NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on Trypanosoma cruzi and Human Enzymes

Article abstract of DOI:10.1021/acs.jmedchem.8b00484

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.

Full text of DOI:10.1021/acs.jmedchem.8b00484

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Brief Article
An NMR biochemical assay for oxidosqualene cyclase: evaluation
of inhibitor activities on Trypanosoma cruzi and human enzymes
Osamu Tani, Yukie Akutsu, Shinji Ito, Takayuki Suzuki, Yukihiro Tateishi, Tomohiko Yamaguchi,
Tatsuya Niimi, Ichiji Namatame, Yasunori Chiba, Hitoshi Sakashita, Tomomi Kubota, Tetsuo
Yanagi, Shusaku Mizukami, Kenji Hirayama, Koji Furukawa, and Kazuhiko Yamasaki
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00484 • Publication Date (Web): 17 May 2018
Downloaded from http://pubs.acs.org on May 17, 2018
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Journal of Medicinal Chemistry
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An NMR biochemical assay for oxidosqualene cyclase: evaluation of
inhibitor activities on Trypanosoma cruzi and human enzymes
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Osamu Tani , Yukie Akutsu , Shinji Ito , Takayuki Suzuki , Yukihiro Tateishi , Tomohiko Yama-
2
guchi ,
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Tatsuya Niimi , Ichiji Namatame , Yasunori Chiba , Hitoshi Sakashita , Tomomi Kubota , Tetsuo
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Yanagi ,
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1,
Shusaku Mizukami , Kenji Hirayama , Koji Furukawa , and Kazuhiko Yamasaki *
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Hi-
1
gashi, Tsukuba, 305-8566, Japan.
2
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, 305-8585, Japan.
3
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Tech-
nology (AIST), 1-1-1 Umezono, Tsukuba, 305-8568, Japan.
4
Department of Immunogenetics, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki,
852-8523, Japan.
ABSTRACT: Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here
we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor
analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causa-
tive of Chagas disease, and compared with their effects on human OSC for selectivity. Among them, one novel inhibitor
showed a significant parasiticidal activity.
INTRODUCTION
Therefore, it is necessary to develop new drugs for Chagas disꢀ
ease. For this purpose, inhibitors were being investigated
against a number of target proteins, such as a cystein protease
Oxidosqualene cyclase (OSC; lanosterol synthase, E.C.
.4.99.7) is a key enzyme in sterol biosynthesis, which catalyzes
5
14
15
cruzain , and a histone deacetylase sirtuin .
cyclization of 2,3ꢀoxidosqualene to lanosterol with the multiꢀ
1, 2
Enzymes in sterol biosynthesis have been also chemotheraꢀ
ring steroid scaffold . Successive enzymatic reactions lead to
16
peutic targets against Chagas disease . The antifungal azoleꢀ
type inhibitors ketoconazole and posaconazole, inhibiting lanosꢀ
terol 14ꢀdemethylase, and azasterol inhibitors against sterol
cholesterol in mammalian systems or ergosterol in fungal and
protozoan systems. Because hypercholesterolemia is one of the
major risks of arteriosclerosis, OSC inhibitors have been develꢀ
oped as candidate for nonstatin drugs to reduce cholesterol .
Also because disturbance of sterol biosynthesis impairs forꢀ
mation of membrane lipid bilayer, OSC is a highly potential
3
ꢀ5
ꢁ
24ꢀmethltransterase (E.C. 2.1.1.43), have been shown to affect
17ꢀ19
growth of T. cruzi
. Similarly, effects of numerous OSC
inhibitors, including those originally developed for human OSC,
6
, 7
were tested for T. cruzi OSC, and IC values of nanomolar–
target of developing antifungal drugs . For these purposes, a
number of promising OSC inhibitors were designed and proꢀ
50
20ꢀ23
micromolar level were obtained . In animal, however, lanosꢀ
8
terol depletion as a result of inhibition or mutational defect of
duced to date . Indeed, established azoleꢀtype antifungal drugs,
24, 25
OSC is known to cause lesions such as cataract
. The imꢀ
e.g., ketoconazole and posaconazole, inhibit lanosterol 14ꢀ
demethylase (E.C. 1.14.13.70), a downstream enzyme of OSC
9,
portance of lanosterol in the lens was clearly demonstrated, in
which treatment of the cataractꢀaffected lens by lanosterol, but
1
0
.
In addition, sterol ꢁ8,7ꢀisomerase (E.C. 5.3.3.5) and sterol
26
not by cholesterol, dissolved crystallin aggregation . Therefore,
a usable inhibitor of T. cruzi OSC as a drug for Chagas disease
should not affect the activity of human OSC, although it is not
ꢁ
14ꢀreductase (E.C. 1.3.1.70) also in the downstream of OSC
are targets of antifungal amorolfine .
1
1
Chagas disease is one of the worst neglected tropical diseases,
with 10,000~14,000 people mostly in Latin America being deꢀ
27
achieved at this moment .
12, 13
To discover T. cruzi OSCꢀspecific inhibitors, we need to evalꢀ
uate a number of candidate compounds and, therefore, should
employ an efficient method to measure enzymatic activity; it
should be rapid and accurate as well. Nonetheless, the generally
used assays for OSC are based on chromatography and radioacꢀ
ceased per year
. This is caused by a protozoan parasite
Trypanosoma cruzi, as transmitted by bloodꢀsucking triatomine
bugs. Two clinically used drugs, benznidazole and nifurtimox,
are effective in acute phase, although they tend to be unsatisfacꢀ
tory in chronic phase, and have numerous toxic side effects
12, 13
.
1, 2, 4, 20, 21, 23, 28
tivity measurements
, which appear to be timeꢀ
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consuming and also are at a risk of radiation exposure. Namely, quantitative analyses (Fig. 2A). In order to evaluate enzymatic
they utilize radioisotopeꢀlabeled substrates or precursors for the activity, reaction times were varied within 10–80 min and timeꢀ
reaction. Then, typically after saponification of other types of dependent changes of the NMR spectra were analyzed. To deal
lipids and extraction by hexane and/or ether, lanosterol was with the entire data points in a selected spectral region, FA was
isolated by thin layer chromatography (TLC) for quantification. used as described previously (see also the Experimental secꢀ
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Alternatively, wholeꢀcell assay using gas chromatographyꢀmass tion)
. By this treatment, loading vectors and score vectors
spectrometry (GCꢀMS) to evaluate inhibitors of the sterol bioꢀ are obtained for the respective “factors”, where the former repꢀ
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synthesis was reported
. This method is especially potential resent spectral profile and the latter represent the time dependꢀ
in identifying the target enzymes of inhibitors, although culture ence in this case. The loading vector of the first factor that corꢀ
of cells takes one day to one week. Nonetheless, GC can be responds to the largest eigenvalue, but not those of the others,
31
used for evaluating activity of recombinant OSC .
exhibited a profile reproducing the NMR peaks of lanosterol
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In the process of exploration of novel inhibitors against anothꢀ (Fig. 2B). Also, the score vector only of this factor showed a
er T. cruzi enzyme, we developed a novel method of biochemiꢀ timeꢀdependent change fitting an exponential curve (Fig. 2C).
1
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cal assay by H NMR measurement and factor analysis (FA)
The first factor, therefore, clearly represents the timeꢀdependent
3
3
1
of the spectral data . The merits of H NMR are that we can use change of the population of lanosterol, that is, the enzymatic
nonꢀlabeled intact molecules for detection, and that we can deal reaction of OSC. The kinetic constant for this reaction was calꢀ
with mixed sample, where individual molecules can be sepaꢀ culated to be 0.083 min for T. cruzi OSC. By the same apꢀ
–1
–1
rately detected as spread peaks over the spectral region. Also, proach, a kinetic constant of 0.017 min was obtained for huꢀ
the merits of the FA treatment are that we can deal with all the man OSC (Fig. S2). Note that the rates depend on the concenꢀ
data points in the spectral region of interest simultaneously, trations of enzymes, which should be set low enough to allow
which is suitable for an automatic process, and that we can isoꢀ evaluation of strong inhibitors.
late meaningful change from baseline instability and/or noise
Evaluation of inhibitors. In the present study, one known
4
and, thereby, obtain more accurate parameters for reaction and inhibitor Ro 48ꢀ8071 (compound 1 in Fig. 3) and three novel
33, 34
inhibition
.
compounds (compounds 2–4 in Fig. 3) were tested for T. cruzi
In the present study, we developed an efficient biochemical and human OSCs. In order to evaluate the inhibitory effects, a
33, 34
assay for OSC, based on the previous NMR/FA method
. In protocol shown in Fig. 1B was carried out, which involves a
the experimental protocol, a detergent as well as organic solꢀ series of reactions with variable concentrations of the inhibitors
vents were used, because OSC, a membraneꢀassociated microꢀ at a constant time. Similarly to the above process for the timeꢀ
35
somal protein , and its substrate/product are insoluble to simple dependent data, FA was applied to these concentrationꢀ
aqueous buffers. Inhibitory activities of one known and three dependent data (Fig. 4, Fig. S3). By fitting the score vector of
new compounds against T. cruzi and human OSCs were evaluꢀ the first factor with equations 3 and 4 in the methods section,
ated and compared for the selectivity. In addition, we present a the inhibitor concentration causing 50% inhibition (IC ) was
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novel highꢀthroughput method, which simultaneously processes calculated. Compound 1 inhibited T. cruzi and human OSCs
data for different compounds by FA. These methods, in combiꢀ with IC50 values of 13 and 7.5 nM, respectively (Table 1). It is
nation, are useful in many stages of drug discovery, e.g., from noteworthy that this inhibitor is more effective to the human
an initial screening stage of fragmentꢀbased drug discovery enzyme than to the T. cruzi enzyme. This selectivity is conꢀ
36
(FBDD) to a final optimization stage.
sistent with a previous study using the two enzymes expressed
23
in Saccharomyces cerevisiae . Namely, the IC values for T.
50
RESULTS AND DISCUSSION
cruzi and human OSCs were 0.90 and 0.17 ꢂM, respectively.
By the same method, three novel compounds (2ꢀ4) were
evaluated and IC50 values were obtained (Table 1). The comꢀ
pound 2, which has an allylmethylamine moiety similar to 1
NMR biochemical assay of OSC. In the assay system for
OSC, we must deal with waterꢀinsoluble substrate and product,
,3ꢀoxidosqualene and lanosterol, respectively. These materials
2
(
Fig. 3) showed 3ꢀfold weaker inhibitory activity against T.
were dissolved in the reaction buffer by detergent Triton Xꢀ100,
which was also necessary to dissolve the membraneꢀassociated
and partly hydrophobic OSC protein. This detergent, however,
disturbed NMR detection, probably because of interaction beꢀ
tween the molecules and the largeꢀmolecular micelle of Triton
Xꢀ100 (data not shown). Addition of organic solvent, therefore,
was necessary to solubilize the molecules freely and to enable
the NMR detection. We thus carried out a protocol shown in
Fig. 1A. Namely, reaction proceeds at 37 °C in the aqueous
cruzi OSC than 1 with an IC50 value of 42 nM, and the selectiviꢀ
ty against human OSC remained unchanged. Compound 3
showed inhibitory activity to T. cruzi OSC with an IC50 value of
44 nM, which was comparable to that of 2. However, compound
3 inhibited human OSC with an IC50 value of 120 nM, that is, 3
is 2.7ꢀfold more selective to T. cruzi OSC than human. Also,
compound 4, which is a dimethylamine analogue of 2, showed
inhibitory activity against T. cruzi and human OSC with IC50
values of 140 and 230 nM, respectively. Considering that 3 and
solution containing detergent, and 10:1 methanol–D O mixture
2
4
showed selectivity to T. cruzi OSC, but allylmethylamines 1
was added to stop the reaction and at the same time dissolve the
substrate and product. Then, NMR measurements were carried
out to detect substrate and/or product. Note that one may use
deuterated methanol to simplify the spectrum, although the
sharp methyl peak of methanol can be effectively reduced by a
presaturation pulse (see Experimental section).
and 2 did not, the dimethylamine moiety may contribute to the
selectivity. While we understand that 3ꢀfold selectivity is not
large and distant from the goal, it can be a clue to acquire highly
T. cruzi OSCꢀselective inhibitors. We should point out that a
compound with a similar structure but lacking the dimethylaꢀ
mine moiety of 3 or 4 (compound 29 in ref. 38) was only effecꢀ
tive to human OSC, but not to T. cruzi OSC.
Because 2 and 3 have stronger activity than 4, we tested their
effects on the proliferation of T. cruzi (Table 1). They indeed
affected proliferation of the parasite in the epimastigote, correꢀ
Although most of the NMR peaks originating from the subꢀ
strate and product fully or partially overlap with many other
peaks including those of detergent, two singlet peaks due to
3
7
methyl groups of lanosterol (19ꢀH and 28ꢀH; see Fig. S1 in the
Supporting Information) were observed separately and used for
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sponding to the insectꢀhosted stage, by IC50 values of 110 and Especially by automatic measurements and FA data proꢀ
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2150 ꢂM for 2 and 3, respectively. Note that the effects are cessing, the NMR assay for OSC is simple and with a considerꢀ
weaker than those against T. cruzi OSC, especially for 3. This is able throughput. Because NMR is most useful in evaluating
3
9, 40
probably because compounds should permeate though the cell biophysical interaction between protein and compounds
membrane to reach OSC, which is a microsomal enzyme locatꢀ may carry out biophysical binding assay by NMR, as well under
ed inside the cell. a comparable condition. Thus, utilizing NMR for screening at
, we
We further accessed the parasiticidal effects on the different criteria will reduce pseudoꢀpositive or pseudoꢀnegative
amastigote, corresponding the intercellular stage, by duration hits, and thereby improves the reliability of drug discovery proꢀ
until appearance of trypomastigote, a form generally seen in cesses.
blood, after treatment of compounds (Table 2). Similarly to the
above, 2 is stronger than 3, with a longer duration of silence. It EXPERIMENTAL SECTION
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should be especially noted that 2 is significantly more effective
than clinically used benznidazole.
Sample preparation. The DNAs that code for oxidosqualene
cyclase (E.C. number 5.4.99.7) of Homo sapiens and T. cruzi
A method suitable for initial screening. In drug discovery, strain CL Brener (NCBI codes AAB36220 and XP_820967.1,
throughput in an NMR analysis is frequently a matter to be disꢀ respectively) were chemically synthesized (Thermo Fisher Sciꢀ
cussed. Indeed the process of obtaining an IC50 value shown in entific Inc., Waltham, MA) with the codon usage optimized for
Fig. 4 involves 12 sets of NMR measurements (a point relevant Pichia pastoris. After PCR amplification, the DNAs were subꢀ
for absence of the inhibitor is not shown in Fig 4C). This reꢀ cloned into expression vector pPICZ B (Thermo Fisher Scienꢀ
quires approximately two hours including setup for the measꢀ tific), with mycꢀepitope and His tag attached at the Cꢀterminus.
6
urement, even if we use automatic measurement protocol, and is The P. pastoris cells (strain GS115) were grown in BMGY meꢀ
not suitable for the initial screening stage of FBDD starting dium [1% yeast extract, 2% peptone, 100 mM potassium phosꢀ
from the fragment library.
phate (pH 6.0), 1.34% yeast nitrogen base, 0.4 mg/L biotin,
To accelerate the drug discovery, we present here a simple 0.5% glucose, 0.2% glycerol] at 30 °C for 3 days. Then, the
protocol to evaluate multiple compounds simultaneously (Fig. proteins were expressed in BMMY medium [1% yeast extract,
1C), in which inhibitor concentration as well as reaction time 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34%
was fixed. Fig. 5 shows an application to the four inhibitory yeast nitrogen base, 0.4 mg/L biotin, 1% methanol] at 25 °C for
compounds on T. cruzi OSC. The NMR data were also proꢀ 2 days.
cessed by FA, with the score vectors corresponding to different
After cells were lysed by highꢀpressure homogenizer Emulsiꢀ
compounds (Fig. 5). It should be noted that the score vector is Flex C5 (Avestin, Ottawa, Canada) in a buffer [50 mM TrisꢀHCl
nearly proportional to IC , having a correlation coefficient of (pH 7.5), 250 mM NaCl, 5 ꢂM pepstatin A (SigmaꢀAldrich), 1
5
0
0
.89. Therefore, this approach is useful in screening the comꢀ mM PMSF], and 0.5–1% Triton Xꢀ100 (Wako, Osaka, Japan)
pound library, in order to pick up candidates of inhibitory comꢀ was added to the crude extract. Proteins were purified by colꢀ
pounds. Once a compound is identified as a candidate of an umn chromatography using HisTrap HP (GE Healthcare), and
inhibitor, the IC50 value should be determined for more accurate Resource Q (GE Healthcare) for T. cruzi OSC, and Superdex
evaluation by the protocol shown in Fig. 1B. Application to 200 (GE Healthcare), in addition to the above, for human OSC.
human OSC is also shown in Fig. S4. Also, correlation between Buffers used are: [20–50 mM TrisꢀHCl (pH 7.5–8.0), 5% glycꢀ
score vector and IC50 value was confirmed with a correlation erol, 0–250 mM imidazole, 0–500 mM NaCl, and 0.2% Triton
coefficient of 0.94.
Xꢀ100 or 0.2–1.2% βꢀoctylglucoside (Nacalai, Kyoto, Japan)]
Assuming that approximately 10 min is required for the for T. cruzi OSC and [20–50 mM TrisꢀHCl (pH 7.5–8.0), 5%
NMR measurements to evaluate a single compound, the initial glycerol, 0–250 mM imidazole, 0–500 mM NaCl, and 0.2%
screening of a fragment library of 1,000 compounds will take Triton Xꢀ100 or 0.2–0.8% βꢀoctylglucoside (Nacalai)] for huꢀ
one weak. When the rate of positive hit is low, we may use a man OSC. The protein concentrations were determined by
mixed sample. Thus, when we use mixtures of e.g., 10 comꢀ Pierce 660 nm protein assay reagent (Thermo Fisher Scienꢀ
pounds each, the time required for the initial screening becomes tific.).
within a single day.
NMR assay for enzymatic activity. The brief protocol was
Advantage of NMR biochemical assay for OSC. Optical illustrated as flow chart in Fig. 1A. The substrate 2,3ꢀ
detection systems including fluorescent system are most suitaꢀ oxidosqualene (SigmaꢀAldrich) was dissolved at the concentraꢀ
ble for biochemical assay in drug discovery in terms of tion of 100 ꢂM in the reaction buffer [10 mM sodium phosphate
throughput, although they are not applicable to OSC currently. (pH 7.0), 0.1% Triton Xꢀ100, 100 ꢂM sodium 2,2ꢀdimethylꢀ2ꢀ
1
In such a situation, H NMR detection is a powerful alternative, silapentaneꢀ5ꢀsulfonate (DSS; SigmaꢀAldrich), and 5% dimeꢀ
which is sensitive to almost all chemical conversions.
thylsulfoxideꢀd6 (DMSOꢀd6; Cambridge Isotope Laboratories
One of the merits of the present NMR method is that we do Inc., Andover, MA)]. Reactions were initiated by adding enꢀ
not need a radioactive substrate, in contrast to the conventional zyme to a final concentration of 19 nM, which were kept at 37
1, 2, 4, 20, 21, 23, 28
assays
. Therefore, experiments can be carried out °C in a thermal cycler (Biometra, Göttingen, Germany) for 10–
without care for exposure to radiation. Also, in an NMR specꢀ 80 min, and stopped by adding 5ꢀfold larger volumes of 10:1
trum, protons of different molecules exhibit peaks in different mixture of methanol and 99% D O (Shoko, Tokyo, Japan).
2
positions, although some of them overlap with each other. As far Samples were set in the cartridge of 96 tubes for a SampleJet
as we identify separate peaks originating from the molecule of automatic sample changer (Bruker BioSpin, Rheinstetten, Gerꢀ
1
interest, we can deal with samples mixed with other molecules, many). H NMR measurements of reaction solutions were carꢀ
such as detergent. Therefore, we do not need chromatographic ried out on an Avance III 500 spectrometer (Bruker BioSpin;
separation or saponification also in contrast with the convenꢀ 500.13 MHz) at 25 °C, automatically by ICONꢀNMR program
1, 2, 4, 20, 21, 23, 28
29ꢀ31
tional assays
or GC(ꢀMS) assays
.
module, where water and methanol signals were suppressed by
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ꢀ9ꢀ19 pulse sequence with the field gradient and presaturaꢀ
For simultaneous evaluation of inhibitory activities of a series
of compounds (Fig. 1C), the spectra obtained for different inhibꢀ
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tion, respectively.
The region of the spectra including the peaks of lanosterol itor compounds were composed as data matrix D and FA calcuꢀ
was processed by means of FA , as described previously . lation was carried, as above.
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33
Briefly, a series of spectral data obtained with different reaction
Antiprofileration and parasiticidal activities. For evaluation
times were composed as data matrix D, with rows representing of antiprofileration effects, one million of epimastigote culture
the chemical shifts and columns representing the reaction times. form of T. cruzi, Tulahuen strain, obtained through NEKKEN
In D, the intensity was meanꢀcentered along with the time axis Bioꢀresource Center (Nagasaki, Japan) in LIT culture medium
and data points with change in the intensity less than twice the supplemented with 10% newborn calf serum were coꢀincubated
rootꢀmeanꢀsquare (rms) noise were excluded. Eigensystem with the test compound. After culture, alamarBlue test solution
analysis of covariance matrix
(Thermo Scientific) was added to the media, for which fluoresꢀ
cence intensity was monitored to evaluate the viability.
For evaluation of parasiticidal activity, purified trypoꢀ
mastigote parasites were applied to a 24ꢀwell plate confluently
covered by the NIH 3T3 fibroblast, and incubated for two days.
T
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4
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6
7
8
9
0
1
2
3
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7
8
9
0
Z = D D
(1)
T
where D is the transposed matrix of D, was carried out in orꢀ
der to decompose D as
D = R C + E
(2)
In equation 2, R is the row matrix, with rows representing the Then the residual floating trypomastigotes were washed out.
chemical shifts and columns representing the factors, C is the After culture for 5 days, the test compound was added and incuꢀ
column matrix with row representing the factors and column bated for 5 days, and for another 10 days with a fresh comꢀ
representing the reaction times, and E is the minimized error pound. After washing out the compound, the duration until a
matrix. Note that matrix C is composed of its row vectors that new trypomastigote emerged from the infected cells was obꢀ
are identical with the eigenvectors of Z. The column vectors of served. As a positive control, benznidazole was used.
R show spectral profile for the factors, here called loading vecꢀ
tors, while the row vectors of C show time dependence of the ASSOCIATED CONTENT
factors, here called score vectors. In Figs 2–4, the score vectors,
which are originally unit vectors, are multiplied by root of the Supporting Information
respective eigenvalues, in order to express the degree of contriꢀ
bution. In order to obtain the kinetic constants of the reaction,
the score vector of the first factor, with the largest eigenvalue,
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
was fitted to an exponential curve.
Inhibitors. Compounds 1–4 were derived from the Astellas’
chemical compound library. The synthetic routes were described
in Supporting Information. The purities of the compounds were
Methods for chemical syntheses and supplementary figures illusꢀ
trating chemical structure and an NMR spectrum of lanosterol (Fig.
S1) and NMR analyses on human OSC (Figs. S2–4) (PDF)
evaluated by ultraꢀhigh performance liquid chromatography to
be higher than 95%, also as described in Supporting Inforꢀ
mation.
Molecular Formula Strings (CSV)
NMR assay for inhibitor activity. The flow chart for the inꢀ
hibitor assay was shown in Fig. 1B. The solutions as described AUTHOR INFORMATION
above except for supplement of various concentrations of inꢀ
hibitors (0–100 ꢂM) were placed in the wells of a 96ꢀwell plate
designed for PCR. Reactions were initiated by adding 19 nM of
enzyme at 37 °C, kept in a thermal cycler (Biometra) for 20 min
for T. cruzi OSC or 90 min for human OSC, and stopped by
Corresponding Author
*
Phone: +81-29-8619473. Fax: +81-29-8612706. E-mail: k-
yamasaki@aist.go.jp
Author Contributions
adding 5ꢀfold larger volumes of 10:1 mixture of methanol and
1
The manuscript was written through contributions of all authors.
9
9% D O (Shoko). 1D H NMR measurements were carried out
2
at 25 °C as above.
The spectra obtained at different inhibitor concentrations were
composed as data matrix D and FA calculation was carried out The authors declare no completing interest.
Notes
as described above. To estimate IC , the score vector with the
5
0
ACKNOWLEDGMENT
largest eigenvalue was fitted simultaneously with
ꢂꢃꢄ
Y = Aꢀꢀꢁ
+ B
(3)
(4)
This project was supported by Leading Engine program for
Accelerating Drug discovery (LEAD) of AIST. We thank mem-
bers of the Consortium for Neglected Tropical Diseases partic-
ipating from Astellas Pharma Inc., Drugs for Neglected Diseas-
and
ꢃ
ꢆ
[ꢉ]
ꢉꢊꢋꢆ
ꢀ
ꢀꢅ = ꢇꢈ
where Y is the intensity in the score vector; V is the rate at difꢀ es initiative (DNDi), Tokyo Institute of Technology, High En-
ferent inhibitor concentrations; t is the fixed reaction time; A ergy Accelerator Research Organization, AIST, Nagasaki Uni-
and B are variables for fitting, together with IC ; V is the rate versity, and University of Tokyo for helpful discussions and
5
0
0
without inhibitor, which was determined in advance, as deꢀ advice.
scribed above; and [I] is the concentration of inhibitor. Note that
4
2
equation 4 is deduced from Cheng and Prusoff . The error level ABBREVIATIONS USED
for IC50 was estimated by a Monte Carlo simulation that ranꢀ
domizes the data as Gaussian distributions with the standard
deviation of the initial fitting residuals.
DMSO, dimethylsulfoxide; FA, factor analysis; DSS, sodium 2,2ꢀ
dimethylꢀ2ꢀsilapentaneꢀ5ꢀsulfonate; FBDD, fragmentꢀbased drug
discovery; GCꢀMS, gas chromatographyꢀmass spectrometry; IC50,
the inhibitor concentration causing 50% inhibition; OSC, oxꢀ
4
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idosqualene cyclase; rms, root mean square; TLC, thin layer chroꢀ (22) Benaim, G.; Sanders, J. M.; GarciaꢀMarchan, Y.; Colina, C.; Lira, R.;
Caldera, A. R.; Payares, G.; Sanoja, C.; Burgos, J. M.; LeonꢀRossell, A.;
matography
1
2
3
4
5
6
7
8
9
Concepcion, J. L.; Schijman, A. G.; Levin, M.; Oldfield, E.; Urbina, J. A.
Amiodarone has intrinsic antiꢀTrypanosoma cruzi activity and acts
synergistically with posaconazole. J. Med. Chem. 2006, 49, 892ꢀ899.
(
23) Balliano, G.; Dehmlow, H.; OliaroꢀBosso, S.; Scaldaferri, M.;
Taramino, S.; Viola, F.; Caron, G.; Aebi, J.; Ackermann, J. Oxidosqualene
1) Corey, E. J.; Russey, W. E.; Ortiz de Montellano, P. R. 2,3ꢀ cyclase from Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis
Oxidosqualene an intermediate in biological synthesis of sterols from carinii and Arabidopsis thaliana expressed in yeast: a model for the
squalene. J. Am. Chem. Soc. 1966, 88, 4750ꢀ4751.
development of novel antiparasitic agents. Bioorg. Med. Chem. Lett. 2009,
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conversion of squalene 2,3ꢀoxide to lanosterol and cholesterol. J. Am. Chem. (24) Pyrah, I. T.; Kalinowski, A.; Jackson, D.; Davies, W.; Davis, S.;
Soc. 1966, 88, 4752ꢀ4754.
Aldridge, A.; Greaves, P. Toxicologic lesions associated with two related
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sterol composition of Trypanosoma (Schizotrypanum) cruzi epimastigotes (36) Erlanson, D. A.; McDowell, R. S.; O'Brien, T. Fragmentꢀbased drug
by delta 24(25)ꢀsterol methyl transferase inhibitors and their combinations discovery. J. Med. Chem. 2004, 47, 3463ꢀ3482.
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Molina, J.; Piras, M.; Piras, R.; Perez, N.; Wincker, P.; Loebenberg, D.
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40) Mayer, M.; Meyer, B. Group epitope mapping by saturation transfer
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(
41) Piotto, M.; Saudek, V.; Sklenar, V. Gradientꢀtailored excitation for evaluation of inhibitors, which yields IC50 values, and (C) simultaꢀ
singleꢀquantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR
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neous evaluation of inhibitory activities of a series of compounds,
applicable to screening of the fragment library.
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(42) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant
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(I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099ꢀ3108.
Table 1: IC50 values of inhibitor compounds against T. cruzi
and human OSCs and proliferation of epimastigote (nM)
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
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Compounds
Source of the enzyme Ratio of IC50
Antiꢀ
proliferaꢀ
tion
T. cruzi
human
(human/
T. cruzi)
a
Figure 2. NMR analysis on the enzymatic activity of T. cruzi OSC.
(A) NMR spectra of reactants after the protocol shown in Fig. 1A.
A spectral region contains the NMR peaks originating from the
product, lanosterol (28ꢀH at 0.984 ppm and 19ꢀH at 0.963 ppm).
Reactions were conducted at 37 °C for 10 (red), 20 (magenta), 30
1
2
3
4
(Ro 48ꢀ8071)
13±3
42±12
44±12
140±50
7.5±0.8
16±3
0.59
0.38
2.7
N. D.
110
120±20
230±40
2150
a
1.6
N. D.
(orange), 40 (yellow), 50 (green), 60 (cyan), 70 (blue), and 80
a
Not determined.
(black) min. (B) FA loading vectors, representing the spectral proꢀ
file, with lengths equal to the root of respective eigenvalues. Those
of factors relevant for the five largest eigenvalues are selected; the
first (black), second (red), third (blue), fourth (green) and fifth (yelꢀ
low) factors ordered by eigenvalue. NMR intensity was normalized
to rms noise. (C) FA score vectors, which represent the time deꢀ
pendence, also with lengths equal to the root of respective eigenꢀ
values. Coloring scheme is the same as in (B). Exponential curve
fitting for that of the first factor yields a kinetic constant of 0.083
Table 2: Parasiticidal activity evaluated by durations until deꢀ
tection of trypomastigote (days)
Compounds
Concentration of compounds
a
b
~
10 ꢂM
~20 ꢂM
2
62±6
21±0
35±8
80±11
29±0.4
53±9
–
1
min .
3
benznidazole
a
9.6, 10.9, and 9.6 ꢂM for 2, 3, and benznidazole, respectively.
Twice the concentration of ~10 ꢂM.
b
Figure 3. Inhibitor compounds evaluated in the present study.
Compound numbers are the same as those in Table 1.
Figure 4. Inhibition of T. cruzi OSC by Ro 48ꢀ8071 (compound 1).
(A) NMR spectra of reactants after the protocol shown in Fig. 1B.
As in Fig. 2, the spectral region containing the NMR peaks of
lanosterol was shown. Reactions were carried out at 37 °C for 20
min in the absence (black) or presence of different concentrations
of compound 1, i.e., 0.0017 (blue), 0.051 (cyan), 0.015 (green),
0.046 (yellow), 0.14 (orange), 0.41 (magenta), and above 1.0 ꢂM
(1.2, 3.7, 11, 33, and 100 ꢂM: red). (B) FA loading vectors repreꢀ
senting the spectral profile. (C) FA score vectors representing the
dependence on inhibitor concentrations. Curve fitting of that of the
Figure 1. Protocols of the NMR biochemical assays for OSC: (A)
evaluation of enzymatic activity, which yields kinetic constants, (B)
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first factor with equations 3 and 4 yields an IC50 value of 13 nM. In
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(B) and (C), schemes for color and normalization of the intensities
were the same as those in Figure 2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
1
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3
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9
0
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3
4
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6
7
8
9
0
Figure 5. A highꢀthroughput analysis on inhibition of T. cruzi OSC
by a series of compounds. (A) NMR spectra of reactants after the
protocol shown in Fig. 1C. Reactions were carried out at 37 °C for
20 min in the absence (black) or presence of either of 4 compounds
shown in Fig. 3 at 0.41 ꢂM (1: red; 2: yellow, 3: green, 4: blue). (B)
FA loading vectors representing spectral profile. (C) FA score vecꢀ
tors indicating inhibition profile of compounds. The compound
numbers are the same as those in Fig. 3 and Table 1. In (B) and (C),
schemes for color and normalization in intensity were the same as
those in Figs. 2 and 4, although the first three factors are shown
here. (D) IC50 values of the same compounds.
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