Structural basis for rational design of inhibitors targeting Trypanosoma cruzi Sterol 14α-demethylase: Two regions of the enzyme molecule potentiate its inhibition

Article abstract of DOI:10.1021/jm500739f

Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global, becoming a new worldwide challenge with no cure available. The disease is caused by the protozoan parasite Trypanosoma cruzi, which depends on the production of endogenous sterols, and therefore can be blocked by sterol 14α-demethylase (CYP51) inhibitors. Here we explore the spectral binding parameters, inhibitory effects on T. cruzi CYP51 activity, and antiparasitic potencies of a new set of β-phenyl imidazoles. Comparative structural characterization of the T. cruzi CYP51 complexes with the three most potent inhibitors reveals two opposite binding modes of the compounds ((R)-6, EC₅₀ = 1.2 nM, vs (S)-2/(S)-3, EC₅₀ = 1.0/5.5 nM) and suggests the entrance into the CYP51 substrate access channel and the heme propionate-supporting ceiling of the binding cavity as two distinct areas of the protein that enhance molecular recognition and therefore could be used for the development of more effective antiparasitic drugs.

Full text of DOI:10.1021/jm500739f

Article
pubs.acs.org/jmc
Open Access on 07/17/2015
Structural Basis for Rational Design of Inhibitors Targeting
Trypanosoma cruzi Sterol 14α-Demethylase: Two Regions of the
Enzyme Molecule Potentiate Its Inhibition
Laura Friggeri,†,# Tatiana Y. Hargrove,† Girish Rachakonda,‡ Amanda D. Williams,‡ Zdzislaw Wawrzak,§
Roberto Di Santo,∥ Daniela De Vita,∥ Michael R. Waterman,† Silvano Tortorella,∥ Fernando Villalta,‡
and Galina I. Lepesheva*,†,⊥
†Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232, United States
‡Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee 37208, United States
§Synchrotron Research Center, Life Science Collaborative Access Team, Northwestern University, Argonne, Illinois 60439, United
States
∥“Istituto Pasteur-Fondazione Cenci Bolognetti”, Department of “Chimica e Tecnologie del Farmaco”, Sapienza University of Rome,
Rome, 00185, Italy
⊥Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232, United States
S
* Supporting Information
ABSTRACT: Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone
global, becoming a new worldwide challenge with no cure available. The disease is caused by the protozoan parasite Trypanosoma
cruzi, which depends on the production of endogenous sterols, and therefore can be blocked by sterol 14α-demethylase (CYP51)
inhibitors. Here we explore the spectral binding parameters, inhibitory effects on T. cruzi CYP51 activity, and antiparasitic
potencies of a new set of β-phenyl imidazoles. Comparative structural characterization of the T. cruzi CYP51 complexes with the
three most potent inhibitors reveals two opposite binding modes of the compounds ((R)-6, EC50 = 1.2 nM, vs (S)-2/(S)-3, EC50
= 1.0/5.5 nM) and suggests the entrance into the CYP51 substrate access channel and the heme propionate-supporting ceiling of
the binding cavity as two distinct areas of the protein that enhance molecular recognition and therefore could be used for the
development of more effective antiparasitic drugs.
lack of awareness and diagnostics in nonendemic areas, often
leading to transfusion of infected blood; transplantation of
infected organs, food, and drink contaminations; as well as HIV
coinfections and congenital transmission.5,6 Thus, recent
estimates indicate that there could be up to 1 million cases of
Chagas disease in the USA, a significant portion of which,
particularly in the southern states, are of autochthonous vector-
borne origin.7,8
INTRODUCTION
■
American trypanosomiasis (Chagas disease) is a vector-borne
anthropozoonosis, the life-long infection caused by the
protozoan pathogen Trypanosoma cruzi.1 For centuries the
disease has been a major cause of mortality and morbidity in
South and Central America, where it still remains endemic in
21 countries, resulting in about 14,000 deaths per year,2 mainly
due to heart failure, that is the most typical pathology of the
chronic form of this infection. During the last years, Chagas
disease has begun receiving attention as an emerging global
medical problem,3,4 predominantly because of human and
insect vector (kissing bug) migration, but also as a result of the
Received: May 12, 2014
Published: July 17, 2014
© 2014 American Chemical Society
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Figure 1. VNI and VNF. (A) Structural formulas. (B) Orientation in the CYP51 active site. Distal P450 view. VNI, VNF, and the T. cruzi CYP51
substrate eburicol are shown in blue, red, and green, respectively. The carbon atoms of the heme are colored in gray. Helix C and the β4-hairpin are
outlined as semitransparent gray ribbons.
Despite the severity of the problem and ∼25 million people
at risk of infection, nifurtimox and benznidazole are the only
drugs available for treatment of Chagas disease. Although
generally rather helpful in the acute stage, they vary significantly
in their efficiency against different T. cruzi strains,9 they have
considerable adverse side effects, and their success in curing
chronic Chagas disease is still debated.10,11 Despite these
limitations, pharmaceutical companies remain reluctant to
invest resources in the development of new antichagasic
chemotherapies, because of the lack of assurances that they can
make a return on their investment, since historically Chagas
disease has been known to mostly affect the poorest. As a result,
the majority of efforts to fill the gap for new antichagasic drugs
have come from academia. Repurposing of antifungal azoles,
the drugs that act via sterol biosynthesis by inhibiting the
cytochrome P450 enzyme sterol 14α-demethylase (CYP51)12
so far have been particularly successful (reviewed in refs 1 and
13). The FDA-approved drug posaconazole and an investiga-
tional prodrug of ravuconazole (Eisai) are presently in phase 2
clinical trials for Chagas.3 Some other azole derivatives, e.g. the
anticancer drug candidate tipifarnib14,15 or (S)-2-(5-((1-
(biphenyl-4-ylmethyl)-1H-imidazol-5-yl)methylamino)-
biphenyl-2-ylcarboxamido)-4-(methylthio)butanoic acid (FTI-
2220),16 were also shown to display potent antiparasitic effects
and are under development. Later, several new experimental
heterocyclic compounds, both azoles17,18 and pyridines19,20
were identified as potent and selective inhibitors of T. cruzi
CYP51 (the protein has less than 25% amino acid sequence
identity to its fungal orthologs21) and structurally characterized
in complex with the target enzyme.18,20,22,23 Most recently, one
of these inhibitors, VNI, has been shown to cure both the acute
and chronic forms of Chagas disease in mice.24
substrate access channel, the 2-ring arm of VNF is positioned
within the deepest segment of the CYP51 binding cavity, the
hydrophobic area that accommodates the aliphatic tail of the
sterol substrate (Figure 1B).
In this work we prepared a set of 12 β-phenyl imidazoles and
analyzed their structure−activity relationship in terms of T.
cruzi CYP51 binding parameters, inhibition of reconstituted
enzymatic reaction in vitro, and antiparasitic effects against
GFP-expressing T. cruzi amastigotes. Three most efficient
compounds were cocrystallized with T. cruzi CYP51, the X-ray
costructures uncovering two basic approaches that can be
utilized to further enhance potencies of CYP51 inhibitors.
RESULTS AND DISCUSSION
■
Medicinal Chemistry. Compound 1 (MW 368, clogP 5.3,
tPSA 41.9 (ChemDraw)) has been previously characterized as a
potential antifungal agent and revealed quite promising
results.25 Its structural resemblance to VNF has prompted us
to expand our work on this inhibitory chemotype by modifying
the chemical structure of 1 as shown in Table 1 followed by
testing the original molecule and its derivatives against T. cruzi
and its potential target enzyme T. cruzi sterol 14α-demethylase.
In all cases the polar linker between the phenethylimidazole
moiety and the opposite arm of the new structures was replaced
with the carbamate group, because it was previously found to
have higher hydrolytic stability in liver microsomes than the
ester group of 1.26 Our major focus on modification of the side
chain arm of the compounds was based on the observation that
variations in the composition of this portion of a β-phenyl
imidazole molecule (a) could be crucial for its potency to
inhibit CYP51 activity17 and (b) may alter its orientation within
the enzyme active site.23 In compounds 2 (MW 384, clogP 4.9,
tPSA 53.9) and 3 (MW 367, clogP 4.3, tPSA 53.9), the side
chain arm is one aromatic ring shorter than it is in 1, and in the
para-position of the β-phenyl ring they have either Cl atom (2)
or smaller and more polar F atom (3). Compound 4 (MW 357,
clogP 1.8, tPSA 69.5), similarly to 3, also has fluorine in the
para-position of the β-phenyl ring; however, its side chain arm,
instead of the bulky aromatic ring, carries a flexible three-
carbon atom aliphatic chain ending with the polar imidazole
ring. The arm of compound 5 (MW 494, clogP 5.6, tPSA
Being the most potent T. cruzi CYP51 inhibitors that we
have discovered,17 VNI and VNF share high structural
similarity. The phenethylimidazole portion of these molecules
is connected via the polar linker (carboxamide fragment) to the
lipophilic arm that consists of either a 2-ring (VNF) or a 3-ring
(VNI) linear polycycle (Figure 1A). In the CYP51 costructures,
VNI and VNF are coordinated to the P450 heme iron through
their imidazole ring nitrogen (N3). The other two portions of
the inhibitor molecules, however, adopt an opposite
orientation:23 while the 3-ring arm of VNI lies in the CYP51
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Table 1. Structural Formulas, T. cruzi CYP51 Spectral Binding Parameters, Inhibition of Enzymatic Activity, and Antiparasitic
a
Effects of Compounds 1−6
a
b
The values represent mean standard deviation from three independent experiments. 1 h reaction (examples of HPLC profiles for compounds 2
c
d
and 6 are shown in Figure 3). The corresponding values for posaconazole are EC50 = 5.0 nM ; inhibition at 10 nM = 64%. Compounds
cocrystallized with T. cruzi CYP51.
105.5) bears two aromatic rings linked via the sulfur atom, with
the distal ring ending with a nitro group in the para-position.
Compound 6 (MW 605, clogP 7.2, tPSA 60.4) is the largest of
the molecules. It has two chlorine substituents in the ortho- and
para-positions of the β-phenyl ring, while its long arm is
composed of three rings linked in a linear sequence, with the
distal aromatic ring being complemented with two chlorine
atoms, in the 3- and 4- positions so that the total length of the
arm (∼18 Å) is close to the length of the CYP51 substrate
access channel.22
In this study the compounds were prepared as (R)- and (S)-
stereoisomers using enantioselective synthesis.26 Briefly, the 2-
(1H-imidazol-1-yl)-1-phenylethanones were obtained by con-
densation of 1H-imidazole and commercial bromoacetophe-
nones25 substituted in the ortho/para position with different
halogens (Scheme 1a). In order to obtain the (S)-2-(1H-
imidazol-1-yl)-1-phenylethanols, the keto group of 2-(1H-
imidazol-1-yl)-1-phenylethanones was reduced to hydroxyl
using as the catalyst RuCl(p-cymene)[(R,R)-Ts-DPEN]
(Scheme 1b). Accordingly, RuCl(p-cymene)[(S,S)-Ts-DPEN]
has been used as the catalyst to prepare the (R)-2-(1H-
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a
Scheme 1. Enantioselective Synthesis of (S)-Stereoisomers of Compounds 2−6
a
Reagents and conditions. (a) DMF, 0 °C, 2 h; (b) CH2Cl2, RuCl(p-cymene)[(R,R)-Ts-DPEN], HCOOH, TEA, N2(g), 40 °C, 26 h; (c) anhydrous
CH3CN, NaH, 2 h, room temperature; (d) anhydrous CH3CN, triphosgene, room temperature; overnight; (e) 4-isopropylphenyl isocyanate, room
temperature, 48 h; (f) TEA, R2-NH2, room temperature, overnight.
a
Scheme 2. Synthesis of the Long Arm of Compound 6
a
Reagents and conditions. (a) CH3CN, reflux, 2 h; (b) MeOH, H2, Pd/C, 50 psi, room temperature, 4 h; (c) MeOH, HCl(g).
.
imidazol-1-yl)-1-phenylethanols. The −OH group was sub-
sequently deprotonated with sodium hydride (c) or activated
with triphosgene (d) in anhydrous CH3CN in order to obtain
sodium alkoxide and chloroformate. Then commercial 4-
isopropylphenyl isocyanate, 2-(1H-imidazol-1-yl)propan-1-
amine and 4-((4-nitrophenyl)thio)aniline have been added
for preparing the side chain arms of compounds 2/3, 4, and 5,
respectively. (Scheme 1 e,f).
The long arm of compound 6 has been synthesized as shown
in Scheme 2. The synthesis was performed by condensation of
1-fluoro-4-nitrobenzene with 1-(3,4-dichlorophenyl)piperazine
(a). Then the nitro group of 1-(3,4-dichlorophenyl)-4-(4-
nitrophenyl)piperazine was converted to an amino group by
reduction with H2 using Pd/C as the catalyst (b) to produce 4-
[4-(3,4-dichlorophenyl)piperazin-1-yl]aniline; and the final
amine was stabilized as 4-[4-(3,4-dichlorophenyl)piperazin-1-
yl] anilinium chloride by HCl(g) flow in the same reaction
environment (c).
T. cruzi CYP51 Spectral Responses vs Inhibition of
Reconstituted Sterol 14α-Demethylase Activity in Vitro.
In the resting ferric form the heme iron of CYP51 enzymes is
present in the low spin state, with a water molecule serving as
its sixth axial (distal) ligand. This results in the P450
absorbance spectrum with the Soret band maximum at around
417 nm. Binding of azoles or other heterocyclic compounds
replaces the water molecule in the heme iron coordination
sphere with the basic nitrogen atom, causing a so-called red
shift in the Soret band maximum. Accordingly, in the difference
spectra a trough and a peak appear on the left and the right
sides of an isosbestic point. These spectral changes, also known
as a type 2 spectral response, are widely used to identify new
P450 binding ligands. However, even very low spectral
dissociation constants do not necessarily reflect the com-
pound’s potency to inhibit CYP51 activity, because during the
reaction many ligands can still be replaced in the enzyme active
center by the substrate.17,20,22
Twelve imidazole derivatives tested in this study act in good
agreement with this observation. As expected, all of them cause
typical type 2 spectral responses in the CYP51 heme iron, with
the apparent spectral dissociation constants (Ks) being mostly
in the nanomolar range, and therefore should be defined as
tight binding ligands (Table 1, Figure 2).
However, particularly in the case of compounds 1−3, the
inhibitory effects of the (R)- and (S)-enantiomers on the
CYP51 activity appeared to be quite “independent” of the
binding parameters. Thus, (S)-2 displayed only 2-fold higher
apparent binding efficiency than (R)-2, yet its inhibitory effect
on the substrate conversion is ∼50-fold stronger (Table 1,
Figure 3A). The same tendency was observed for 1 and 3. In all
these three cases, the (S)-enantiomers are much more potent as
T. cruzi CYP51 inhibitors than the (R)-enantiomers. The (R)-
and (S)- enantiomers of compounds 4−6, on the opposite, do
not differ that drastically in their potencies to inhibit T. cruzi
CYP51 reaction (Table 1, Figure 3B), although significant
variations can be seen in the apparent Ks’s, particularly for 4
((S)- ≫ (R)-). Only the enantiomers of compound 6 presented
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Figure 2. Spectral responses of T. cruzi CYP51 to the binding of (A) (R)- and (S)- enantiomers of compound 2; [P450] = 1.1 μM and (B) (R)- and
(S)- enantiomers of compound 6; [P450] = 0.9 μM. Optical path length 5 cm. Upper: absolute absorbance spectra, the Soret band maximum shifts
to the right (from 418 to 425 nm). Lower: difference spectra upon titration with the ligands (titration step 0.2 μM). Insets: titration curves showing
absorbance changes per 1 cm optical path/1 nmol P450 upon increasing ligand concentrations (processed with the Morrison equation).
rather good correlation between the CYP51 inhibition and
binding parameters. Nevertheless, their spectral dissociation
constants are either higher ((S)-, Ks = 0.123 μM) or within
((R)-, Ks = 0.064 μM) the range of the values calculated for the
(R)- enantiomers of compounds 1−3 (weak T. cruzi CYP51
inhibitors).
potencies of CYP51 inhibitors as well as to shed light on the
features that may have caused the observed peculiarities in their
binding behavior, we crystallized T. cruzi CYP51 in the
presence of (S)-2, (S)-3, and (R)-6, which are among the
strongest inhibitors of the enzyme identified in this study
(Table 1), and we determined the X-ray structures of their
complexes. Table 2 summarizes the diffraction and refinement
statistics. As expected, (R)-6 (ligand PDB ID LFD, molecular
volume 1,040 Å3, surface area 880 Å2) adopts a VNI-like
orientation, with its longer arm protruding ∼7 Å further toward
the entrance into the substrate access channel (Figure 5A),
while the vectors of (S)-2 (ligand PDB ID LFT, molecular
volume 870 Å3, surface area 725 Å2) and (S)-3 (ligand PDB ID
LFS, molecular volume 849 Å3, surface area 709 Å2) follow that
of VNF, being directed toward helix C (Figure 5B). The amino
acid residues that contact each inhibitor are listed in Table 3,
and their location in the T. cruzi CYP51 structure is seen in
Figure 5.
Another unusual observation, which we encountered upon
titration of T. cruzi CYP51 with compound 6 (both (R)- and
(S)-enantiomers), was the very slow increase in the amplitude
of the type 2 spectral response over time. This increase was
most pronounced when the enzyme concentration considerably
exceeded the concentration of the ligand. To our knowledge,
such an increase has not been previously reported for any other
CYPs. Since none of the other compounds tested in this work
produced such an effect, we conducted the same experiments
with other known T. cruzi CYP51 inhibitors, and we found that
a somewhat similar pattern is also produced by ketoconazole,
another CYP51 inhibitor with a rigid long arm but not by VNI
(Figure 4). The current lack of any information on the topic
might be related to the fact that traditionally the measurements
of P450 spectral responses have been mostly conducted at
higher ligand/P450 molar ratios.
Overall, the structures suggest that the potency of LFD (20
inhibitor contacting residues) must be enhanced by the
formation of the surface binding subsite (Figure 6A−C,
Supporting Information Figure S1), the rearrangement that
was first observed in the structure of posaconazole-bound T.
cruzi CYP51 [3K1O].23 When two molecules of CYP51-LFD
Crystallographic Analysis. In order to expand our
understanding of the molecular basis that underlies the
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Figure 3. HPLC profiles of eburicol conversion by T. cruzi CYP51 in the presence of a 2-fold molar excess of the inhibitor over the enzyme. 1 h
reaction. (A) (R)- and (S)-Enantiomers of compound 2; (B) (R)- and (S)-Enantiomers of compound 6. S, substrate (4,4,14-trimethylergosta-8-en-
3ß-ol); I1, 14α-carboxyalcohol intermediate; I2, 14α-carboxyaldehyde intermediate; P, 14α-demethylated product (4,4-dimethylergosta-8,14-dien-
3ß-ol).
(this structure has two molecules in the asymmetric unit (Table
2)) are superimposed with CYP51-LFT and CYP51-LFS, it is
clearly seen that in the complex with LFD the secondary
structural elements that form the entrance into the CYP51
substrate access channel are getting better organized,
particularly helix F″, whose density in the complexes of T.
cruzi CYP51 with smaller molecules is either missing (VNF
[3KSW],23 fluconazole [3KHM],23 NEU [4H6O]18) or
appears to be more looplike (LFT and LFS (Figure 6C)).
Moreover, some residues, e.g. F214, P210 (F″), I45 (A′), I72
(β1-1), move 1−1.5 Å closer toward the LFD 1,2-
dichlorobenzene ring (Figure 6B), which, as a result, forms
van der Waals contacts with 8 amino acids around the channel
entrance (a possible example of a “localized” induced fit in
CYP51). These multiple contacts between the enzyme and
inhibitor are clearly decreasing the entrance flexibility
(Supporting Information Figure S2A) and may prevent the
CYP51 channel from opening, making it harder for the
substrate molecule to replace the inhibitor, e.g. upon P450
reduction. On the other hand, LFD does not induce any
significant rearrangements around the CYP51 heme binding
area, which might explain why the (R)- and (S)-enantiomers of
this compound would have similar inhibitory potencies and
display similar apparent binding efficiencies. The observed
time-dependent increase in the spectral response of the P450
heme iron to the binding of the LFD nitrogen might be
connected with some repositioning of the proximal to the heme
portion of the inhibitor molecule, as even in the crystal lattice
the two medium rings of this compound display obvious
differences in their conformations (Figure 6B,E).
Although only 16 of the T. cruzi CYP51 amino acid residues
lie at the distance <4.5 Å from LFT and LFS (Figure 5B, Table
3), which are much smaller molecules than LFD, their
complexes with the enzyme are strengthened by the H-bonds
with Y116. In both these structures the side chain hydroxyl of
Y116 looses its contact with the heme ring D propionate,27
shifts 1.5 Å toward the inhibitor, and interacts with the O and
N atoms of its carbamic fragment (Figure 6 E,F). This change
in the Y116 position increases the flexibility of the heme
(Supporting Information Figure S2B) and allows the p-cymene
moiety of the inhibitors to protrude deeper into the CYP51
binding cavity. Enhancement of the LFT/LFS complexes with
CYP51 by the H-bond formation elucidates the importance of
(S)-stereochemistry for the inhibitory potency of compounds 2
(LFT) and 3 (LFS), because the opposite orientation of the
carbamic fragment in the (R)-enantiomers is not favorable for
hydrogen bonding. It also provides a possible explanation for
the observed “discrepancies” between the spectral binding
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Figure 4. Time course of spectral responses of T. cruzi CYP51 (1.0 μM) to the addition of 0.2 μM of the heme-coordinating ligands.
parameters and inhibition (Table 1), since the H-bond
formation might not influence the spectral response of the
P450 heme iron to the imidazole nitrogen coordination, which
apparently occurs fast and easily for both (R)- and (S)-
enantiomers. Finally, combination of biochemical and structural
data implies that compounds 4 and 5 are most likely to bind to
T. cruzi CYP51 in the LFD orientation: (1) the arm of 5 (∼15
Å) is probably too long and the imidazole ring of 4 is too polar
to fit well into the hydrophobic deepest segment of the cavity,
(2) relatively small differences between the potencies of their
(R)- and (S)-enantiomers suggest the lack of H-bonding.
Antiparasitic Effects in T. cruzi Cells. T. cruzi is a
unicellular eukaryotic parasite with the complex life cycle
involving insect vectors and mammalian hosts. In insects,
multiplying midgut epimastigotes transform into metacyclic
trypomastigotes that infect humans. In humans, T. cruzi exists
either as infective bloodstream trypomastigote or as multiplying
intracellular amastigote (mostly in the heart muscles, but also in
lungs, liver, spleen, and other organs and tissues). In this work
the antiparasitic effects of the CYP51 inhibitors were analyzed
in T. cruzi amastigotes within cardiomyocytes. Because
compound 4 did not inhibit CYP51 activity in vitro, it was
not selected for cellular experiments. For the majority of the
other compounds, a very good correlation between their
potencies to inhibit T. cruzi CYP51 enzyme in the reconstituted
reaction in vitro and antiparasitic effects against the pathogen
cells has been observed (Table 1, Figure 7), although quite
pronounced activity of 5, both the (R)- and (S)- enantiomers,
might suggest that this structure can potentially have alternative
target(s), better cellular permeability, or perhaps even an
additional benznidazole-like mode of action (oxidative stress)
due to the presence of the reactive nitro group.28 Overall,
strong antiparasitic activity combined with simple and cost-
effective syntheses make these compounds, particularly T. cruzi
CYP51 inhibitors (S)-2 (LFT) and (R)-6 (LFD) as well as
compound (S)-5 (e.g., as an option for combination therapy)
potentially promising new antichagasic drug candidates, which
are worth testing in animal models.
CONCLUSIONS
■
By now T. cruzi sterol 14α-demethylase has been validated as
essential for survival and multiplication of T. cruzi. The
antifungal drugs posaconazole and ravuconazole may well pass
clinical trials and reinforce the scarce arsenal of antichagasic
chemotherapy. However, posaconazole is too expensive
(>1,000 euro per patient), and ravuconazole still has unresolved
problems with its bioavailability, which are most likely the
major reason for its suppressive but not curative effect.24 We
hope that VNI, or one of its derivatives,29 will also eventually
proceed to translational research. Nevertheless, additional
options for Chagas disease treatment are still highly needed,
and therefore better understanding of the target CYP51
structure/function and inhibition should be greatly advanta-
geous, particularly if the parasite acquires drug resistance upon
treatment or if humans are infected with strains of T. cruzi that
display natural drug resistance.30
This work, in addition to identifying new potential drug
candidates, also provides the structural rationale for the
compounds’ inhibitory potencies and offers two alternative
approaches that can be followed for further CYP51 structure-
guided drug design and development. One approach is to use
the high rigidity of the CYP51 substrate binding cavity. This
feature is best displayed by the LFT/LFS costructures. Both
these small molecules ensure their tight fit into the deepest
segment of the CYP51 cavity via formation of the hydrogen
bonding that not only strengthens the enzyme/inhibitor
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Table 2. Data Collection and Refinement Statistics
a
b
c
T. cruzi CYP51−inhibitor complex
(R)-6 (ligand PDB ID LFD)
(S)-2 (ligand PDB ID LFT)
(S) 3 (ligand PDB ID LFS)
Data Collection
Wavelength, Å
0.9787
0.9787
0.9787
Space group
P22(1)2(1)
P3(1)21
P3(1)21
Cell dimensions
a, b, c, Å
59.900; 137.180; 152.430
90.00, 90.00, 90.00
2
62.953; 62.953; 222.435
90.00, 90.00, 120.00
1
62.582; 62.582; 221.068
90.00, 90.00, 120.00
1
α, β, γ, deg
No. of molec in asymm. unit
Solvent content, %
Resolution (last shell), Å
Rmerge (last shell)
I/σ (last shell)
58.2
48.8
47.9
100−2.61 (2.67−2.61)
0.049 (0.558)
39.3 (2.9)
30−2.6 (2.69−2.6)
0.052 (0.675)
31.0 (3.0)
30−2.7 (2.8−2.7)
0.05 (0.583)
31.1 (3.4)
Completeness (last shell), %
Redundancy (last shell)
99 (99)
99 (99)
99.5 (100)
7.2 (5.6)
7.2 (7.3)
7.0 (7.2)
Refinement
Resolution, Å
30.0−2.62
0.235
30.0−2.74
0.272
28.8−2.7
0.263
0.284
13738
5.0
R-factor
R-free
0.286
0.296
Reflections used
36336
5.0
13383
5.2
Test set size, %
rms deviations from ideal geometry
Bond lengths, Å
0.005
1.14
0.001
0.93
0.003
1.28
Bond angles, deg
Ramachandran plot
Residues in favorable regions (%)
Residues in allowed regions (%)
Outliers (%)
95.3
99.8
0.2
95.6
100
0
94.2
100
0
Model
No. of atoms (mean B-factor, Å)
7377 (69.7)
3562 (90.5)
A
3541 (90.9)
A
Number of residues per molecule
A/B
Protein
Heme
Ligand
Water
449 (71.8)/450 (71.5)
1 (30.2)/(30.1)
434 (88.1)
1 (67.5)
434 (91.1)
1 (56.2)
LFS 1 (72.7)
4 (68.1)
LFD 1 (75.9)/1 (81.9)
24 (55.6)
LFT 1 (57.8)
25 (75.6)
a
b
c
PDB code 4CK8. PDB code 4CK9. PDB code 4CKA.
Figure 5. T. cruzi CYP51 active site illustrating interactions with LFD (cyan carbon atoms) and LFT (green carbon atoms). (A) Superimposition of
CYP51 complexes with LFD and VNI (blue); (B) Superimposition of CYP51 complexes with LFT and VNF (red).
interaction but also disrupts the heme support from the protein
moiety, thus affecting the environment (and most likely redox
potential) of the catalytic iron. The second approach is to block
the entrance into the CYP51 substrate access channel by
building a surface binding subsite (as, e.g., in the T. cruzi
complex with LFD or posaconazole31), since even weak van der
Waals contacts between the enzyme and inhibitor in this area
appear to prevent the substrate access channel from opening,
the feature that must be functionally essential for sterol 14α-
demethylase catalysis and therefore for the possibility of the
substrate to replace the inhibitor in the enzyme active site.
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Table 3. T. cruzi CYP51 Substrate Binding Cavity and Inhibitor-Contacting Residues
T. cruzi CYP51 active site
inhibitor-contacting residues (<4.5 Å)
amino acid
residues
posaconazole
(3K10)23
(R)-6 [LFD]
(4CK8)
(S)-2 [LFT]
(4CK9)
(S)-3 [LFS]
(4CKA)
VNF
secondary structural elements
(3KSW)23
A′ helix (substrate access channel)
I45
V46
I45
V46
F48
G49
I45
F48
F48
G49
K50
P52
G49
β1-1/β1-2 (substrate access
channel)
I70
I72
I72(4.8 Å)
V77
B′ helix (substrate binding cavity)
V102
Y103
I105
M106
F110
V114
A115
Y116
V102(4.7 Å)
Y103
Y103
I105
Y103
Y103
M106
F110
M106
F110
M106
F110
M106
F110
M106
F110
B′/C loop (substrate binding
cavity)
A115
Y116
M123
Q126
L127
L130
A115
Y116
M123
Q126
L127
L130
A115
Y116
Y116
L127
Y116
L127
C helix (substrate binding cavity)
F″ helix (substrate access channel)
I helix (substrate binding cavity)
Q126
L127
L130
P210
A211
V213
F214
M284
I285
Q126
L127
L130
P210
A211
V213
F214
P210
A211
V213
F214
M284
M284
M284
V286
A287
F290
A291
H294
T295
L356
L357
M358
V359
M360
R361
Y457
H458
T459
M460
V461
V462
A287
F290
A291
A287
F290
A291
A287
F290
A291
A287
F290
A291
A287
A291
T295
L356
L357
M358
T295
L356
T295
L356
T295
L356
T295
L356
β4-1 (substrate binding cavity)
β4 hairpin (substrate access
channel)
Y457
H458
T459
M460
M460
M460
M460
16
M460
V461
Total no. of residues
25
19(20)
16 (17)
14
with an ESI source and a syringe pump; the flow rate was 5 μL min−1.
The examined compounds were dissolved in methanol (10−5 M), and
aqueous HCl was added just before the injection. The molecular peaks
(m/z) have been observed as [M + H]+. The stereochemistry of the
(R)- and (S)-enantiomers as well as their enantiomeric excess (e.e.)
was evaluated by chiral HPLC using a 250 mm × 4.6 mm i.d. Chiralcel
OD column (Chiral Technologies Europe, Illkirch, France).26 The
HPLC apparatus consisted of a PerkinElmer (Norwalk, CT, USA) 200
LC pump equipped with a Rheodyne (Cotati, CA, USA) injector, a 20
mL sample loop, an HPLC Dionex CC-100 oven (Sunnyvale, CA,
USA), and a Jasco (Jasco, Tokyo, Japan) model CD 2095 Plus UV/
CD detector.
EXPERIMENTAL SECTION
■
Chemical Synthesis. All reagents and solvents were of high
analytical grade and were purchased from Sigma-Aldrich (Milano,
Italy). Melting points were determined on a Tottoli apparatus (Buchi)
and are uncorrected. Infrared spectra were recorded on a Spectrum
One ATR Perkin Elmer FT-IR spectrometer. 1H and 13C NMR
spectra were acquired on a Bruker AVANCE-400 spectrometer at 9.4
T, in CDCl3, CD3OD, or DMSO-d6, at 27 °C; chemical shift values are
given in δ (ppm) relative to TMS as internal reference. Coupling
constants are given in Hz. Mass analysis was carried out with a 2000 Q
TRAP instrument (Applied Biosystems), a commercial hybrid triple-
quadrupole linear ion-trap mass spectrometer (Q1q2QLIT), equipped
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Figure 6. Two regions of the T. cruzi CYP51 molecule that strengthen the enzyme interaction with LFD (A−C) and LFT/LFS (D−F). Coloring:
(yellow, cyan) complexes with LFD, molecules A and B, respectively; (green) complex with LFT; (pink) complex with LFS. (A,D) Semitransparent
surface representation (P450 distal view). (B) Structural elements forming the substrate access channel entrance. (C) F″-helix. (E) Structural
elements forming the deepest portion of the CYP51 binding cavity. (F) H-bonds between Y116 and the carbamic fragments of LFT and LFS.
Figure 7. Fluorescence microscopic observation of T. cruzi multiplication inside cardiomyocytes treated with 4 nM of CYP51 inhibitors. Drugs were
added to infected cardiomyocyte monolayers at 24 h of infection, and amastigote multiplication was observed at 72 h of infection. GFP-expressing T.
cruzi amastigotes are green, cardiomyocyte nuclei are blue, and cardiomyocyte actin myofibrils are red.
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Declaration of Purity. The purity of all the compounds,
determined by combustion elemental analysis and reverse-phase
HPLC, was >95%. Elemental analysis was performed using a PE
2400 (PerkinElmer) analyzer, and the analytical results were within
0.4% of the theoretical values. The reverse-phase HPLC system was
equipped with a dual-wavelength UV 2489 detector (Waters) set at
250 and 205 nm and a Symmetry C18 (3.5 μm) 4.6 mm × 75 mm
column. The mobile phase was 55% 0.015 M potassium phosphate
(pH 7.4) and 45% acetonitrile (v/v) with an isocratic flow rate of 1.0
mL/min.
137.9, 137.2, 133.0 (J = 2 Hz), 129.3, 128.9, 128.0 (J = 8 Hz), 119.8,
118.8, 115.8 (J = 22 Hz), 74.05, 51.9, 44.4, 38.2, 31.2. Anal. Calcd for
C18H20FN5O2: C, 60.49; H, 5.64; N, 19.60. Found: (S)-form: C, 60.49;
H, 5.65; N, 19.61; (R)-form: C, 60.60; H, 5.59; N, 19.63. MS-ESI+
358.13 [M + H]+.
Compound 5. The (R)- and (S)-enantiomers of 1-(4-chlorophen-
yl)-(1H-imidazol-1-yl)ethyl 4-(4-nitrophenylthio)phenylcarbamate
were prepared by suspending 1 mmol of (R)- or (S)-1-(4-
chlorophenyl)-2-(1H-imidazol-1-yl)ethanol, respectively, in 5 mL of
anhydrous CH3CN. Then 0.5 mmol of triphosgene was added and the
solution was treated as described above except that 3 mmol of TEA
and 0.8 mmol of 4-((4-nitrophenyl)thio)aniline were added to 1-(4-
chlorophenyl)-2-(1H-imidazol-1-yl)ethyl chloroformate. The crude
product was purified by silica gel column chromatography using
CHCl3/MeOH (9:1). The yellow solid was obtained with the yield
Compound 2. The (R)- and (S)-enantiomers of 1-(4-chlorophen-
yl)-2-(1H-imidazol-1-yl)ethyl-4-isopropylphenylcarbamate were syn-
thesized using the (R)- and (S)-enantiomers of 1-(4-chlorophenyl)-2-
(1H-imidazol-1-yl)ethanol, respectively. One mmol of NaH was used
to deprotonate 1 mmol of 1-(4-chlorophenyl)-2-(1H-imidazol-1-
yl)ethanol suspended in 5 mL of anhydrous CH3CN. The reaction
mixture was stirred for 2 h at room temperature, and then 1.5 mmol of
4-isopropylphenyl isocyanate was added and stirred for 24 h at room
temperature. The solvent was evaporated under reduced pressure, and
the residue was washed with MeOH (3 × 3 mL). The mother liquor
was dried and purified by silica gel column chromatography using
CH2Cl2/MeOH (9:1) as eluent. The white solid was obtained with
1
85% (e.e. > 98%). Mp = 100−102 °C; IR: 1727 cm−1; H NMR
(CD3OD): δ 8.07 (d, 2H, J = 9.05 Hz), 7.57 (m, 3H), 7.49 (d, 2H, J =
8.8 Hz), 7.38 (m, 4H), 7.18 (m, 3H), 6.96 (s, 1H), 6.04 (t, 1H, J = 6.2
Hz), 4.49 (m, 2H); 13C NMR (DMSO-d6): δ 152.6, 149.2, 145.3,
141.1, 138.4, 137.1, 136.5, 133.4, 128.9, 128.7, 128.6, 126.4, 124.7,
122.2, 120.5, 120.1, 74.6, 50.7. Anal. Calcd for C24H19ClN4O4S: C,
58.24; H, 3.87; N, 11.32. Found: (S)-form: C, 58.34; H, 3.88; N,
11.33; (R)-form: C: 58.01; H, 3.99; N, 11.30. MS-ESI+ 495.07 [M +
H]+.
1
45% yield (e.e. > 99%). Mp = 162−4 °C; IR 1722 cm−1; H NMR
(DMSO-d6): δ 9.75 (s, broad, 1H), 7.55 (s, 1H), 7.43 (d, 2H, J = 8.4
Hz), 7.36 (d, 2H, J = 8.4 Hz), 7.30 (d, 2H, J = 8.4 Hz), 7.15 (s, 1H),
7.12 (d, 2H, J = 8.4 Hz), 6.84 (s, 1H), 5.94 (m, 1H), 4.41 (m, 2H),
Compound 6. The (R)- and (S)-enantiomers of 1-(2,4-
dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-[4-(3,4-dichlorophenyl)-
piperazin-1-yl]phenylcarbamate were prepared using 1 mmol of (R)-
or (S)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol to obtain
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl chloroformate fol-
lowing the procedure described for compound 5. The synthesis of
4-[4-(3,4-dichlorophenyl)piperazin-1-yl] anilinium chloride was car-
ried out by condensation of 1 mmol of 1-(3,4-dichlorophenyl)-
piperazine dissolved in 5 mL of CH3CN and 2.5 mmol of 1-fluoro-4-
nitrobenzene (see Scheme 2). After refluxing for 2 h, the solvent was
removed and the crude residue was purified by silica gel column
chromatography, using CH2Cl2/MeOH (9:1) as eluent. 1-(3,4-
Dichlorophenyl)-4-(4-nitrophenyl)piperazine was obtained with 80%
yield. Then 1-(3,4-dichlorophenyl)-4-(4-nitrophenyl)piperazine was
suspended in 90 mL of MeOH and reduced to amine by
hydrogenation in the presence of 10% Pd/C as catalyst at room
temperature, 50 psi for 4 h. The catalyst was removed by filtration, and
the solution was bubbled with gaseous HCl for 1 h at 0 °C. The
solvent was evaporated under reduced pressure to give 4-[4-(3,4-
dichlorophenyl)piperazin-1-yl] anilinium chloride with 90% yield. 0.8
mmol of 4-[4-(3,4-dichlorophenyl)piperazin-1-yl] anilinium chloride
and 3 mmol of TEA was added to the (R)- or (S)-1-(2,4-
dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl chloroformate dissolved in
5 mL of anhydrous CH3CN. The reaction was stirred overnight at
room temperature, the crude mixture diluted with H2O, and the
aqueous layer extracted with CH2Cl2 (3 × 10 mL). The combined
organic layers were dried under anhydrous sodium sulfate, and the
solvent was evaporated. The crude product was purified by silica gel
column chromatography using CH2Cl2/MeOH (9.5:0.5) as eluent.
The white solid was obtained with 70% yield (e.e. > 98%). Mp = 220−
13
2.80 (m, 1H), 1.14 (d, 6H, J = 6.8 Hz); C NMR (DMSO-d6): δ
152.2, 144.3, 138.5, 135.3, 135.2, 134.2, 129.1, 127.4, 126.8, 126.4,
120.2, 119.1, 73.6, 52.4, 33.5, 24.0; Anal. Calcd for C21H22ClN3O2: C,
65.71; H, 5.78; N, 10.95. Found: (S)-form: C, 65.48; H, 5.78; N,
10.93; (R)-form: C, 65.80; H, 5.75; N, 10.91. MS-ESI+ 384.8 [M +
H]+.
Compound 3. The (R)- and (S)-enantiomers of 1-(4-fluorophen-
yl)-2-(1H-imidazol-1-yl)ethyl-4-isopropylphenylcarbamate were pre-
pared using the corresponding enantiomers of 1-(4-fluorophenyl)-2-
(1H-imidazol-1-yl)ethanol following the procedure described for
compound (R)-2 and (S)-2. The white solid was obtained with 30%
1
yield (e.e. > 99%). Mp = 200−202 °C; IR: 1715 cm−1; H NMR
(DMSO-d6): δ 9.74 (s, broad, 1H), 7.55 (s, 1H), 7.42 (m, 2H), 7.31
(d, 2H, J = 8.5 Hz), 7.22 (m, 2H), 7.15 (s, 1H), 7.13 (d, 2H, J = 8.5
Hz), 6.84 (s, 1H), 5.94 (t, 1H, J = 5.4 Hz), 4.43 (m, 2H), 2.80 (m,
1H), 1.16 (d, 6H, J = 6.9 Hz); 13 C NMR (CD3OD): δ 162.7 (J = 245
Hz), 153.0, 143.8, 137.8, 136.0, 133.5 (J = 3 Hz), 128.0 (J = 9 Hz),
127.5, 126.3, 120.2, 118.8, 115.1 (J = 21 Hz), 74.2, 51.3, 33.4, 23.1;
Anal. Calcd for C21H22FN3O2: C, 68.65; H, 6.04; N, 11.44. Found:
(S)-form: C, 68.39; H, 6.05; N, 11.42; (R)-form: C, 68.61; H, 6.15; N,
11.63. MS-ESI+ 368.4 [M + H]+.
Compound 4. The (R)- and (S)-enantiomers of 1-(4-fluorophen-
yl)-2-(1H-imidazol-1-yl)ethanol were also used for synthesis of (R)-
and (S)-1-(4-fluorophenyl)-2-(1H-imidazol-1-yl)ethyl 3-(1H-imidazol-
yl)propylcarbamate. 0.5 mmol of 1-(4-fluorophenyl)-2-(1H-imidazol-
1-yl)ethanol was suspended in 5 mL of anhydrous CH3CN, and then
0.25 mmol of triphosgene was added and the solution was stirred
overnight at room temperature. The reaction mixture was treated with
Et2O, producing a white precipitate of 1-(4-fluorophenyl)-2-(1H-
imidazol-1-yl)ethyl chloroformate. The solvent was removed by
decantation; the precipitate was washed with Et2O (2 × 5 mL) and
dissolved in anhydrous CH3CN. Then, 1.6 mmol of TEA and 0.8
mmol of 3-(1H-imidazol-1-yl)propan-1-amine were added to the
solution. The reaction was stirred overnight at room temperature. The
crude mixture was diluted with H2O (5 mL), and the aqueous layer
was extracted with CHCl3 (3 × 10 mL). The combined organic layers
were dried under anhydrous sodium sulfate, and the solvent was
evaporated under reduced pressure. The crude product was purified by
silica gel column chromatography using CH2Cl2/MeOH (8:2). The
white solid was obtained with the yield 80% (e.e. > 98%). Mp = 101−
1
225 °C; IR: 1720 cm−1; H NMR (DMSO-d6): δ 9.75 (s broad, 1H),
7.69 (d, 1H, J = 2.0 Hz), 7.54 (s, 1H), 7.44−7.39 (m, 2H), 7.29−7.18
(m, 4H), 7.11 (s, 1H), 6.98 (dd, 1H, J = 8.9 Hz, J = 2.8 Hz), 6.93 (d,
2H, J = 8.4 Hz), 6.86 (s, 1H), 6.11 (m, 1H), 4.46 (m, 2H), 3.38 (m,
4H), 3.36 (m, 4H); 13C NMR (CD3OD): δ 150.5, 148.7, 138.8, 135.1,
133.0, 132.9, 132.0, 130.5, 129.6, 129.2, 128.1, 127.9, 125.5, 122.5,
120.4, 120.3, 117.5, 117.2, 116.3, 115.5, 70.9, 50.6, 49.8, 48.9; Anal.
Calcd for C28H25Cl4N5O2: C, 55.56; H, 4.16; N, 11.57. Found: (S)-
form: C, 55.65; H, 4.16; N, 11.56; (R)-form: 55.68; H, 4.15; N, 11.69.
MS-ESI+ 605.98 [M + H]+.
CYP51 Expression and Purification. For functional studies,
including ligand binding and enzymatic activity assays, we used the
full-length Tulahuen T. cruzi CYP51 (GenBank IDs AY856083)
expression construct, designed and purified as previously described in
ref 21. For crystallization purposes, we utilized the T. cruzi CYP51 N-
terminal truncated construct where the membrane anchor sequence
1
103 °C; IR: 1710 cm−1; H NMR (CD3OD): δ 7.61 (s, 1H), 7.56 (s,
1H), 7.36 (m, 2H), 7.11 (m, 4H), 6.96 (s, 1H), 6.94 (s, 1H), 5.92 (m,
1H), 4.40 (m, 2H), 4.00 (t, 2H, J = 6.8 Hz), 3.05 (m, 2H), 1.93 (m,
2H, J = 6.8 Hz); 13C NMR (CDCl3): δ 162.8 (J = 247 Hz), 155.2,
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(first 31 residues upstream of P32) was replaced with the 5-amino acid
sequence fragment MAKKT-.23 In all cases the CYP51 gene was His-
tag-engineered at the C-terminus, subcloned into the pCW expression
vector, and expressed in the E. coli strain HMS174(DE3) (Novagen).
CYP51 Spectral Ligand Binding Assay. Changes in the T. cruzi
CYP51 Soret band absorbance spectra upon titration with each
compound were recorded at 25 °C using a dual beam Shimadzu UV-
240IPC spectrophotometer. The P450 concentration was determined
from the Soret band intensity of the reduced CO complexes using
fluorimetically quantified as Relative Fluorescence Units (RFU)
using a Synergy HT fluorometer (Biotek Instruments).23,34 For
fluorescence microscopy observation, the infection assays were
performed in 8-well LabTech tissue culture chambers in triplicate;
after 72 h of infection the cardiomyocyte monolayers were fixed with
2.5% paraformaldehyde and stained with 4′,6-diamidino-2-phenyl-
indole, to visualize DNA, and with Alexa fluor 546 phalloidin
(Invitrogen) to visualize cardiomyocyte actin myofibrils as described.24
X-ray Crystallography. The initial screening of crystallization
conditions was performed using Hampton Research crystallization kits.
Twenty mM stock solutions of compounds (S)-2, (S)-3, and (R)-6 in
DMSO were added at 2-fold molar excess to the 350 μM solution of T.
cruzi CYP51 in 20 mM K-phosphate buffer, pH 7.2, containing 200
mM NaCl, 0.1 mM EDTA, 10% glycerol, and 0.048 mM n-tridecyl-β-
d-maltoside. Crystals were grown at 23 °C using the hanging-drop
vapor diffusion method, cryoprotected by plunging them into a drop
of reservoir solution supplemented with 20% glycerol, flash-frozen in
liquid nitrogen, and then prescreened on a Bruker Microstar
microfocus rotating-anode X-ray generator/Proteum PT135 CCD
area detector. Crystals that diffracted to ∼3.0 Å resolution were
subsequently used for the data collection at the synchrotron
(Advanced Photon Source, Argonne National Laboratory, IL) on
beamline 21ID-F at 100 K. The diffraction images were integrated
using Mosflm and scaled with Aimless (CCP4 Program Suite 6.3.035)
in the P22(1)2(1) space group in the case of LFD (to resolution of
2.61 Å) and in the P3(1)21 space group in the cases of LFT (2.60 Å)
and LFS (2.70 Å). Solvent content was estimated with a Matthews
probability calculator. The crystal structures were determined by
molecular replacement in PhaserMR using the atomic coordinates of
the posaconazole-bound T. cruzi CYP51 structure (PDB code 3K1O)
as the search model. In each case Phaser found a single solution, with
one protein molecule in the asymmetric unit in the cocrystals of T.
cruzi CYP51 with (S)-2 and (S)-3 and with two protein molecules in
the asymmetric unit in the cocrystals of T. cruzi CYP51 with (R)-6.
The models were built and refined with COOT and REFMAC5,
respectively, from the CCP4 Program Suite 6.3.0.35 Data collection
and refinement statistics are shown in Table 2. The electron densities
for each inhibitor were well-defined in all cases, showing full
occupancy and opposite orientation of short (S)-2/(S)-3 versus long
(R)-6 (the 2Fo − Fc electron density maps for (R)-6 and (S)-3
weighted at 1.5σ are shown in the graphical abstract).
visible absorption difference spectroscopy, Δε450−490 = 91 mM−1
32
cm−1
.
Spectral titrations were performed at ∼1 μM P450
concentration in a 20 mM K-phosphate buffer, pH 7.4, containing
200 mM NaCl and 0.1 mM EDTA. The optical path length was 5 cm.
Ligand binding was monitored as a red shift in the Soret band
maximum reflecting coordination of the heterocyclic nitrogen to the
P450 heme iron.17 Difference spectra were generated by recording the
P450 absorbance in a sample cuvette versus the absorbance in a
reference cuvette, both containing the same amount of the protein.
Compounds were added to the sample cuvette in the concentration
range 0.2−2.0 μM from 0.4 mM stock solutions in DMSO. The
titration step was 0.2 μM. At each step, the corresponding volume of
DMSO was added to the reference cuvette. The apparent dissociation
constants of the enzyme−inhibitor complexes (spectral dissociation
constants (Ks)) and the magnitude of spectral binding (binding
efficiency (ΔAmax/Ks)) were calculated with GraphPad Prism (Graph-
Pad Software, La Jolla, CA) using a quadratic function for tight binding
ligands by fitting the data for the ligand-induced spectral change (peak
to trough absorbance changes in the difference spectra (ΔA)) versus
total ligand concentration to the following equation:
ΔA = (ΔAmax/2E)((L + E + Ks)
− ((L + E + Ks)2 − 4LE)0.5
)
where [L] and [E] are the concentrations of the ligand and the
enzyme used for the titration, respectively.20
CYP51 Activity Assay. The enzymatic activity of T. cruzi CYP51
was reconstituted in vitro as described previously using eburicol (24-
methylenedihydrolanosterol) as the substrate.21 Briefly, the reaction
mixture contained 1 μM CYP51, 2 μM cytochrome P450 reductase
(CPR), 100 μM dilauroyl-α-phosphatidylcholine, 0.4 mg/mL iso-
cytrate dehydrogenase, and 25 mM sodium isocitrate in 20 mM
MOPS (pH 7.4), 50 mM KCl, 5 mM MgCl2, and 10% glycerol. After
addition of the [3H]-radiolabeled sterol substrate (∼2,000 cpm/nmol,
final concentration 50 μM), the mixture was preincubated for 5 min at
37 °C; the reaction was initiated by addition of 100 μM NADPH and
stopped by extraction of the sterols with ethyl acetate. The reaction
products were analyzed by a reverse-phase HPLC system (Waters)
equipped with a β-RAM detector (INUS Systems, Inc.) using a Nova
Pak C18 column. The potencies of the compounds to inhibit T. cruzi
CYP51 activity were compared as inhibition of the substrate
conversion in a 1 h reaction at molar ratio enzyme/substrate/inhibitor
= 1:50:2, as these conditions were shown previously to allow
distinguishing the most potent compounds.17,20,23
T. cruzi Cellular Growth Inhibition Assay. A cellular T. cruzi
infection assay was performed using highly invasive 20A clone of the
Tulahuen strain of T. cruzi, which was shown to infect >98% of
exposed cardiomyocytes.33 T. cruzi trypomastigotes expressing green
fluorescent protein (GFP) were generated as described.23 Trypomas-
tigotes were used to infect cardiomyocyte monolayers in 96-well tissue
culture plates and in 8-well LabTech tissue culture chambers in
triplicate at the ratio of 10 parasites per cell as described.24,34 Cultures
were incubated with DMEM supplemented with 10% fetal bovine
serum (FBS) as described.24 Unbound trypomastigotes were removed
by washing the cellular monolayers with DMEM; and infected
monolayers were exposed to several concentrations of the inhibitors
(from 1 nM to 25 nM), dissolved in DMSO/DMEM free of phenol
red in triplicate at 24 h of infection, and cocultured in DMEM + 10%
FBS for 48 h to observe parasite multiplication. Posaconazole was used
as a control. At 72 h of infection, the cardiomyocyte monolayers were
washed with phosphate-buffered saline, and the infection was
ASSOCIATED CONTENT
* Supporting Information
■
S
Two figures showing (1) surface binding subsites formed in the
complexes of T. cruzi CYP51 with LFD and with posaconazole
and (2) the influence of binding of LFD and LFT on T. cruzi
CYP51 flexibility. This material is available free of charge via
the Internet at http://pubs.acs.org.
Accession Codes
The coordinates and structure factors of T. cruzi CYP51 in
complex with compounds (R)-6 (ligand PDB ID LFD), (S)-2
(LFT), and (S)-3 (LFS) have been deposited in the Protein
Data Bank, PDB codes 4CK8, 4CK9, and 4CKA, respectively
(http://www.rcsb.org).
AUTHOR INFORMATION
Corresponding Author
■
*Vanderbilt University, 622 RRB, 23rd@Pierce, Nashville, TN,
37232, USA. Tel: 615-343-1373, e-mail: galina.i.lepesheva@
vanderbilt.edu.
Present Address
#(L.F.) Department of “Chimica e Tecnologie del Farmaco”,
Sapienza University of Rome, Rome 00185, Italy.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by funding from the National
Institutes of Health (GM067871, G.I.L.) and in part by
AI080580, AI007281, and U54 MD007593 (F.V.). The
Confocal Microscopy Facility at Meharry was supported by
G12MD007586. Organic synthesis was funded by grants from
Sapienza University of Rome “Progetti di Ricerca di Ateneo”,
̀
and from Ministero dell’Istruzione, dell’Universita e della
Ricerca. Use of the Advanced Photon Source operated by
Argonne National Laboratory was supported by the U.S. DOE
under Contract No. DE-AC02-06CH11357. Use of the LS-
CAT Sector 21 was supported by the Michigan Economic
Development Corporation and the Michigan Technology Tri-
Corridor (Grant 085P1000817).
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Courtemanche, G.; Devine, W.; Erath, J.; Juda, C. E.; Wawrzak, Z.;
Wood, J. T.; Lepesheva, G. I.; Rodriguez, A.; Pollastri, M. P.
Antitrypanosomal lead discovery: identification of a ligand-efficient
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Chem. 2013, 56, 2556−2567.
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P.; Ridenour, W.; Uddin, M. J.; Caprioli, R. M.; Marnett, L. J.; Nes, W.
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ABBREVIATIONS USED
■
CYP, cytochrome P450; CYP51, sterol 14α-demethylase; T.
cruzi, Trypanosoma cruzi; EC50, drug concentration that gives
half-maximal response in cellular growth reduction; GFP, green
fluorescent protein; RuCl(p-cymene)[(S,S)-Ts-DPEN], (S,S)-
N-(p-toluenesulfonyl)-1,2-diphenylethanediamine(chloro)(p-
cymene)ruthenium(II); VNI, ((R)-N-(1-(2,4-dichlorophenyl)-
2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)-
benzamide); VNF, ((R)-N-(2-(1H-imidazol-1-yl)-1-phenyleth-
yl)-4′-chlorobiphenyl-4-carboxamide)
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