R -configuration of 4-aminopyridyl-based inhibitors of CYP51 confers superior efficacy against Trypanosoma cruzi

Article abstract of DOI:10.1021/ml500010m

Sterol 14α-demethylase (CYP51) is an important therapeutic target for fungal and parasitic infections due to its key role in the biosynthesis of ergosterol, an essential component of the cell membranes of these pathogenic organisms. We report the developm

Full text of DOI:10.1021/ml500010m

Letter
pubs.acs.org/acsmedchemlett
R‑Configuration of 4‑Aminopyridyl-Based Inhibitors of CYP51
Confers Superior Efficacy Against Trypanosoma cruzi
Jun Yong Choi,† Claudia M. Calvet,‡,§,∥ Debora F. Vieira,‡,§ Shamila S. Gunatilleke,‡,§,#
Michael D. Cameron,⊥ James H. McKerrow,‡,§ Larissa M. Podust,*,‡,§ and William R. Roush*,†
†Department of Chemistry, Scripps Florida, Jupiter, Florida 33458, United States
‡Center for Discovery and Innovation in Parasitic Diseases and §Department of Pathology, University of California San Francisco, San
Francisco, California 94158, United States
∥Cellular Ultra-Structure Laboratory, Oswaldo Cruz Institute (IOC), FIOCRUZ, Rio de Janeiro, RJ 21040-362, Brazil
⊥Department of Molecular Therapeutics, Scripps Florida, Jupiter, Florida 33458, United States
S
* Supporting Information
ABSTRACT: Sterol 14α-demethylase (CYP51) is an important therapeutic target for fungal
and parasitic infections due to its key role in the biosynthesis of ergosterol, an essential
component of the cell membranes of these pathogenic organisms. We report the development
of potent and selective D-tryptophan-derived inhibitors of T. cruzi CYP51. Structural
information obtained from the cocrystal structure of CYP51 and (R)-2, which is >1000-fold
more potent than its enantiomer (S)-1, was used to guide design of additional analogues. The
in vitro efficacy data presented here for (R)-2−(R)-8, together with preliminary in vitro
pharmacokinetic data suggest that this new CYP51 inhibitor scaffold series has potential to
deliver drug candidates for treatment of T. cruzi infections.
KEYWORDS: T. cruzi, CYP51, R-configuration, inhibitors
he sterol biosynthesis pathway has been identified as an
Timportant therapeutic target for antifungal agents due to
the different set of sterols that occur in fungal membranes as
opposed to mammalian cell membranes.1,2 Sterol 14α-
demethylase (CYP51) catalyzes the oxidative removal of the
14-methyl group of lanosterol. The resulting Δ14,15-unsaturated
intermediate is subsequently converted to ergosterol, an
essential fungal cell membrane component. CYP51 is also a
potential chemotherapeutic target for the treatment of infection
Figure 1. Comparison of the binding modes of (S)-1 (blue) and
by the protozoan parasite Trypanosoma cruzi because T. cruzi’s
membrane sterol components are similar to those of fungi, and
the organism utilizes an analogous pathway for ergosterol
biosynthesis.3 Thus, antifungal azole drugs have been
repurposed as antiparasitic agents for treatment of Chagas
disease.4−8 These agents, as well as the new T. cruzi CYP51
inhibitors tipifarnib, NEU321, and VNI analogues utilize azoles
as a heme-binding unit. Consequently, resistance is a potential
issue since resistance to azole drugs in cell culture and in T.
cruzi infected mice has been reported.9 Consequently, an
important objective for development of drugs to treat T. cruzi
infection is to identify potent and safe CYP51 inhibitors based
on nonazole chemotypes.
posaconazole (yellow) in the active site of T. brucei CYP51. Protein is
shown as solvent accessible surface with the M460 side chain present
in 2×2N (a) or omitted (b) from the 4BJK coordinates.
new inhibitors ((R)-2, derived from D-tryptophan) in the
cocrystal structure with T. cruzi CYP51 is quite different from
that of its S-enantiomer ((S)-1, derived from L-tryptophan).10
Relationships between the binding poses and inhibitory
potential toward T. cruzi are presented and analyzed herein.
The nonazole CYP51 inhibitor LP10, which was initially
identified by HTS,11 possesses moderate inhibition potency
(EC50 = 680 nM) against T. cruzi in infected mammalian cells.
However, its closely related analogues have poor stability in
liver microsome preparations (t1/2 = <7 min) and poor
In this letter we describe the structure-guided development
of a series of potent and selective N-indolyl-oxopyridinyl-4-
aminopropanyl D-tryptophan-derived inhibitors of T. cruzi
CYP51 that utilize a pyridine ring to coordinate to the heme
group. The binding pose of the R-enantiomer of one of these
Received: January 9, 2014
Accepted: January 15, 2014
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of Key Intermediates 9, 10, 11, 15, 18, and 20 and Inhibitors (R)-3−(R)-8
a
Reagents and conditions: (a) arylboronic acid, 5 mol % Pd2(dba)3, 10 mol % PCy3, 2 M K3PO4, dioxane, 100 °C (microwave), 1 h, ca. 90%; (b)
H2SO4/MeOH (1/10), 70 °C, 24 h, 91%; (c) H2 (balloon), Pd/C, MeOH−acetone, 23 °C, 24 h, 92%; (d) 2,4-difluorobenzyl bromide, K2CO3,
acetone, 70 °C, 5 h, 95%; (e) 1-(3-(trifluoromethyl)phenyl)piperazine, Pd(OAc)2, P(o-tolyl)3, Cs2CO3, toluene, 50 °C, 48 h, 88%; (f) 10% NaOH
(aq), MeOH, THF, 70 °C, 2 h, 99%; (g) 4-fluorobenzyl bromide, K2CO3, acetone, 50 °C, 12 h, 91%; (h) 4-carboxy-3-fluorophenylboronic acid, 5
mol % Pd2(dba)3, 10 mol % PCy3, 2 M K3PO4, dioxane, 110 °C (microwave), 1 h, 30%; (h) 9, 10, 11, 15, 18, or 20 (as appropriate), PyBOP, HOBt,
Et3N, CH2Cl2, 23 °C, 1 h, ca. 70%.
selectivity against human metabolic cytochrome P450 (CYP)
enzymes (92, 77, and 75% inhibition of CYPs 2C9, 2D6, and
3A4 at 1 μM, respectively).10 Despite LP10s suboptimal
pharmacokinetic properties, its promising efficacy in an animal
model of acute T. cruzi infection7 encouraged to pursue
structure-aided hit-to-lead optimization studies.
accessible area, they protrude through different hydrophobic
tunnels (Figure 1). The ample void space surrounding the
biaryl unit of (S)-1 and the tail unit of posaconazole suggested
the possibility that the (R)-enantiomer of (S)-1, specifically
inhibitor (R)-2, could adopt a different binding orientation
compared to (S)-1.
We recently reported results of first generation structure-
based optimization of LP10.10 Initially, the S-enantiomers of
LP10 analogues (deriving from L-tryptophan) were pursued
because of better binding affinity and inhibitor potency
compared to those with R-configuration at the tryptophan
center. However, in spite of considerable effort, we were unable
to identify CYP51 inhibitors with EC50s less than 10 nM.5
During the course of these studies the X-ray cocrystal structure
of the biaryl inhibitor (S)-1 complexed with T. brucei CYP51
was determined to a resolution of 2.7 Å.5 From an in-depth
comparison of the binding modes of (S)-1 and posaconazole in
the active site of CYP51,10,12 we identified several critical
features that we used in the design of the next generation of
substantially improved CYP51 inhibitors that we report herein.
Specifically, although the biaryl unit of (S)-1 and the tail
portion of posaconazole are oriented toward a solvent-
To test this assumption, (R)-2 and a series of additional
analogues were synthesized starting from D-tryptophan, using
the general procedure used previously for (S)-1 (Scheme 1).10
Briefly, 4-bromo-2-fluorobenzoic acid was subjected to
palladium-mediated coupling reactions13−16 with various aryl
boronic acids to provide biaryl intermediates such as 9, 10, 11,
and 12. Carboxylic acid 12 was converted to methyl ester 13,
and then the benzyl group was replaced with a 2,4-
difluorobenzyl unit through catalytic hydrogenation (Pd/C
and H2) followed by O-alkylation with 2,4-difluororbenzyl
bromide. Pd(0)-mediated coupling17−19 of N-aryl piperazine
with 16 provided 17, which was then hydrolyzed to give
carboxylic acid 18. O-alkylation of commercially available 4-
bromo-2-(trifluoromethyl)phenol with 4-fluorobenzyl bromide
followed by Suzuki coupling with 4-carboxy-3-fluorophenylbor-
onic acid generated carboxylic acid 20. All carboxylic acid
B
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Table 1. Efficacy, Microsome Stability, and CYP Inhibition Profile of New Key CYP51 Inhibitors
a
EC50 of compounds as determined vs. T. cruzi-infected mouse C2C12 myoblasts (see Supporting InformatioI for details and Figure S1 for dose−
response curves). EC50 values were used to assess inhibitor potency since the UV−vis CYP51 binding assay is incapable of discriminating between
b
compounds with IC50 < 10 nM (ref 10). Stability of compounds in human (h), rat (r), and mouse (m) liver microsomes as evaluated in comparison
c
to the Sunitinib reference (see Supporting Information). Inhibition of CYPs as evaluated in human liver microsomes using selective marker
d
e
substrates for each CYP (see Supporting Information). Reported previously (ref 11). Reported previously (ref 10).
Figure 2. Binding and anti-T. cruzi activity of (S)-1 and (R)-2. (a) Dose−response curves for (S)-1 (blue) and (R)-2 (red) show >1000-fold
difference in potency (T. cruzi cell-based assay). (b) A fragment of the 2Fo − Fc electron density map (blue mesh) contoured at σ = 1.0 corresponds
to (R)-2 (red). Heme is shown in gray spheres. (c) (R)-2 viewed through the opening of the hydrophobic tunnel in 4BY0. Protein is represented by
solvent accessible surface. Binding pose of posaconazole (yellow sticks) is derived from the superimposed 2×2N structure. (d) Overlap between (S)-
1 (blue) and (R)-2 (red) derived from the superimposed structures, 4BJK and 4BY0, respectively. Heme is shown in semitransparent spheres.
C
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intermediates were coupled with D-tryptophan derivative 215 to
partially due to flexibility of the M460 side chain (which is not
resolved in the X-ray structure of T. brucei CYP51 complexed
with (S)-1) (Figure 1B). When (R)-2 is bound, the interactions
between M460 and the central phenyl ring of the inhibitor are
established and partially block the entrance to the active site
(Figure 2C).
provide the final compounds 3−8.20
As shown in Table 1 and Figure 2A, the potency of (R)-2 as
an inhibitor of T. cruzi in infected cells increased >1000-fold
compared to (S)-1, making (R)-2 the first pM inhibitor derived
from LP10. The new inhibitor (R)-2 retained good microsome
stability and had an acceptable profile for inhibition of human
CYPs.
The X-ray cocrystal structure of (R)-2 with T. cruzi CYP51
was determined to a resolution of 3.1 Å (PDB ID 4BY0) (See
Supporting Information for details). The diffraction data and
refinement statistics are shown in Table 2. Despite a relatively
On the basis of the TcCYP51-(R)-2 cocrystal structure, 17
hydrophobic amino acid residues, F48, Y103, I105, M106,
F110, Y116, P210, V213, F214, A287, F290, A291, T295, L356,
M358, M360, M460, and V461, constitute the binding site
within 5 Å of (R)-2 (Figure 3). No direct H-bonds between
(R)-2 and the TcCYP51 target are suggested by the cocrystal
structure, with the qualification that water molecules, which
might potentially mediate such contacts, may not be resolved at
3.1 Å. The amino acids I45, F48, I72, V213, L357, M358, and
M360 surround the terminal 3-fluorophenyl ring of (R)-2
(Figure 3), providing space to explore a variety of ring
substituents. By applying structure-based molecular design
considerations,21,22 a series of (R)-2 analogues were synthesized
with the objective to increase potency against T. cruzi.
Specifically, additional substituents on the terminal phenyl
ring were introduced to fill the hydrophobic pocket identified in
the cocrystal structure. In addition, a flexible piperazine ring
was inserted between the aromatic rings of the rigid biaryl unit
to probe binding interactions in the hydrophobic pocket
projecting toward the solvent accessible area. A diverse set of
new inhibitors was synthesized, among which (R)-3−(R)-8
(see Scheme 1 for syntheses of these compounds) are
particularly potent (Table 1). The (S)-enantiomers of several
of these and of other inhibitors were also synthesized and in all
cases were found to be considerably less potent than the
corresponding (R)-enantiomers (see Table S1 and Figure S2,
Supporting Information).
Table 2. X-ray Data Collection and Refinement Statistics
protein
inhibitor
PDB ID
T. cruzi CYP51
(R)-2 (small molecule code 5PS)
4BY0
Data Collection
space group
P3221
cell dimensions
a, b, c (Å)
124.2, 124.2, 119.8
α, β, γ (deg)
90.0, 120, 90.0
molecules in AU
wavelength
2
1.11587
3.10
resolution (Å)
Rsym or Rmerge (%)
I/σI
a
15.6 (134.3)
9.6 (1.5)
completeness (%)
redundancy
100.0 (100.0)
8.2 (8.4)
Crystallization Conditions
0.2 M ammonium sulfate
0.1 M Bis-Tris
pH 6.5
25% PEG 3350
The 3,4-difluoro analogue (R)-4 gained an order of
magnitude in potency compared to (R)-2, while potency of
the 2,5-analogue (R)-3 dropped an order of magnitude. The
potency gain by (R)-4 is likely due to hydrophobic interactions
between the 4-fluoro substituent and the side chain of F48
(Figure 3), which is otherwise missing from the electron
density. Increasing the size of the substituent from 4-fluoro in
(R)-4 to 4-trifluoromethyl in (R)-5 resulted in a ca. 600-fold
loss in potency, while its microsome stability and selectivity to
human CYP enzymes were significantly improved compared to
(R)-4. Insertion of a piperazine ring between the aryl groups
yielded (R)-6, which was only 4-fold less potent than (R)-2.
Both (R)-7 and (R)-8 with terminal fluorine-substituted benzyl
ethers maintained EC50 potency comparable to (R)-2 and (R)-
5, respectively. Two of these compounds, (R)-5 and (R)-8, had
substantially improved microsome stability compared to (R)-2,
and (R)-8 in particular had a substantially improved inhibition
profile vs human CYP enzymes (Table 1). The binding poses
of inhibitors were predicted by docking studies using Glide (see
Figure S3, Supporting Information, for details). The terminal
phenyl ring of (R)-6 fits well into the hydrophobic pocket near
to the biaryl ring of (R)-2. In addition, the benzyl moiety of
(R)-7 or (R)-8 is oriented toward the solvent accessible area or
the hydrophobic pocket, respectively.
Refinement
no. reflections
18756
R
work/Rfree (%)
23.2/29.7
no. atoms
protein
6663
86
heme
ligand
74
solvent
13
mean B value
B-factors
protein
78.2
81.4
62.0
72.0
52.1
heme
ligand
solvent
rmsd
bond lengths (Å)
bond angles (deg)
0.011
1.664
a
Values in parentheses are for highest resolution.
low resolution, electron density unambiguously defined (R)-2
in the active site (Figure 2B), indicating that the biaryl unit of
(R)-2 is oriented toward the hydrophobic tunnel that is utilized
by the tail of posaconazole (Figure 2C). (R)-2 is distinguished
by an L-shape in the active site of T. cruzi CYP51, while (S)-1 is
more linear (Figure 2D). The different binding modes of (S)-1
and (R)-2 affect the conformations of interacting amino acid
residues, thus changing the landscape of the CYP51 binding site
for the two compounds. The entrance from the solvent-
accessible area remains quite open when (S)-1 is bound,
In summary, a new series of 4-pyridinyl-based CYP51
inhibitors has been developed by using structure-guided
molecular design methodology. Starting with the cocrystal
structure of (S)-1 bound to T. brucei CYP51, we significantly
improved inhibitor potency by switching to the R-configuration
of the inhibitor scaffold. Compared to the S-enantiomer series,
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ACS Medicinal Chemistry Letters
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Figure 3. Stereogram of the (R)-2 binding site in TcCYP51. Amino acid residues (green) are shown within 5 Å of (R)-2 (red), plus those
constituting a cavity surrounding the terminal 3-fluorophenyl ring of (R)-2. The side chains of I45 and F48, disordered in the X-ray structure, are
reconstituted by molecular modeling. Heme is in gray spheres.
Funding
the antiparasite efficacy of some R-inhibitor isomers increased
>1000-fold in the cell-based assay, with concomitant increases
in microsome stability and selectivity against human drug
metabolizing CYPs. The cocrystal structure of (R)-2 with the
ultimate therapeutic target, T. cruzi CYP51, aided in the design
of the next generation of inhibitor analogues, one of which (R)-
4 is a ca. 20 pM inhibitor of T. cruzi in cell culture, while the
less potent analogue (R)-8 has excellent in vitro PK properties.
Collectively, this work demonstrates that the R-series of N-
indolyl-oxopyridinyl-4-aminopropanyl inhibitors has consider-
able promise for the development of anti-T. cruzi therapeutics.
Further studies toward this goal will be reported in due course.
This research was generously supported by NIH R01 grant
AI095437, and C.M.C was supported by Conselho Nacional de
Desenvolvimento Cientifico e Tecnologico (CNPq) and
FIOCRUZ, Brazil. The Advanced Light Source is supported
by the Director, Office of Science, Office of Basic Energy
Sciences, of the U.S. Department of Energy under Contract No.
DE-AC02-05CH11231.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the staff members of beamline 8.3.1, James Holton,
George Meigs, and Jane Tanamachi, at the Advanced Light
Source at Lawrence Berkeley National Laboratory, for
assistance with data collection, Claudia Ruiz (Scripps Florida)
for microsome stability assays, and Potter Wickware for proof
reading of the manuscript.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for T. cruzi cell-based assay, X-ray
structure analysis, hepatic microsome stability and CYP
inhibition assays, chemical synthesis of the inhibitors, and the
dose−response curves and binding poses of the compounds
predicted by molecular docking. This material is available free
of charge via the Internet at http://pubs.acs.org.
ABBREVIATIONS
T. cruzi, Trypanosoma cruzi; CYP51, cytochrome P450 isoform
51; EC50, half-maximal effective concentration
■
Accession Codes
The atomic coordinates and structure factors (PDB ID codes
4BY0) have been deposited in the Protein Data Bank, Research
Collaboratory for Structural Bioinformatics, Rutgers University,
New Brunswick, NJ (http://www.rcsb.org/).
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AUTHOR INFORMATION
■
Corresponding Authors
*(W.R.R.) E-mail: roush@scripps.edu.
*(L.M.P.) E-mail: larissa.podust@ucsf.edu.
Present Address
#College of Science and Engineering, Seattle University, Seattle,
Washington 98122, United States
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