Exploring Leishmania major Inositol Phosphorylceramide Synthase (LmjIPCS): Insights into the ceramide binding domain

Article abstract of DOI:10.1039/c0ob00871k

The synthesis of set of ceramide analogues exploring hydrophobicity in the acyl chains and the degree and nature of hydroxylation is described. These have been assayed against the parasitic protozoan enzyme LmjIPCS. These studies showed that whilst the C-3 hydroxyl group was not essential for turnover it provided enhanced affinity. Reflecting the membrane bound nature of the enzyme a long (C₁₃) hydrocarbon ceramide tail was necessary for both high affinity and turnover. Whilst the N-acyl chain also contributed to affinity, analogues lacking the amide linkage functioned as competitive inhibitors in both enzyme and cell-based assays. A model that accounts for this observation is proposed.

Full text of DOI:10.1039/c0ob00871k

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PAPER
Exploring Leishmania major Inositol Phosphorylceramide Synthase
(LmjIPCS): Insights into the ceramide binding domain†
John G. Mina,a,b Jackie A. Mosely,a Hayder Z. Ali,a Paul W. Denny*a,b and Patrick G. Steel*a
Received 12th October 2010, Accepted 13th December 2010
DOI: 10.1039/c0ob00871k
The synthesis of set of ceramide analogues exploring hydrophobicity in the acyl chains and the degree
and nature of hydroxylation is described. These have been assayed against the parasitic protozoan
enzyme LmjIPCS. These studies showed that whilst the C-3 hydroxyl group was not essential for
turnover it provided enhanced affinity. Reflecting the membrane bound nature of the enzyme a long
(C13) hydrocarbon ceramide tail was necessary for both high affinity and turnover. Whilst the N-acyl
chain also contributed to affinity, analogues lacking the amide linkage functioned as competitive
inhibitors in both enzyme and cell-based assays. A model that accounts for this observation is proposed.
which is then reduced to dihydrosphingosine 2. Subsequently,
Introduction
there is evolutionary divergence in the pathway. In mammals,
dihydrosphingosine is acylated to produce dihydroceramide which
is then desaturated to ceramide 3, a key bioactive molecule. In
contrast, fungi and plants first generate hydroxylated sphinganine
before acylation to form phytoceramide 4, Fig. 1.11,12 Like mam-
mals Leishmania spp. also predominantly synthesise ceramide.13
Beyond this point in the biosynthetic pathway a further dichotomy
emerges in the synthesis of the predominant phosphosphingolipids
(PSLs). Utilising sphingomyelin synthase (SMS) mammalian cells
transfer phosphorylcholine from phosphatidylcholine (PC) to
ceramide to give sphingomyelin 5.14 In contrast, but like fungi and
plants, Leishmania spp. (and other trypanosomatids) synthesise
inositol phosphorylceramide (IPC) 6 as their primary PSL. One
notable difference is that in trypanosomatids the final product
formed 6 is IPC derived from ceramide 3 whereas in plants and
fungi the dominant SL used is phytoceramide 4 leading to the
formation of IPC 7. Whilst the essential fungal enzyme catalysing
this reaction, AUR1p or IPC synthase (IPCS), has long been
characterised as a novel target for anti-fungals,12,15 until recently,
the trypanosomatid (and plant)16 orthologues of this protein
remained unknown. However, using complementation strategy we
have isolated the gene encoding IPCS in L. major (LmjIPCS).17
Moreover, closely related orthologues are apparent in the genome
sequence databases of the other parasitic trypanosomatids, Try-
panosoma cruzi and T. brucei.17–19
Protozoan parasites of the order Trypanosomatidae cause a
range of human diseases, including the leishmaniases and hu-
man African trypanosomiasis (HAT).1–3 These infections are
of increasing prevalence, particularly in developing countries,
and have been classified by the World Health Organisation as
Category I: emerging or uncontrolled diseases.3 Moreover the
spread and severity of leishmaniasis is exacerbated by its status
as an important co-infection of AIDS patients and the overlap
in prevalence of HIV and Leishmania spp.4 The treatment of
trypanosomatid infections is difficult with the most serious visceral
form of leishmaniasis often requiring a long and costly course of
drug therapy. The challenge presented by these disease states is
heightened by the fact that the few efficacious drugs available often
exhibit serious, potentially fatal, side-effects. Moreover, reports of
resistance to even the newer drugs are emerging.5,6 This situation
renders the discovery and validation of new therapeutic targets a
priority in these organisms.
Sphingolipids are essential components of eukaryotic cell mem-
branes having critical roles in a variety of cell processes including
signal transduction, intracellular membrane trafficking and the
regulation of cell growth and survival.7–10 The de novo biosynthesis
of these lipid species is initiated by the condensation of palmitoyl
CoA 1 with L-serine via the ubiquitous eukaryotic enzyme
serine palmitoyltransferase (SPT) to produce 3-ketosphinganine
IPCS catalyses the transfer of the phosphorylinositol group
from phosphatidylinositol (PI) to ceramide or phytoceramide with
the concomitant release of diacylglycerol (DAG). Consequently,
in addition to producing the major PSL this reaction is also
important in maintaining homeostasis in the levels of the key
signaling components ceramide and DAG. Since the former is
pro-apoptotic and the latter mitogenic,20 modulating the activity
of this enzyme can have catastrophic effects on cell architecture
and function. Reflecting this the Trypanosoma brucei orthologue
aCentre for Bioactive Chemistry, Biophysical Sciences Institute, Department
of Chemistry and School of Biological Sciences, Durham University,
Science Laboratories, South Road, Durham, UK DH1 3LE. E-mail:
p.w.denny@durham.ac.uk, p.g.steel@durham.ac.uk; Tel: +44 (0)191 334
3983 or +44 (0)191 334 2131
bSchool of Medicine and Health, Durham University, Queen’s Campus,
Stockton-on-Tees, UK TS17 6BH
† Electronic supplementary information (ESI) available: Synthetic proce-
dures and characterisation data, biological screening methods and MS
data. See DOI: 10.1039/c0ob00871k
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drug targets is essential. Like all other PSL synthases, LmjIPCS is
an integral membrane protein with six predicted transmembrane
spanning domains.19 This makes structure-function studies using
protein crystallisation or spectroscopy a significant challenge.
In addition, whilst similarities with related enzymes, notably
the lipid phosphate phosphatases (LPPs),14,17,21,22 have enabled
the identification of a conserved active site triad incorporating
two histidines and one aspartate residue,17 the substrate binding
sites have not been identified. Consequently, we have initiated a
chemical biology approach to explore the enzyme with the aim of
developing a model of the active site that may ultimately inform
the design of effective inhibitors. To this end we have established a
microtitre plate-based assay and delineated the kinetic parameters
and mode of action of LmjIPCS.23 These studies revealed that
the enzyme follows a ping-pong bi-bi mechanism and that, of
the two substrates, ceramide has a higher affinity for the enzyme
than does PI. This observation, combined with the fact that PI is a
relatively abundant substrate in most membraneous environments,
suggested that ceramide is the rate-limiting substrate in the in situ
IPCS reaction. On this basis we opted to explore the binding of this
substrate to LmjIPCS and in this report we describe the synthesis
and evaluation of a set of ceramide analogues.
Results and Discussion
Synthesis of ceramide analogue library
Even excluding functional group variations, simple analysis of
the ceramide structure revealed many possible points of variation
including the degree of hydroxylation, stereochemistry at the two
stereogenic centres and the nature of the fatty acid component.
Moreover we have previously demonstrated that, whilst N-
acetyl sphingosine 8 was an acceptable substrate for LmjIPCS,
sphingosine 9 was not and functioned as a competitive inhibitor.23
Consequently, all the structures initially targeted as potential
substrate probes retained an amide linkage. With this requirement
we opted to explore the length and substitution in the sphingosine
tail, stereochemistry and, more drastically, the presence of the C-1
and C-3 hydroxy groups, Fig. 2.
Fig. 1 Divergent pathways in phosphosphingolipid biosynthesis (SPT,
serine palmitoyl transferase; 3-KSR, 3-ketosphinganine reductase; DHCS,
dihydroceramide synthase; DHCD, dihydroceramide desaturase; PS,
phytoceramide synthase; SMS sphingomyelin synthase; IPCS inositol
ceramide synthase; PC phosphatidylcholine; PI phosphatidylinositol).
Fig. 2 The selected structural features of the ceramide molecule.
Whilst many different routes to substituted sphingolipids have
been reported, including asymmetric strategies,24–45 we sought
routes that would minimise the need for individual compound
purification strategies. Recognising that variation in the sphingo-
sine and fatty acid alkyl chains could be introduced by simple
has recently been shown to be essential for the pathogenic
bloodstream form stage of the lifecycle.18,19 Given the global
impact of these diseases, further study of these putative protozoan
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cross metathesis and amine acylation respectively,46 the problem
simplified to generation of a set of core hydroxybutenyl amine
scaffolds, which in turn could be accessed from readily available
a-amino acids, Scheme 1.26–34
Scheme 1 Retrosynthetic Analysis of the common scaffold.
Synthetic work commenced by exploring routes to the fully
substituted ceramide core as represented by ‘Scaffold 1’. This was
achieved following an approach developed by Katsumura,31 based
on the stereoselective reduction of the vinyl ketone 12, Scheme 2.
Whilst, in our hands, protection of N-Boc Ser 10 as the TBS
ether followed by direct addition of a vinyl nucleophile proved
not to be viable, a stepwise strategy proceeding via the Weinreb
amide proved efficient providing ketone 12 in good yield (74%).
Reduction with LiAl(OtBu)3H was highly selective affording the
desired (2S, 3R) alcohol 14 with only trace amounts (<1%) of
the undesired diastereoisomer being detected in the crude reaction
mixture. Selective silyl group deprotection was then achieved using
dilute aqueous acid to afford the first core structure 15. We then
explored methods to allow a ceramide array to be constructed in a
time efficient fashion minimising chromatography where possible.
Cross-metathesis using the Grubbs’ second-generation catalyst
with a variety of terminal alkenes afforded N-Boc sphingosine
analogues. Deprotection of the Boc group could be achieved using
either TFA-DCM or HCl-dioxane mixtures although the former
led to variable amounts of the corresponding trifluoroacetamide.
Following removal of volatiles, direct treatment of the crude
reaction mixture with the various acid chlorides in the presence of
NaHCO3 (pH = 8) afforded the desired ceramide analogues 17 in
good chemical and stereochemical purities. Whilst final products
were purified by chromatography where needed, given the array
nature of this synthesis, no attempt was made to optimise reactions
in which low conversions were obtained. Importantly, with a view
towards future larger library generation, it also proved possible
to conduct the last three steps (cross-metathesis, deprotection
and acylation) with minimal chromatographic purification. In
Scheme 2 The synthesis and elaborations of ‘Scaffolds 1 & 2’.
this case, partition of the final crude reaction mixture between
dichloromethane and dilute aqueous acid (pH 5) by passage
through a ‘hydrophobic’ filter tube (Whatman PTFE) provided
product of sufficient purity (≥85% 1H NMR) to permit screening.
In an identical fashion, commencing from N-Boc Ala 11 allowed
the synthesis of ceramide analogues lacking the primary hydroxy
group, ‘Scaffold 2’, as well as a homologated series derived
from allyl ketone 19, Scheme 2. Whilst, in this latter series the
synthesis could be further shortened by the direct addition of
allylmagnesium bromide to the starting N-Boc protected amino
acid,47 the reduction of the ketone 19 to the amino alcohol 20
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was somewhat less selective producing an 84 : 16 mixture of the
(2S,3R) and (2S,3S) diastereoisomers respectively. These proved
trivial to separate by standard column chromatography and the
major isomer was taken through the metathesis and acylation steps
as for the other analogues.
In order to explore the relevance of the secondary hydroxyl
group in ceramide we then prepared the corresponding series of
compounds in which this group was lacking, ‘Scaffold 3’. These
could be accessed by asymmetric alkylation of the benzophenone
imine of glycine following the precedents established by Corey,
Lygo and others, Scheme 3.48,49Thus reaction of the glycine imine
with allyl bromide and KOH in the presence of 5 mol% of the
cinchona catalyst 29 derived from cinchonidine afforded the allyl
glycine derivative 23 in good yield. Following exchange of the
nitrogen protection group and reduction of the ester, metathesis
and acylation, as before, afforded the desired set of ceramide
analogues 27. Replacing the phase-transfer catalyst in the initial
alkylation with that 28 derived from cinchonine provided access
to the enantiomeric amino alcohol in similarly good yields. Cross-
metathesis with 1-alkenes and acylation as before then afforded
the enantiomeric analogues 26.
Biological Evaluation
All members of the synthesized analogue library were then
evaluated for their ability to inhibit the synthesis of fluorescently
labelled IPC using our established microtitre plate-based assay.23
In this system each compound was incubated with the labelled
acceptor substrate NBD-C6-ceramide, and the donor substrate PI
in the presence of LmjIPCS. The amount of labelled product,
inositol phosphoryl-NBD-C6-ceramide (NBD-C6-IPC), formed
was then quantified. The ratio between this value and that
for a control reaction, without the addition of an analogue,
provided a measure of competitive binding/inhibition. Notably,
the calculated Z-factor for this system is > 0.5 rating the assay as
statistically valid for screening purposes.50
This screen identified 24 compounds that reduced the quantity
of LmjIPCS synthesized NBD-C6-IPC by > 40%. Of these, 13
compounds reduced formation of labelled product by > 50%
including 3 by > 75%, (Fig. 3 & Table 1). The results indicated
a proportional increase in the inhibitory effect of the ceramide
analogues based on their hydrophobicity. With the exception of
three derivatives (Table 1 entries 24, 28 and 48), all compounds that
exhibited greater than 40% inhibition of LmjIPCS with respect
to formation of NBD-C6-IPC, contained a long hydrophobic
sphingosine tail (R1 = C13H27). This observation strongly suggests
that the chain length of the sphingosine tail is crucial for binding to
the enzyme, as might be expected given the hydrophobic nature of
LmjIPCS as an integral membrane enzyme. Similarly, increasing
the hydrophobicity of the N-acyl moiety appeared to favour its
affinity for LmjIPCS as determined by the reduction in NBD-C6-
IPC synthesis. However, the effect was smaller and less consistent
than that seen when increasing hydrophobicity of the sphingosine
residue (Fig. 3, n to x). Consistent with these observations, analysis
of the N-Boc protected cross-metathesis products produced as
synthetic intermediates showed similar trends (Table 1 entries 6-
16). Whilst those compounds containing a short chain (C4H9) or
an aromatic residue (CH2Ph) in the sphingosine backbone showed
little or no inhibition (£ 16%) of LmjIPCS mediated NBD-C6-IPC
Scheme 3 Synthesis and elaboration of ‘Scaffold 3’.
formation, those with a long alkyl tail (C13H27) exhibited moderate
levels of inhibitory effect (29–59%).
Switching from a D4,5 to a D5,6 alkene only had a small effect
except when in conjunction with a long chain N-acyl unit where
this change resulted in a twofold decrease in inhibition of NBD-
C6-IPC production (Table 1 entries 32 & 44). Previous work
has indicated that the trans double bond exerts a considerable
effect on the hydrogen bonding interactions of the 3-OH group
in ceramide.51 Whilst the loss of this effect may account for the
observed reduction in apparent affinity for LmjIPCS in series 21,
it is possible that a simple conformational change enforced by
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Fig. 3 Graphical representation of screening of ceramide analogues against LmjIPCS. Bars show level of inhibition of NBD-C6-IPC production against
control. (R1: a = C13H27, b = C4H9 and c = CH2Ph; R2: n = NH2, x = C7H15, y = CH2Ph and z = CH3).
the alkene position is also responsible. In support of the latter
suggestion, when comparing 1,3-dihydroxy analogues (series 17)
with derivatives lacking the 3-OH group (series 26 and 27), it
is clear that the 3-hydroxyl group exerts a minimal influence on
substrate binding.
for effective substrate binding and IPC synthesis. Moreover,
incorporation of steric bulk into either the sphingosine backbone
or the N-acyl moiety appears to result in these compounds acting
as true enzyme inhibitors (compare 27a or 26ay with 26ax). In
this respect it is pertinent to note that a-branched N-pivaloyl
phytoceramide analogues have been shown to exhibit relatively
high inhibition of S. cerevisiae IPCS turnover. However, in this
case it was not stated whether these were acting as true inhibitors
or alternative substrates.29
To further investigate the interaction of ceramide with LmjIPCS
four of the most active true-inhibitors were analysed in dose-
inhibition assays and the respective IC50 values determined (Fig. 4).
The most effective inhibitor as identified in the screen (Table 1),
N-octanoyl 1-deoxyceramide (18ax), had an IC50 of 4.79 mM is,
not surprisingly, structurally the closest to the natural substrate.
Consistent with the initial screening data incorporation of steric
bulk into the N-acyl unit led to higher IC50 values (Fig. 4 C&D)
Previously we have demonstrated that whilst N-acetyl-
D-erythro-sphingosine was an acceptor substrate, D-erythro-
sphingosine was not.23 Similarly here, MS analyses revealed that
whilst N-octanoyl-3-deoxyceramide (26ax) was turned over by the
enzyme, its derivative containing a free amine (26an) was not, and
functioned as an inhibitor with an IC50 of 14.7 mM (Fig. 4B).
Collectively, these results suggest that the free-amino group of
sphingosine contributes strongly to the observed inhibition by
these compounds potentially through an electrostatic interaction
with a positively charged ammonium salt that would be formed at
physiological relevant pH.23
Whilst a number of analogues significantly reduced the level of
NBD-C6-IPC production in the assay, this could arise through
one of two functions; either true inhibition of LmjIPCS or
compounds behaving as an alternative competitive substrates
and being processed to non-labelled IPC analogues. In order to
determine the extent of each possibility a small subset of the assay
reactions, involving both good and poor modulators of NBD-
C6-IPC formation, were analysed in greater detail using mass
spectrometry to look for the formation of the correspondingly
unlabelled but modified sphingolipid. Following reaction and frac-
tionation as described, organic phases were subjected to positive
and negative ion mass spectrometry. The resultant MS spectra (see
ESI) were searched for the mass peaks [M+241] corresponding to
the hypothetical PSL products. Whilst compounds 17ax, 26af,
and 26ax all showed signals for the phosphorylated product
indicating that these were viable substrates, no evidence for
phosphosphingolipid formation could be detected for compounds
18ax, 21bx, 17bx, 27a, 26an and 26ay suggesting that these
functioned as true inhibitors of the enzyme.
These observation are consistent with the predicted reaction
mechanism,23 in that analogues that lack the primary (C1)
hydroxyl group (e.g. 18ax) showed no evidence of phosphorylation
and the presence of an inositol head group. However, these
compounds do function as effective inhibitors suggesting that
the oxygen atom is non-essential for binding (comparing series
17 with 18). Not surprisingly, the N-octanoyl ceramide analogue,
17ax, functioned as an alternative substrate as did its derivative
26ax which lacked a 3-hydroxy group further confirming that this
latter group is not essential for turnover by LmjIPCS. Additionally,
although ceramide analogues with a shorter sphingosine backbone
(R1 = C4H9) resulted in moderate inhibition suggesting competitive
binding to LmjIPCS, such analogues (e.g. 17bx) were not processed
by LmjIPCS indicating the importance of a long backbone
Similar observations have been previously reported, e.g. the
inhibitory effect of sphingosine analogues on the S. cerevisiae
phosphatidate phosphatase,52 a member of the LPP enzyme
superfamily believed to share a mechanism of action with the
sphingolipid synthases.14 Moreover, Sigal et al.21 have proposed a
generalised hypothetical mechanism of action for such phosphoryl
transferases, Fig. 5B. This mechanism, in addition to the catalytic
triad, involves one lysine and two arginine residues. These three
residues are presumed to be in a protonated state and are therefore
able to stabilise the transition state structure during phosphate
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Table 1 Screening of ceramide analogues against LmjIPCS
the nucleophilic histidine residue of the active site. Investigation
of the LmjIPCS sequence identified Arg262 (in LmjIPCS) as a
conserved residue in all the identified orthologous IPC synthases
in the TriTryp genome,17 Fig. 5A.17 Consequently, Arg262 can be
presumed a potential candidate residue involved in the stabilisa-
tion of the transition state during the phosphorylinositol group
transfer by LmjIPCS, Fig. 5C. This speculation is consistent with
the hypothesis that the protonated amino group of sphingosine can
electrostatically interfere with the protonated Arg262 resulting in
inefficient stabilisation of the transition state and inhibition of the
catalytic transfer mechanism.
Significantly, analyses of the efficacy of the four compounds
above against cultured wild type L. major promastigotes identified
the free amino-derivative (26an) as the only one to reproducibly
demonstrate significant anti-protozoal effects at the concentra-
tions analysed. Under the experimental conditions described
maximal cytotoxicity was recorded at 12.5 mM, where cell viability
was assessed as 4.6 0.5 and 3.1 0.7% of an untreated control
in 2 independent experiments. In contrast, equivalent analyses
of a L. major serine palmitoyltransferase (SPT) mutant line53
demonstrated cell viability to be 14.6 0.02 and 13.8 0.7%
of the control. Loss of SPT function, which catalyses the first
and rate limiting step in sphingolipid biosynthesis pathway, is
tolerated by insect stage L. major promastigotes and renders
LmjIPCS redundant.53 Therefore, this line will be resistant to
specific LmjIPCS inhibitors. A similar strategy has been used to
identify specific S. cerevisiae IPCS inhibitors.54 Although these
mutants remain sensitive to 26an indicating off target effects,
potentially an S1P ripple response,55–57 they are 3–4 fold less
sensitive than the wild type line. This suggests that targeting
LmjIPCS is a viable strategy for anti-parasitic agents.
Entry R1
R2
R3
R4
n
No
Inh SE (%)
9.0 17
25
11 19
0
24
34.0
0.0
0
1
H
H
H
H
H
C13H27
C4H9
CH2Ph
C13H27
C4H9
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
H
OH OH
H
H
OH
OH
OH OH
OH OH
OH OH
H
H
H
H
H
H
0
0
1
0
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
15
2
OH
OH
H
16
8
3
20
4
24
1
5
H
25
1
6
17a
17b
17c
18a
18b
18c
21a
21b
21c
26a
27a
8
7
5
12
6
8
9
OH
OH
OH
OH
OH
OH
H
0
0
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
3
1
CH2Ph
C13H27
C4H9
29
16
15
55 15
59
3
6
9
CH2Ph
C13H27
C13H27
C13H27
C13H27
C13H27
C13H27
C4H9
OH
OH
OH OH
OH OH
H
4
17an 46 10
COCH3
COCH2Ph OH OH
COC7H15
H
COCH3
COCH2Ph OH OH
COC7H15
H
COCH3
COCH2Ph OH OH
17az 64
17ay 60
17ax 87
17bn 35
17bz 29
17by
17bx 56
17cn
17cz 14
17cy 29
17cx 45
18an 36
18az 19
5
1
4
3
3
OH OH
OH OH
OH OH
C4H9
C4H9
C4H9
0
3
OH OH
OH OH
OH OH
1
10
8
2
4
4
7
CH2Ph
CH2Ph
CH2Ph
CH2Ph
C13H27
C13H27
C13H27
C13H27
C4H9
0
COC7H15
H
COCH3
COCH2Ph
COC7H15
H
COCH3
COCH2Ph
COC7H15
H
COCH3
COCH2Ph
COC7H15
H
COCH3
COCH2Ph
COC7H15
H
COCH3
COCH2Ph
COC7H15
H
COCH3
COCH2Ph
COC7H15
H
COCH3
COCF3
OH OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
18ay 45 11
18ax 79
18bn 19
18bz 13
2
8
6
Conclusion
C4H9
C4H9
C4H9
A library of ceramide derivatives built around a set of hydroxy-
butenyl amine cores has been prepared exploring variations in the
sphingosine tail, N-acyl unit and the degree of hydroxylation.
The ability of these compounds to perturb the conversion of
NBD-C6-ceramide to NBD-C6-IPC mediated by LmjIPCS has
been assessed using a microtitre plate-based assay. The dominant
factor for effective binders, as determined by the reduced levels of
NBD-C6-IPC produced, was the possession a long chain lipophilic
sphingosine tail. Competitive substrates and inhibitors could be
distinguished by MS analyses of the reaction products. Whilst the
presence of the hydroxyl groups and a long chain N-acyl unit were
beneficial for activity, they were not essential for binding to the
active site of LmjIPCS. Notably, a free amino group conferred
a true inhibitory effect (rather than function as an alternative
substrate) and this is consistent with previously reported models
of the mechanism of action of this class of enzymes. Furthermore,
this class of analogue demonstrated anti-leishmanial activity in
cellulo with a significant proportion of this activity indicated to be
due to on-target effects.
18by
0
2
9
18bx 32
18cn 32
5
8
CH2Ph
CH2Ph
CH2Ph
CH2Ph
C13H27
C13H27
C13H27
C13H27
C4H9
18cz
0
18cy 10
18cx 10
21an 40
21az 39
21ay 55
21ax 45
21bn 38
21bz 20
21by 15
21bx 42
8
9
2
6
5
4
1
9
6
4
C4H9
C4H9
C4H9
CH2Ph
CH2Ph
CH2Ph
CH2Ph
C13H27
C13H27
C13H27
C13H27
C13H27
C13H27
C13H27
C13H27
C13H27
21cn
21cz
21cy 38 17
21cx 32
26an 57
26az 52
26af 65
26ay 76
26ax 57
27an 48
1
0
6
1
H
8
7
1
5
4
4
6
OH
OH
OH
H
H
H
H
H
H
H
H
COCH2Ph OH
COC7H15
H
COCH3
COCH2Ph OH
COC7H15 OH
OH
OH
OH
27az 42 11
27ay 50
27ax 46
These SAR studies will contribute to the development of
a pharmacophore model of the active site of this membrane
bound enzyme and help guide the design of future inhibitors
of this essential enzyme as potential new drug treatments for
leishmaniasis. In this respect it is pertinent to note that through
these studies the microtitre plate-based assay has been statistically
5
3
group transfer. Moreover, Sigal demonstrated that one of these
arginine residues is conserved across different families of enzymes
and organisms. This residue is located close (5 amino acids away) to
1828 | Org. Biomol. Chem., 2011, 9, 1823–1830
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Fig. 4 Inhibition curves and IC50 values of selected inhibitors. Activity refers to percentage activity relative to untreated control. Calculated IC50 values
A (18ax) 4.8 mM; B (26an) 14.7 mM; C (26ay) 13.2 mM; D (27a) 15.0 mM.
Fig. 5 A. Sequence alignment of active site residues of trypanosomatid IPCSs. B. Proposed mechanism of action of phosphoryl transferases adapted
from Sigal et al. (ref. 21). C. Proposed mechanism of action of LmjIPCS.
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validated for a future high-throughput screening purposes with a
Z-value > 0.5. Studies in this direction are in progress and will be
reported in due course.
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We thank the BBSRC (BB/D52396X/1 to PWD), Royal Society
(2005/R1 to PWD), Wolfson Research Institute and Durham
University (Overseas Research Student Award to JGM) for
financial support; Dr A. M. Kenwright (Durham University) for
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