Discovery of plasmodium vivax N -myristoyltransferase inhibitors: Screening, synthesis, and structural characterization of their binding mode

Article abstract of DOI:10.1021/jm300040p

N-Myristoyltransferase (NMT) is a prospective drug target against parasitic protozoa. Herein we report the successful discovery of a series of Plasmodium vivax NMT inhibitors by high-throughput screening. A high-resolution crystal structure of the hit com

Full text of DOI:10.1021/jm300040p

Brief Article
pubs.acs.org/jmc
Discovery of Plasmodium vivax N-Myristoyltransferase Inhibitors:
Screening, Synthesis, and Structural Characterization of their Binding
Mode
Victor Goncalves,† James A. Brannigan,‡ David Whalley,§ Keith H. Ansell,§ Barbara Saxty,§
Anthony A. Holder,∥ Anthony J. Wilkinson,‡ Edward W. Tate,*,† and Robin J. Leatherbarrow*,†
†Department of Chemistry, Imperial College London, London SW7 2AZ, United Kingdom
‡Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom
§Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, United Kingdom
∥MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
S
* Supporting Information
ABSTRACT: N-Myristoyltransferase (NMT) is a prospective drug target against parasitic protozoa. Herein we report the
successful discovery of a series of Plasmodium vivax NMT inhibitors by high-throughput screening. A high-resolution crystal
structure of the hit compound in complex with NMT was obtained, allowing understanding of its novel binding mode. A set of
analogues was designed and tested to define the chemical groups relevant for activity and selectivity.
compound collection was sourced by Medical Research Council
Technology, largely from commercial suppliers. In total
∼59500 compounds were screened against PvNMT. A
confirmed hit rate of 0.30% was observed and, in total, 107
compounds were found to inhibit NMT activity with IC50 in
the range 2−50 μM. The structure of some hit compounds and
their respective activity against NMT are reported in Figure 1.
INTRODUCTION
■
Plasmodium vivax is the most widely distributed cause of
malaria in the world and is endemic in large parts of Asia and
the Americas. Because P. vivax kills rarely and is not amenable
to continuous in vitro culture, it has so far been relatively little
studied in comparison to Plasmodium falciparum.1 Yet, the
overall burden, morbidity, and socioeconomic impact caused by
vivax malaria make the search for efficient treatments against
this infection essential.2 N-Myristoyltransferase (NMT) is a
ubiquitous enzyme in eukaryotes that catalyzes the covalent
attachment of the saturated 14-carbon fatty acid, myristate, to
the N-terminal glycine of susceptible proteins by amide bond
formation.3 NMT has been found to be essential in eukaryotes,
and the sequence variation between parasite and host NMTs
should permit the development of selective inhibitors.4−6
Therefore, targeting N-myristoylation in P. vivax might con-
stitute an attractive strategy for the treatment of vivax malaria.
However, in order to validate this approach, inhibitors of
P. vivax NMT (PvNMT) must first be identified.
In the present paper, we report the discovery, by high-
throughput screening, of a new small-molecule inhibitor of
PvNMT: 3-butyl-4-((2-cyanoethyl)thio)-6-methoxy-2-methyl-
quinoline. We describe a first study of the structure−activity
relationships of this new series and establish its binding mode
by resolving the high-resolution X-ray structure of this com-
pound in complex with PvNMT.
Figure 1. Structure and activity of some hit compounds.
At the end of the selection process, we decided to focus our
efforts on compound 1 (MRT00057965), which displays micro-
molar IC50 against P. vivax NMT.
Binding Mode of Hit Compound 1. To reveal the basis of
high affinity binding, we determined the first reported crystal
structure of PvNMT, in a ternary complex with compound 1
and a nonhydrolyzable myristoyl-coenzyme A analogue,
RESULTS AND DISCUSSION
■
Screening. The screening assay was adapted from a recently
reported 96-well plate fluorogenic assay for NMT.7 A number
of potential substrates were tested, with Homo sapiens
pp60src
(GSNKSKPKDASQRRR-NH2) being chosen as
(2−16)
the most suitable substrate for the PvNMT assay, with appar-
Received: January 11, 2012
Published: March 22, 2012
ent Km (Kmapp) of 5.71 0.6 μM in assay conditions. The
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
NHM.8 X-ray diffraction data extending to a spacing of 1.55 Å
were collected on synchrotron beamline ID14−4 (λ = 0.9393 Å)
at the ESRF (Grenoble).
Details of structure solution and a table of the data collection
and refinement statistics can be found in Supporting
Information. The core of the structure is an 11-stranded β-
sheet which is twisted so as to form an extended substrate
binding groove on either side of which NHM and compound 1
are bound (Supporting Information Figure 1). The mode of
binding of compound 1 is well-defined by the electron den-
sity maps (Figure 2A). Compound 1 is bound in PvNMT such
that ∼90% of its surface area is buried, and it forms interactions
with the side chains of a number of aromatic residues. Adjacent
residues on one face of a β-strand, Phe103 and Phe105, pack
onto opposite faces of the quinoline ring, forming π−π stacking
interactions. Meanwhile the phenolic ring of Tyr211 packs
against the nitrile of the exocyclic 2-cyanoethylthioether group.
Polar interactions are formed between the quinoline nitrogen of
compound 1 and the hydroxyl of Ser319 and between the
nitrile nitrogen of the ligand and the imidazole ring of His213.
There are additional apolar contacts to the side chains of
Leu330, Val96, and Asp98. Finally, a quintet of water molecules
clusters in the neighborhood of 1, one of which forms a
hydrogen bond with the sulfur of the 2-cyanoethylthioether
group. The large number of interactions between the enzyme
and compound 1 could certainly account for the observed
inhibitory activity.
Comparison of the structure of the PvNMT complex with
that of a ternary complex of Saccharomyces cerevisiae NMT
(ScNMT) with myristoyl-coenzyme A and the octapeptide
(GLYASKLA)9 suggests that 1 is a competitive inhibitor that
binds in the peptide binding groove of PvNMT occupying
volume corresponding to that filled by Ala4 and Ser5 of the
peptide in ScNMT (Figure 2B). In this superposition, the plane
of the bicyclic ring in 1 is approximately perpendicular to the
direction of the peptide. The binding mode of 1 (Figure 2C)
differs from that described previously for potent inhibitors
of Candida albicans NMT (CaNMT) and Trypanosoma brucei
NMT (TbNMT).6,10 In particular, 1 does not make any
interaction with the C-terminal NMT carboxylate that is a key
characteristic of those other inhibitors. Recently, Brand et al.
reported cocrystal structures of hit compounds against TbNMT
in complex with Leishmania major NMT (PDB accession codes
4A2Z and 4A30).11 These inhibitors present a binding mode
comparable to 1 (i.e., no interaction with the NMT C-terminus
and H-bonding with Ser319), but the bulkiness of the quinoline
ring and the presence of the n-butyl chain in 1 appear to induce
significant widening of the binding pocket.
Chemistry and Structure−Activity Relationships. A set
of compounds was prepared to address the key structure−
activity relationships of the quinoline scaffold. First, a series of
3-butyl-6-methoxy-2-methylquinoline derivatives was synthe-
sized according to Scheme 1. Condensation of 4-methoxyani-
line with an appropriate β-ketoester under acidic conditions led
to the formation of the 4-hydroxyquinoline core 1a, which
could then be converted into its 4-mercaptoquinoline derivative
1c in two steps.12 1c was S-alkylated with an appropriate halo-
genated molecule to give the hit compound 1 and its analogues
12−16 (Table 1). Next, our efforts focused on positions 2-, 3-,
and 6- of the quinoline scaffold. A series of 4-hydroxyquinoline
derivatives was obtained by a Conrad−Limpach or Gould−
Jacobs synthesis (Scheme 2).13,14 The 4-hydroxy position was
Figure 2. (A) View of 1 in chain A of PvNMT (cyan ribbon) in
cylinder format and colored by atom, carbon (green), oxygen (red),
nitrogen (blue), sulfur (yellow). The side chains (labeled) of
surrounding protein residues are similarly colored but with carbons
in gray. Electron density (2Fo − Fc) associated with 1 contoured at 1 σ
is displayed. Polar interactions with the protein and solvent are shown
as dashed lines. Y211 labeled in italics is shown in two alternate
conformations. (B) 1 (green) from the PvNMT ternary complex
superposed with residues GLYASK of the peptide ligand from the
crystal structure of the ScNMT ternary complex.9 (C) 1 (green) from
the PvNMT ternary complex superimposed with two inhibitors
described previously for CaNMT10 (orange) and TbNMT6 (blue).
The C-terminal residue of PvNMT (stick format) is labeled.
subsequently chlorinated and substituted with 3-mercaptopro-
panenitrile to provide final compounds 7−11 and 20−24.
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Synthetic Pathway to Compound 1
a
Reagents and conditions: (i) PTSA·H2O, toluene, reflux, 16 h; (ii) Ph2O, reflux, 1 h; (iii) SOCl2, DMF, 80 °C, 1 h; (iv) NaSH, DMF, reflux, 4 h;
(v) 3-bromopropanenitrile, K2CO3, DMF, 110 °C, 1 h, then, HCl 2 N in Et2O.
a
Table 1. Biochemical Activity of Tested Compounds against PvNMT and Human NMTs
Kiapp (μM)
c
d
e
compd
R2
R3
nBu
R4
R6
PvNMT
HsNMT1
HsNMT2
cLog P
LE
LipE
1
Me
Me
Me
H
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CCH
S-(CH2)3-CN
S-CH2-CN
MeO
MeO
MeO
MeO
H
1.73 0.16
3.23 0.19
>100
1.69 0.22
15.63 1.37
>100
11.39 2.11
23.23 2.53
>100
4.9
3.9
2.5
3.0
2.7
3.2
3.8
4.3
5.4
5.3
4.6
5.0
6.7
3.7
4.6
5.5
2.9
3.2
3.5
2.0
2.8
0.36
0.37
0.9
1.6
b
5
Et
6
H
7
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
nBu
4.74 0.24
24.42 1.79
24.37 0.53
60.63 4.72
>100
19.43 0.86
>100
>100
0.33
0.31
0.30
0.26
2.4
1.9
1.4
0.5
8
H
>100
9
Me
Et
H
10.80 0.91
47.92 7.84
>100
41.53 6.23
94.17 6.23
>100
10
11
12
13
14
15
16
17
H
nPr
Me
Me
Me
Me
Me
Me
Me
Me
H
H
MeO
MeO
MeO
MeO
MeO
H
11.60 1.39
35.23 7.64
>100
>100
>100
0.31
0.26
−0.5
−0.8
nBu
47.60 3.51
46.06 13.04
nBu
nBu
S-(CH2)3-OH
S-(CH2)2-Ph
NH-(CH2)-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
S-(CH2)2-CN
17.35 0.84
>100
>100
>100
0.30
−0.2
nBu
CO2Et
nBu
>100
b
18
b
H
2.46 0.l6
21.55 2.38
7.77 0.38
6.97 0.54
10.39 0.80
>100
>100
>100
>100
>100
0.38
0.30
0.33
0.33
0.32
1.0
−0.9
2.2
19
nBu
Br
20
21
22
23
24
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
F
33.48 9.61
27.49 2.40
>100
37.52 10.69
>100
H
Me
1.9
H
Cl
>100
1.5
H
acetyl
H
CH3-CH(OH)
>100
a
Apparent Ki values of tested compounds on the enzymatic activity of recombinant P. vivax NMT and Homo sapiens NMT isoforms 1 and 2. Each Ki
b
c
is the mean SD from duplicates. Purchased compound from Interbioscreen Ltd. Purity >85% based on RP-HPLC/MS analysis. cLog P values
d
were calculated with ChemDraw for Excel version 12.0.2. LE: ligand efficiency (PvNMT); LE = −RT ln(Kiapp)/N where N is the number of non-
e
hydrogen atoms of the compound. LipE: lipophilic efficiency (PvNMT); LipE = pIC50 − cLog P.
a
Scheme 2. Synthetic Pathways to Derivatives 7−11 and 20−24
a
Reagents and conditions: (i) 140 °C, 1 h; (ii) Ph2O, reflux, 2 h; (iii) NaH, N,N-dimethylacetamide, 120 °C, 20 min; (iv) POCl3, 110 °C, 20 min;
(v) 3-mercaptopropanenitrile, K2CO3, THF, reflux, 4 h.
These molecules were evaluated for their inhibitory potency
against P. vivax NMT as well as for their selectivity versus
human NMT isoforms 1 and 2 (HsNMT1 and HsNMT2,
respectively) (Table 1).
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Reducing the length of the n-alkyl chain in position 3
(compounds 1, 5, and 6) resulted in a progressive loss of
activity toward PvNMT as chain length was shortened.
However, the replacement of this aliphatic chain by an ethyl
ester (compound 7) greatly improved solubility and selectivity,
particularly over HsNMT2, while retaining a good activity
versus PvNMT. The n-alkyl group is enclosed in an apolar
pocket although there is the potential for the ester group to
form polar interactions with residue Asn365. Extension of
the alkyl chain in position 2 of the quinoline scaffold (entries
9−11) led to a dramatic loss of activity against PvNMT. Con-
sistent with these observations, the 2-methyl group in the
parent compound fits snugly into a protein pocket in which the
binding of bulkier substituents would lead to steric hindrance.
Interestingly, compound 8, in which the 2-methyl group is
deleted, displayed a similar potency to 9 but with an improved
selectivity over human NMTs.
Crystallization of this compound with PvNMT revealed the
binding mode of this competitive inhibitor. A variety of small
molecules based on the hit compound structure were synthe-
sized and revealed some key attributes of 1. These findings
constitute a starting point for the development of potent NMT
inhibitors as potential therapeutics for vivax malaria and for
target validation studies.
EXPERIMENTAL SECTION
■
Chemistry. All chemicals were purchased from Sigma-Aldrich Ltd.
(UK), Acros Organics (Belgium), or VWR (UK). Reagents and
solvents were used without any further purification. Purity of tested
compounds was determined by RP-HPLC and was superior to 95%
unless specified. 1H and 13C NMR spectra were recorded on a Bruker
̈
AV-400 (400/100 MHz) spectrometer. Mass spectra and accurate
mass data were obtained by J. Barton at the Chemistry Department
Mass Spectrometry Service (Imperial College London) by electrospray
ionization. The synthesis and structural characterization of all
compounds is described in the Supporting Information.
Subtle modifications to the length as well as the composition
of the 4-substituent chain (compounds 12−14) resulted in loss
of activity. Attempts to establish π−π stacking interactions with
the nearby aromatic residues Tyr211 and Tyr334 by intro-
ducing a phenyl ring in position 4 (compound 16), or to
establish H-bonds with the His213 and Asn365 side chains by
introducing a hydroxyl group in place of the nitrile (compound
15), failed to improve activity. Similarly, substitution of the
thioether linker with an amine (17) induced a complete loss
of activity; unfortunately, the corresponding ether derivative
could not be synthesized due to its chemical instability. The
polarity and length of the 2-cyanoethylthioether moiety appear
to be important as this group extends toward the side chains of
Asn365, His213, and Tyr211. Finally, a series of quinolines
modified at position 6- were evaluated. While small substituents
(entries 1, 18, and 20−22) were tolerated by PvNMT, the
presence of bigger or branched side chains (19, 23, and 24)
abolished inhibitory activity. In particular, the introduction of
polar substituents (23 and 24), with the potential to establish
polar contacts with the protein carboxamide backbone, caused a
dramatic loss of activity. These results suggest lack of flexibility
of the protein around this position. Indeed, clashes can be
predicted from the protein structure in this region although
there is potential for alterations as the methoxy group is
oriented back toward the cofactor binding site and there are
three water molecules within 4 Å of the methyl carbon, suggest-
ing that certain modifications at this position might be tol-
erated. Interestingly, direct comparison of compounds 1 and 18
(as well as 7 and 8) shows that the removal of the 6-methoxy
group induces a significant improvement of inhibition selec-
tivity over HsNMT1 and HsNMT2. This result cannot be
easily rationalized on the basis of the structure reported here
because the residues of PvNMT in contact with the inhibitor 1
are fully conserved in the human NMTs. The differences in
observed activity probably arise from subtle changes in chain
flexibility and residue orientation.
ASSOCIATED CONTENT
* Supporting Information
Detailed high throughput screening protocol, expression and
purification of PvNMT, X-ray data collection and statistics, syn-
thesis details and structural characterization of all compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
Accession Codes
The coordinates and structure factor files have been deposited
in the Protein Data Bank under the accession code 4a95.
AUTHOR INFORMATION
Corresponding Author
*For E.W.T.: phone, +44 (0)20-7594-3752; fax, +44-(0)20-
7594-1139; E-mail, e.tate@imperial.ac.uk. For R.J.L.: phone,
+44 (0)20-7594-5752; fax, +44-(0)20-7594-1139; E-mail, r.
leatherbarrow@imperial.ac.uk.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Deborah Smith (University of York) and co-
workers for helpful discussions and Marek Brzozowski for expert
crystal handling. We acknowledge the European Synchrotron
Radiation Facility for provision of excellent synchrotron radiation
facilities. This work was supported by the Medical Research
Council (MRC; grants G0900278 and U117532067), the
Wellcome Trust (grant 087792), and the Biotechnology and
Biological Sciences Research Council (David Phillips Research
Fellowship to E.W.T., grant BB/D02014X/1).
ABBREVIATIONS USED
■
These optimizations led to the identification of compound 7,
which exhibits micromolar activity against PvNMT, some
selectivity over human NMT isoforms, and improved lead-like
properties (cLog P = 3.0; LipE > 2; MW = 316) compared to
the initial hit 1 (Table 1).15
Ca, Candida albicans; DMF, N,N-dimethylformamide; ESRF,
European Synchrotron Radiation Facility; Hs, Homo sapiens;
NHM, S-(2-oxo)pentadecyl-coenzyme A; NMT, N-myristoyl-
transferase; PTSA, p-toluenesulfonic acid; Pv, Plasmodium
vivax; Sc, Saccharomyces cerevisiae; SD, standard deviation;
siRNA, small interfering RNA; Tb, Trypanosoma brucei
CONCLUSION
■
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