Peptidomimetic inhibitors of N-myristoyltransferase from human malaria and leishmaniasis parasites

Article abstract of DOI:10.1039/c4ob01669f

N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex. This journal is

Full text of DOI:10.1039/c4ob01669f

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Peptidomimetic inhibitors of N-myristoyltransferase
from human malaria and leishmaniasis parasites†
Cite this: DOI: 10.1039/c4ob01669f
Tayo O. Olaleye,a James A. Brannigan,b Shirley M. Roberts,b Robin J. Leatherbarrow,a
Received 6th August 2014,
Accepted 10th September 2014
Anthony J. Wilkinsonb and Edward W. Tate*a
DOI: 10.1039/c4ob01669f
www.rsc.org/obc
N-Myristoyltransferase (NMT) has been shown to be essential in
N-Myristoyltransferase (NMT), an enzyme that modifies
Leishmania and subsequently validated as a drug target in Plasmo- protein substrates by attaching myristate (14 : 0) to an N-term-
dium. Herein, we discuss the use of antifungal NMT inhibitors as a inal glycine via an amide bond, has been proposed as a poten-
basis for inhibitor development resulting in the first sub-micro- tial therapeutic target in both malaria and leishmaniasis5,6
molar peptidomimetic inhibitors of Plasmodium and Leishmania and has recently been validated as viable drug target for
NMTs. High-resolution structures of these inhibitors with Plasmo- human malaria.7 Catalysis is thought to commence with
dium and Leishmania NMTs permit a comparative analysis of ordered binding of S-myristoyl-coenzyme A (Myr-CoA) followed
binding modes, and provide the first crystal structure evidence for by the protein substrate, transfer of myristate and ordered
a
ternary NMT-Coenzyme A/myristoylated peptide product release of myristoylated protein and free coenzyme A.8 Myris-
complex.
toylation is important for protein–protein and protein–mem-
brane interactions; the essentiality of NMT in the viability of
both fungal and protozoan organisms9,10 makes NMT an inter-
esting target for the development of antifungal and anti-para-
sitic drugs.11,12
Malaria and leishmaniases, infectious diseases caused by Plas-
modium and Leishmania respectively, rank among the world’s
most important public health challenges. These diseases
result in high mortality and morbidity; moreover, they impose
a severe economic burden on affected countries, mainly in
Africa. Of the Plasmodium genus, P. falciparum is the most
deadly, accounting for over 1 million deaths in 2010, and
P. vivax is the most widespread.1 Although malaria-related
deaths have reduced by 30% in recent years, resistance to
current anti-malarial drugs presents an ongoing challenge,
highlighting the need to identify and develop safer and prefer-
ably less resistance-prone treatments.2 Visceral leishmaniasis,
the most deadly of the leishmaniases, is caused by L. donovani,
and accounts for ∼50 000 deaths per annum.3 Currently avail-
able treatments can be expensive, possess a relatively narrow
therapeutic window or are subject to resistance, highlighting
the need for better drugs.4
Peptidomimetic inhibitors based on peptide substrates of
NMT have previously been developed against Candida albicans
NMT (CaNMT),13,14 but have yet to be reported in the context
of parasitic NMT inhibition. CaNMT shares 44% and 43%
sequence identity with P. vivax and L. donovani NMTs (PvNMT,
LdNMT) respectively; we reasoned that inhibitors of Plasmo-
dium and Leishmania NMTs might be acquired through a
‘piggy-back’ approach, using CaNMT peptidomimetics as a
platform.15 Reported CaNMT peptidomimetic inhibitors were
based on residues 1–7 at the N-terminus of C. albicans ADP
ribosylation factor protein, GLYASKL. Subsequently, the
N-terminal amine and Ser5-Lys6 dipeptide, a motif also seen in
known substrates of Plasmodium and Leishmania NMTs, were
identified as making important binding contributions.5,7 We
therefore chose to employ a similar peptidomimetic scaffold
based on the Ser-Lys motif, substituting the first four amino
acids with an alkyl chain capped by a group that mimics the
N-terminal amine, and the C-terminal leucine with a hydro-
phobic motif (Fig. 1). Our inhibitor library design incorporated
modifications at the C- and N-termini with the objective of
exploring contacts at both ends of the scaffold. Peptidomi-
metics were synthesized through a combination of solid and
solution phase chemistries. N-Boc-protected amino and 1H-
imidazol-1-yl acids were coupled to an O-t-butyl serine/N-Boc
lysine dipeptide linked via a chlorotrityl (Route A, Scheme 1)
aDepartment of Chemistry, Imperial College London, London, SW7 2AZ, UK.
E-mail: e.tate@imperial.ac.uk; Tel: +44 (0)20 7594 3752
bStructural Biology Laboratory, Department of Chemistry, University of York, York,
YO10 5DD, UK
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, characterization of intermediates and target compounds, description of
biological assays, and crystallographic information are available in the Sup-
plementary Information. The coordinates and structure factor files have been de-
posited in the Protein Data Bank with the accession codes: 4c68 (PvNMT-NHM-
10), 4c7h (LmNMT-MyrCoA-10) and 4c7i (LmNMT-MyrCoA-46). See DOI:
10.1039/c4ob01669f
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Fig. 1 Peptidomimetic scaffold targeting parasite NMTs. R1 and R2 represent points of variation at the N- and C-termini.
Scheme 1 Synthetic routes to peptidomimetics. Reagents and conditions. (a) Fmoc-Ser(t-Bu)-OH, HBTU, DIPEA, DMF; (b) 20% piperidine–DMF;
(c) R3COOH, HATU, DIPEA, DMF; (d) 0.5% TFA in DCM; (e) R4NH2, HATU, DIPEA, DMF; (f) TFA–TIPS–H2O (95 : 2.5 : 2.5); (g) Fmoc-Lys(Boc)-OH,
HBTU, DIPEA, DMF; (h) Cu(OAc)2, pyridine, DCM, R4NH2.
or hydrazinobenzoyl linker (Route B, Scheme 1) to polystyrene (pKa ∼ 8.0).19 To cover a range of basicity, imidazolyl (pKa ∼
resin. In the case of chlorotrityl resins, intermediates were 7.0) and amine analogues (pKa ∼ 10.0) were also synthesized,
cleaved from the resin with 0.5% TFA–DCM and coupled to each exploring a range of chain lengths (n) (Table 1). To probe
the requisite amine (Scheme 1). C-terminal amide and acid versatility at the C-terminus, primary amides, carboxylic acids,
analogs were synthesized using similar chemistry on Rink 2-cyclohexylethyl and 2-(1-cyclohexenyl)ethyl amides were
amide and Wang resins, respectively.
incorporated. Using a previously reported fluorogenic assay,20
Switching to a hydrazinobenzoyl linker eliminated the need inhibitors were tested against the parasitic enzymes and
for solution phase chemistry to couple the C-terminal HsNMT1 in order to explore their selectivity against a relevant
amine;16 thus one-pot treatment of the peptidyl-resin in the host enzyme. Previously reported imidazolyl-derived peptido-
presence of copper(II) acetate and excess of the appropriate mimetics (1–4) shown to possess low-µM activities against
amine led to simultaneous cleavage of the peptide from the CaNMT14 displayed little or no activity against the parasitic
resin and formation of the corresponding protected enzymes used in this study. Slightly improved potency was
peptidomimetic.
observed for 2-methylimidazolyl analogues (5–8) across all
Final products were deprotected by treatment with TFA and chain lengths tested, and particularly 6, which provided sub-
purified by semi-preparative RP-HPLC-MS to give a small micromolar inhibition for L. donovani NMT. However, amine 9
library of peptidomimetic inhibitors. Modifications at the showed markedly improved inhibition against the NMTs of
N-terminus were approached by two distinct structural permu- P. vivax (PvNMT), L. donovani (LdNMT) and H. sapiens
tations: varying the N-terminal motif (R1, Fig. 1) to optimize (HsNMT1) (Table 1). Reduction of the alkyl chain length from
basicity, and varying the alkyl linker length (n) to determine n = 10 to 9 gave compound 10, which is the most potent
the optimal distance between the N-terminal amide of the L. donovani NMT inhibitor reported to date (LdNMT IC50 = 24
serine and the N-terminal nitrogen atom of R1 (Fig. 1). The nM). It also showed somewhat lower activity against HsNMT1
N-terminal motif of the peptidomimetic was expected to inter- (IC50 = 60 nM) and PvNMT (680 nM). Further reduction of the
act directly with the C-terminal carboxylate leucine of the chain length (11 and 12, n = 8 and 7, respectively) led to loss of
enzyme, a critical residue for catalytic activity responsible for detectable activity against Plasmodium NMTs and significant
the activation of the N-terminal amine of the protein/peptide loss of activity against LdNMT and HsNMT1. Comparing
substrate.17
N-terminal variations with similar chain length, the potency of
2-Methylimidazolyl analogues were synthesized building on amine 10 against LdNMT was over 400- and 20-fold higher
the hypothesis that 2-methylimidazole (pKa ∼ 7.9)18 is a than 2 (1H-imidazol-1-yl) and 6 (2-methyl-1H-imidazol-1-yl),
reasonable mimetic for N-terminal Gly in the peptide substrate respectively.
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Table 1 Structures and enzyme affinities for peptidomimetics synthesized in this studya
IC50 (µM)
LdNMT
R1
n
R2
PvNMT
PfNMT
HsNMT1
1
2
3
4
5
6
7
8
1H-Imidazol-1-yl
1H-Imidazol-1-yl
1H-Imidazol-1-yl
1H-Imidazol-1-yl
2-Methyl-1H-Imidazol-1-yl
2-Methyl-1H-Imidazol-1-yl
2-Methyl-1H-Imidazol-1-yl
2-Methyl-1H-Imidazol-1-yl
H2N–
H2N–
H2N–
H2N–
MeNH–
H3C–
H2N–
H2N–
1H-Imidazol-1-yl
1H-Imidazol-1-yl
2-Methyl-1H-Imidazol-1-yl
2-Methyl-1H-Imidazol-1-yl
H2N–
H2N–
H2N–
H2N–
H2N–
H2N–
10
9
8
7
10
9
8
7
10
9
8
7
9
0
10
9
10
9
10
9
10
9
8
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-Cyclohexylethanamine
2-(1-Cyclohexenyl)-ethanamine
2-(1-Cyclohexenyl)-ethanamine
2-(1-Cyclohexenyl)-ethanamine
2-(1-Cyclohexenyl)-ethanamine
2-(1-Cyclohexenyl)-ethanamine
2-(1-Cyclohexenyl)-ethanamine
–NH2
>100
>100
>100
>100
>100
>100
>100
>100
1.04 0.01
0.68 0.08
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
24.3 3.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25.8 8.2
10.6 1.6
16.7 2.5
34.8 3.7
>100
0.63 0.01
3.42 0.34
1.46 0.22
0.14 0.01
0.024 0.003
2.01 0.30
1.39 0.18
0.21 0.01
>100
6.60 1.28
0.44 0.04
>100
>100
>100
28.0 2.5
13.3 2.0
1.36 0.20
27.1 2.6
61.9 4.7
86.7 10.4
>100
47.6 3.8
44.0 7.1
>100
>100
21.8 0.8
7.92 0.83
49.9 10.6
82.2 14.0
0.34 0.03
0.06 0.003
7.68 0.86
6.75 0.45
0.73 0.10
>100
2.30 0.14
0.67 0.04
>100
>100
>100
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
>100
12.9 1.07
3.55 0.38
>100
>100
>100
>100
>100
22.9 3.5
>100
>100
>100
5.40 0.41
1.36 0.29
33.1 2.5
>100
39.2 2.0
92.1 17.6
–NH2
–NH2
–NH2
–OH
7
10
9
>100
>100
–OH
a Enzymatic activities of recombinant P. vivax, P. falciparum and L. donovani NMT in the presence of peptidomimetic inhibitors expressed as IC50
values. These values are a mean of duplicate or triplicate experiments.
We next probed the SAR around the amino group of 10, and potent combination irrespective of the enzyme tested, and at
found that N-methylation (to give 13) led to significant the N-terminus, inhibitor potencies increased in the order:
reduction in potency, whilst replacing the flexible N-terminal 1H-imidazol-1-yl < 2-methyl-1H-imidazol-1-yl < –NH2.
chain with an acetyl group (to give 14) resulted in no observa-
ble activity.
To determine the binding mode of 10, a ternary complex
with PvNMT and S-(2-oxo)pentadecyl-CoA (NHM, a non-hydro-
We further probed the importance of charge at the N-termi- lysable Myr-CoA analogue) was crystallized and solved to a
nus by substituting a hydroxyl for the amine and observed a high resolution of 1.38 Å (accession code: 4c68, Fig. 2A). 10
more modest loss in activity of >100 and 1000 folds in Leishma- sits in the peptide-binding pocket, and mimics the key recog-
nia and Human NMTs respectively (46, ESI,† accession code: nition elements involved in binding the parent peptide
4c7i). These observations are consistent with our expectation (GLYASKL, Fig. 2B and C). The N-terminal amine electrostati-
that the N-terminal moiety of the inhibitor is involved in a cally interacts with the carboxylate of Leu410 whilst the
strong electrostatic interaction with the C-terminal carboxylate hydroxyl group of the serine hydrogen bonds to the His213
of the enzyme, an interaction likely to be sensitive to changes side chain. In addition, there are ionic interactions between
in inhibitor structure and charge.21 Amongst inhibitors with a the amino group of the lysine and three neighboring aspartic
C-terminal 2-(1-cyclohexenyl)ethanamide (15–20, Table 1), 16 acid residues (Asp98, Asp100, Asp385), all of which are con-
showed fair activity against LdNMT, HsNMT1 and PvNMT, served in equivalent residues CaNMT. The aliphatic chain on
whilst others showed little (15) or no activity (17–20) against the N-terminus and the N-terminal amino group of the inhibi-
the tested enzymes up to the highest concentration tested tor are guided by the peptide binding channel to the C-term-
(100 µM). This 10–20 fold drop in activity relative to 2-cyclohexyl- inal residue of PvNMT–Leu410. For comparison, a ternary
ethanamide suggests that the presence of a single unsaturated structure of 10 with the native substrate, Myr-CoA, in L. major
bond in the pocket occupied by the cyclohexenyl ring deters NMT (97% sequence identity to L. donovani) was solved and
important interactions with the enzyme, presumably by modi- refined to a resolution of 1.41 Å (accession code: 4c7h). As
fying ring conformation. Inhibitors with C-terminal carbox- expected, the mode of binding of 10 in L. major is very similar
amides and carboxylic acids (21–26) showed minimal activity to that in P. vivax.
across the enzymes tested, with the exception of 22 (Table 1) with
Unexpectedly, the electron maps indicated a mixture of
an n = 9 chain length. Overall, an ideal chain length of n = 9 ligand structures such that ∼20% of the electron density rep-
and a C-terminal cyclohexyl ring was observed to be the most resents a N-myristoylated 10 product complex with the CoA by-
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Fig. 2 (A) Co-crystal structure of PvNMT (chain C; grey surface) with bound NHM (magenta) and 10 (green), colored by atom. (B) Superimposition
of peptide substrate (GLYASKL, blue) with 10 (green, 4c68) in PvNMT. The peptide was modeled to maximize overall structural similarity while main-
taining peptide geometric restraints. (C) Ternary structure of 10 (green, sticks) and non-hydrolysable Myr-CoA (partially shown; magenta; sticks) in
PvNMT (4c68) showing main recognition interactions between 10 and the enzyme. Residues within 4 Å of 10 are shown in blue. Polar interactions
and their distances (in Å) are shown as dashed lines. (D) Refined electron density map showing the mixture of structures of 10 and myr-10 (yellow) in
a ball and stick representation observed in LmNMT (4c7h). The figure shows the mixture of ligands present in the final refined model (80% reactants
– purple and red and 20% products – green and yellow) shown in a ball-and-stick representation to aid identification. Electron density figures were
made using the program CCP4 mg.22 See Fig. S2 (ESI†) for more details.
product in LmNMT (Fig. 2D). This is the only structural evi- NMT substrates, N-terminal Gly is preferred to the exclusion of
dence for a product complex in any N-myristoyltransferase, all other residues, with relatively non-sterically demanding
and captures and supports the proposed mechanism of action residues such as alanine. The substrate properties of 27 and 28
of NMT just prior to regeneration of the active enzyme. Kinetic suggest a highly peptidomimetic binding conformation, and
experiments were performed to study 10 both as a potential sub- imply that steric factors play a key role in the progression of
strate and as an NMT inhibitor. As anticipated from the struc- the catalytic cycle, dictating the approach of the N-terminal
tures above, 10 appeared to behave as a peptide-competitive amine to the C-terminal leucine of the enzyme.
inhibitor but no substrate characteristics were detected in the
The potent inhibitors discovered in this study showed low
assay up to 40 µM (Fig. S3 and S4, ESI†). The N-myristoylation specificity for the Plasmodium NMTs in comparison to Leish-
of 10 observed above therefore presumably occurs within the mania and human NMTs. A sequence alignment of residues
crystal where all reagents are present at high effective concen- within 6 Å of the catalytic sites of Leishmania and Human
trations, and turnover is blocked by limited egress of products.
NMTs (see ESI†) showed a 74% similarity between both
To probe the degree to which 10 acts as a true peptidomi- enzymes. In comparison, a similar alignment of Human and
metic, we investigated the potential to switch from inhibitor to Plasmodium NMTs showed a 53% similarity between residues.
a peptidomimetic substrate. A glycine (27) or alanine (28) The catalytic site of Leishmania NMT is, therefore, thought to
residue was incorporated in the N-terminal extension; 27 was show more comparability to the Human enzyme than the Plas-
found to be a substrate for Leishmania NMT with KM = 1.3 µM, modium NMTs. Additionally, there are key residues, such as
which compares favorably with the affinity of a CaNMT octa- Arg231 (LdNMT) and Lys308 (Human NMT), capable of
peptide substrate (GLYASKLS-NH2, KM = 0.6 µM)13 and the forming hydrogen bond interactions which would otherwise
model peptide substrate used for inhibitor assays be impossible in the Plasmodium NMTs (Gly225). It is thought
src
(2–9)
(GSNKSKPK-NH2 H. sapiens p60
, KM = 22.6 µM), although that the presence of such basic centres could increase the
with a considerably reduced Vmax (Fig. S4, ESI†). 28, on the binding and subsequently, potencies of these inhibitors for
other hand, showed no detectable substrate activity. In native Leishmania and human NMTs.
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Unusually, the inhibitors in this study showed a higher
specificity for P. vivax NMT in comparison to P. falciparum
NMT. An alignment of the primary sequences of both enzymes
(see ESI†) highlighted four differing residues within 6 Å of the
peptide binding pocket: Ile102, Tyr212, Ser228, Tyr334
(PvNMT) and Val102, Phe212, Cys228 and Phe334 (PfNMT). Of
these differences, the replacement of Y334 to F334 in PfNMT
constitutes a structural change in the peptide pocket that
could lead to a loss of potential hydrogen bond interactions
within the catalytic site. The reason for the specificity for
PvNMT is unclear; however, we propose that the loss of such
vital hydrogen bonds in PfNMT could be responsible for this
observation.
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Conclusions
Using antifungal NMT inhibitors as a platform, we developed
inhibitors of Plasmodium and Leishmania NMTs, resulting in
the identification of an inhibitor, 10, with sub-micromolar
potency against P. vivax and L. donovani NMTs, and the most
potent Leishmania donovani NMT inhibitor reported to date.
High-resolution ternary structures of compound 10 were
achieved in both PvNMT and LmNMT, with the latter structure
revealing the first structural evidence supporting the proposed
catalytic cycle of NMT. Although 10 shows moderate selectivity
against human NMT, the structures presented here may serve
as a useful tool to enable a structure-guided design of more
potent and selective parasitic inhibitors.
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Funding sources and
acknowledgements
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). We are also grateful to Imperial
College for an Imperial College President and Rector’s award
and the Student Opportunities Fund for funding. We are grate-
ful to Prof. Deborah Smith (University of York), Dr J. Hutton
(Imperial College) and colleagues for helpful discussions and
Diamond Light Source (Harwell, UK) for provision of excellent
synchrotron radiation facilities.
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