrepancy between the expected activity of the
compounds in the low micromolar range, as we inferred from
the A. thaliana IspD results, and the low nanomolar EC50
values against Plasmodium suggest a different or additional
mechanism of action. Consequently, we computationally
predicted potential macromolecular targets of lead structure
(Æ)-18. We used a self-organizing map (SOM)[15] that was
trained to cluster 11827 drugs and bioactive lead compounds
that are known to bind to a total of 673 macromolecular
targets comprising 212 target and activity families.[16–18] The
SOM groups molecules with similar pharmacophoric features
into clusters of functionally related compounds.[19,20] Com-
pound (Æ)-18 was projected onto this map, and from the
known targets of the co-clustered drug molecules we statisti-
cally inferred its potential targets. Following this concept, we
previously identified new targets for old drugs,[21,22] and
successfully “deorphaned” combinatorial compound libra-
The measured human kinase inhibition offers only an
indirect explanation for the observed antiplasmodial activity,
as plasmodial homologues of the inhibited kinases are yet
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search tool (BLAST).[27] We focused on local sequence motifs
in plasmodial proteins that were hit by all of the four query
kinase sequences to probe for a common binding site. This
consensus approach retrieved a total of 36 plasmodial
subsequences from 30 proteins with statistically significant
maximal expect (E)-values < 10À5 for all four searches (E-
value: the number of hits that can be expected to be seen by
chance). Remarkably, half of the hits belong to kinase families
that were previously reported as essential for the parasite,
confirmed in knockout studies, and suggested as targets for
innovative antiplasmodial therapy, for example, NimA
(“never in mitosis” A)-related, calcium-dependent and
cyclin-dependent kinases.[28]
As a consequent extension of the initial sequence-based
analysis, we performed a structure-based search for matching
protein surface cavities. As a consequence of the observed
multikinase inhibition, we focused on the nucleotide-binding
pockets from the four human kinases that were fully inhibited
by compound (Æ)-18 in the panel assay (PDB IDs:[29] 3omv
for RAF-1, 4aoj for TrkA, 4ibm for IRK, 2src for Src), and all
seven P. falciparum kinases, for which X-ray structures were
available from the PDB (PDB IDs: 1ltk, 1v0p, 1z6g, 2w41,
2wwf, 2yog, 3q5i). The software PoLiMorph[30] compares the
shape and properties of protein surface cavities by converting
the pocket volumes to branched graphs (so-called pocket
graphs) that are labeled by the local hydrogen-bonding,
hydrophobic, and electrostatic potentials, and their buried-
ness and surface accessibility. These graphs are compared by
applying a deterministic matching algorithm to obtain pair-
wise similarity scores.[31] Significant pocket similarity scores
were observed for PfPK5[32] matching with human RAF-1,
TrkA, and IRK. Subsequently, we compared PfPK5 to its
human homologue CDK2 in both the cyclin-activated (PDB
ID: 1fin) and the inactive (PDB ID: 4fko) state (Supporting
Information, Figure S3). Both comparisons yielded significant
similarity scores, and both CDK2 pockets exhibited compa-
rable screening results against the previously selected human
kinase panel (Supporting Information, Table S15).
Table 3: Inhibition of human kinases by pyrrolopyrazines 6, 8, 16, and
(Æ)-18 at a concentration of 10 mm (n=2).[a]
Kinase
Compound number
6
8
16
(Æ)-18
Abl
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
78
92
76
71
–
97
82
–
91
77
–
80
81
77
–
–
74
–
Akt1/PKBa
CDK2
CHK1
CHK2
CK1a
c-Met
EGFR
EphA2
FGFR2
FGFR3
IKKa
interf.
–
–
–
–
91
73
88
88
–
–
interf.
–
–
–
–
–
–
–
95
70
94
99
interf.
78
88
92
92
96
83
95
100
91
91
99
99
IRK (InsR)
JAK3
Lck kinase
MKK6
MNK2
MST4
Pim2
–
–
-
–
interf.
–
interf.
–
–
–
–
–
–
–
80
78
–
59
–
84
–
95
98
PKA
PKCb2
RAF-1
SGK1
SIK
Src
TrkA
[a] Targets are listed for which more than 50% inhibition was observed
(interf.: interference with the assay read-out).
Because of this putative pharmacophoric pocket similar-
ity, the retrieval of PfPK5 in the sequence-based search, and
the observed moderate inhibitory activity of compounds 16
and (Æ)-18 (76% and 26% inhibition at 10 mm, n = 2) against
the human homologue CDK2, we selected compound (Æ)-18
and its analogues 8 and 16 for a competitive PfPK5 binding
assay. Both 16 and (Æ)-18 exhibited moderate but significant
binding to PfPK5 at a concentration of 30 mm, while the
negative control 8 showed no effect (Figure 2B). Of note, all
three compounds did not bind to PfCDPK1 in a counterscreen
(data not shown). This result validates the computational
target identification and suggests binding of our lead structure
to the active site of PfPK5. Evidently, these preliminary
binding data do not warrant the relevance of PfPK5 inhibition
by compound (Æ)-18 in vivo. Quantification of histone H1
phosphorylation in parasites together with in vitro evolution
and comparative genomic analysis might help to obtain
further evidence.[33]
Figure 2. A) Concentration-dependent inhibition of human kinase
activity by compound (Æ)-18 (n=2); positive control: staurosporin.
B) qPCR signal of DNA-labeled P. falciparum protein kinase 5 (PfPK5)
after adding 30 mm test compound (n=2 technical replicates) to
compete with immobilized reference ligand.
unknown. At the same time, one might consider antikinase
activity a possible explanation for the observed cytotoxicity
(EC50 = 2 mm for (Æ)-18; Table 1), as rat myoblasts contain
homologues of the strongly inhibited kinases with more than
90% sequence identity, and we observed lower cytotoxicity
for selected derivatives that do not show measurable kinase
inhibition. In an effort to elucidate potential P. falciparum
target kinases of inhibitor (Æ)-18, we firstly performed
a rigorous sequence-based computational search (Supporting
Information, Table S14). To this end, we locally aligned the
complete amino acid sequences of the four most strongly
inhibited human kinases (IRK, RAF-1, Src, TrkA) with all
plasmodial proteins annotated in PlasmoDB (v9.3) for P.
falciparum strains 37D and IT using the basic local alignment
The pyrrolopyrazine, which was initially discovered in
a high-throughput screen on the herbicidal target IspD,
proved to show remarkable antiplasmodial activity. Optimi-
4
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[6] F.-J. Gamo, L. M. Sanz, J. Vidal, C. Cozar, E. Alvarez, J.-L.
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compound (Æ)-18. Advanced ligand-, protein sequence-, and
pocket-based computational studies were applied in concert,
and the results suggested essential plasmodial kinases as
potential targets of this compound class. In fact, multiple-
target inhibition has been proposed as a resistance-robust
antiplasmodial strategy,[34] and the here identified kinase
PfPK5 has previously been suggested as a candidate target of
efficient multi-target drugs.[7] Our study demonstrates that
chemistry-based target inference ideally complements and
inspires biochemical proteome mining and chemical genetics.
By pointing out subtle functional relationships between
ligand classes, the prediction method supplements other
computational tools and provides a fresh view on ligand-
target association.
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Experimental Section
Ligand-based target prediction: Drug molecules (collection of
bioactive reference compounds, COBRA v.11.10)[16] and lead com-
pound (Æ)-18 were pre-processed using the “wash” function within
the molecular operating environment (MOE 2011.10),[35] with options
“disconnect salts”, “remove ion pairs”, “deprotonate strong acids”,
“remove minor components”, “protonate strong bases”, and “add
hydrogen”. Molecules were subsequently represented by topological
pharmacophores (“CATS2” descriptor) and clustered by SOM train-
ing using our own software tools, as previously described.[17,20]
Computational analysis investigated similarities of drugs to (Æ)-18
in terms of their pharmacophoric features, and transformed them into
false-positive probabilities using background statistics of distances
between molecules of the COBRA database annotated to bind
different targets. Calculation of average false-positive probabilities
led to a rank ordering according to prediction confidence.[22]
Kinase assays: In vitro data for human kinases were obtained at
Cerep (86600 Celle lꢀEvescault, France; http://www.cerep.fr) on a fee-
for-service basis. Binding affinities of selected compounds for PfPK5
were obtained at DiscoverX (San Diego, CA USA; http://www.
discoverx.com) in their KINOMEscan assay on a fee-for-service basis.
[19] G. Schneider, W. Neidhart, T. Giller, G. Schmid, Angew. Chem.
1999, 111, 3068 – 3070; Angew. Chem. Int. Ed. 1999, 38, 2894 –
2896.
Received: December 23, 2013
Revised: March 6, 2014
Published online: && &&, &&&&
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Keywords: computer-assisted drug design ·
.
isoprenoid synthase · kinases · medicinal chemistry ·
target prediction
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Angew. Chem. Int. Ed. 2014, 53, 1 – 7
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Angewandte
Chemie
Communications
Medicinal Chemistry
Pyrrolopyrazines show strong activity
against Plasmodium falciparum parasites
and Plasmodium berghei liver schizonts.
In-depth bioinformatical analysis and
target panel screening, suggesting IspD
and multi-kinase inhibition, revealed their
likely mode of action. The composite
computational approach provides
a unique access to deciphering poly-
pharmacological effects of new bioactive
chemical agents.
D. Reker, M. Seet, M. Pillong, C. P. Koch,
P. Schneider, M. C. Witschel,
M. Rottmann, C. Freymond, R. Brun,
B. Schweizer, B. Illarionov, A. Bacher,
M. Fischer, F. Diederich,
G. Schneider*
&&&& — &&&&
Deorphaning Pyrrolopyrazines as Potent
Multi-Target Antimalarial Agents
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!