Structure-Guided Optimization of Inhibitors of Acetyltransferase Eis from Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acschembio.0c00184

The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis (Mtb) is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of Mtb, we designed and optimized structurally unique thieno[2,3-d]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3-d]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors in vitro as well as their ability to restore the activity of kanamycin in a resistant strain of Mtb, in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds in vitro. This study showcases how structural information can guide Eis inhibitor design.

Full text of DOI:10.1021/acschembio.0c00184

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Structure-Guided Optimization of Inhibitors of Acetyltransferase Eis
from Mycobacterium tuberculosis
Ankita Punetha,⊥ Huy X. Ngo,⊥ Selina Y. L. Holbrook, Keith D. Green, Melisa J. Willby,
Shilah A. Bonnett, Kyle Krieger, Emily K. Dennis, James E. Posey, Tanya Parish, Oleg V. Tsodikov,*
and Sylvie Garneau-Tsodikova*
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ABSTRACT: The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis (Mtb) is a versatile acetyltransferase
that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of
promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of Mtb, we
designed and optimized structurally unique thieno[2,3-d]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination
therapy. We obtained 12 crystal structures of enzyme−inhibitor complexes, which guided our rational structure-based design of 72
thieno[2,3-d]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors in vitro as well as their
ability to restore the activity of kanamycin in a resistant strain of Mtb, in which Eis was upregulated. Furthermore, we evaluated the
metabolic stability of 11 compounds in vitro. This study showcases how structural information can guide Eis inhibitor design.
KAN, a hallmark of extensively drug-resistant (XDR) TB
(along with resistance to a fluoroquinolone), has also emerged
and spread. XDR TB has a high mortality rate and requires
prolonged treatment that can last as long as 2 years, even if
successful. A clinically significant mechanism of KAN
resistance in TB is KAN acetylation by the acetyltransferase
Eis as a result of point mutations in the eis promoter or the 5′
untranslated region of the gene encoding a transcription factor
WhiB7.3−5
We previously showed that Eis transfers an acetyl group
from acetyl coenzyme A (AcCoA) to one or more amino
groups of all clinically relevant aminoglycosides,6−10 abolishing
their antibacterial activity. In addition, Eis can acetylate the
INTRODUCTION
■
The advent of antibiotics, including antitubercular agents, has
been one of the most powerful technological advances
responsible for the reduction of mortality due to bacterial
infections.1 Unfortunately, in the last few decades, due to the
overuse and misuse of the same antibiotics and the paucity of
new antibacterial agents, resistance to antibiotics has emerged
as a global crisis. There is a pressing need for discovery of new
molecules and strategies to combat antibiotic resistance. In
2018, tuberculosis (TB), caused by infections with Mycobacte-
rium tuberculosis (Mtb), killed 1.5 million people, which is
more than any other infection worldwide, and caused 10
million new cases.2 Misuse of anti-TB drugs, poor treatment
compliance, and over 50 years of using many of the same
antibiotics to fight this disease have led to the emergence and
spread of Mtb strains resistant to one or more of these agents.
Multidrug-resistant (MDR) Mtb strains that cause MDR TB
are, by definition, resistant to first-line drugs isoniazid and
rifampin, taken orally. A second-line antibiotic, aminoglycoside
kanamycin (KAN) is used to treat MDR TB, but resistance to
Received: March 12, 2020
Accepted: May 4, 2020
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peptide antibiotic capreomycin.11 This versatility is apparently
enabled by a large substrate binding site that is lined largely by
acidic and hydrophobic residues.6,8,12 The acetylation reaction
proceeds via a random sequential mechanism, where either
AcCoA or the aminoglycoside substrate can bind first to a free
enzyme.13
Two orthogonal approaches to overcoming Eis-mediated
aminoglycoside resistance have been considered: (1) chemical
modifications of existing aminoglycosides or development of
novel aminoglycosides that would not succumb to acetylation
by Eis, and (2) development of Eis inhibitors as adjuvants of
the clinically used aminoglycosides. The first approach has
been used successfully to design β-lactam antibiotics that are
not susceptible to inactivation by β-lactamases.14 Because of
the very broad substrate versatility of Eis, an analogous
approach of developing aminoglycosides that would not be
susceptible to acetylation by Eis does not appear to be
promising. Instead, our groups have focused on development
of potent small molecule Eis inhibitors for their therapeutic use
as aminoglycoside adjuvants against MDR TB.15−21 We have
discovered by high-throughput screening (HTS) Eis inhibitors
of several structural families. Optimization of these molecules
by classical medicinal chemistry has yielded several compounds
that displayed potency (nanomolar in some cases) in inhibiting
Eis in the test tube and in overcoming KAN resistance of a
relevant resistant Mtb strain in culture.15−20 In these studies,
the crystal structures of Eis in complexes with several of these
inhibitors showed that these inhibitors occupied the amino-
glycoside binding site, in agreement with steady-state kinetic
studies. These structures also established the molecular basis
behind the inhibitor specificity and explained the structure−
activity relationships (SAR).
In the current study, we report the discovery of Eis
inhibitors with a chemical scaffold that is distinct from the
previously reported ones. Here, crystal structures of Eis−
inhibitor complexes are used to guide synthetic efforts in
generating derivatives for further preclinical testing.
Figure 1. Cone diagram showing the evaluation and triage of ∼23 000
structurally diverse compounds.
RESULTS AND DISCUSSION
■
Thieno[2,3-d]pyrimidine-Containing Eis Inhibitor. To
identify potential Mtb Eis inhibitors, HTS of ∼23 000
structurally diverse compounds was carried out using our
previously established aminoglycoside acetylation assay with
Mtb Eis (Figure 1; step 1).15 The HTS assay had a Z′ score22
of 0.65, indicating its robustness. Among the ∼23 000
molecules tested, compound 2i containing a thieno[2,3-
d]pyrimidine moiety was an HTS hit (Z = 4.9, Figure 1;
step 2). Eis inhibitors with this structural core have not been
characterized previously.
effect of our previously reported Eis inhibitors.16−20 We
observed that when tested at 100 μM, 2i restored, within a 2-
fold dilution, the sensitivity of Mtb K204 to KAN to the level
of the parent strain H37Rv (MICKAN = 2.5 μg/mL against Mtb
K204 in the presence of 2i).
Structural Basis for Lead Compound Binding and
Analogue Design. The HTS library contained 70 other
molecules with a thieno[2,3-d]pyrimidine moiety, which were
not identified as hits (Figure S223). These inactive compounds
aided our analogue design by demonstrating what chemical
modifications to avoid (e.g., aromatic groups in the thioether
side chain; heteroatoms in ring A; or substituents on the
primary amine of ring C; see Figure 2 for ring labeling). To
guide our inhibitor design, we determined a crystal structure of
Mtb Eis in a complex with lead inhibitor 2i (Figure 2A,B; PDB
ID 6VUT; Table S1). As a reference structure, we chose the
crystal structure of Eis in complex with inhibitor 39b from a
previous study (Figure 2C; PDB ID 6B3T).20 Inhibitor 39b
contains a 1,2,4-triazino[5,6b]indole-3-thioether core that is
partially isosteric with the tricyclic moiety of 2i. Both 2i and
39b contain flexible substituents on the same side of the
tricyclic moieties, each on a nitrogen-containing ring. Similar
to 39b and all the other previously reported inhibitors, 2i is
In order to validate 2i as an Eis inhibitor, we measured the
dose response of the acetylation in the same assay (Figure 1,
step 3) using a purchased fresh powder of 2i. We then
resynthesized 2i and all analogues (Figure 1, step 4;
characterization of all analogues in this study is shown in
Figures S1−S222). We observed robust inhibition of Eis in this
dose−response experiment, with an IC50 of 1.6 0.2 μM. To
test and validate the mechanism of action of 2i as an Eis
inhibitor in the Mtb cell, we determined the effect of 2i on the
MIC of KAN for the model strain Mtb K204, which bears a
single clinically relevant mutation in the eis promoter resulting
in Eis overexpression and KAN resistance3 (MICKAN in K204
is >10 μg/mL) in the background of strain H37Rv (MICKAN
1.25 μg/mL). Mtb K204 was used extensively to observe the
=
B
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Figure 2. Crystal structures of Eis (one monomer is shown) in complex with inhibitors. (A) Crystal structure of Eis−2i complex. Eis is shown in
cyan and 2i as orange sticks. (B) Zoomed-in view of the Eis−2i interface. The polder omit map for the inhibitor is contoured at 4σ and is shown by
the brown mesh. The amino acid residues interacting with compound 2i are shown by dark turquoise sticks. The nitrogen atoms are in light blue,
oxygen atoms in red, sulfur atoms in yellow, and fluorine atoms in cyan. The C-terminus is labeled “C-ter”. (C) Previously reported crystal structure
of Eis−39b complex (PDB ID 6B3T).20 A water molecule in the active site is shown as a red sphere. The chemical structures of the bound
inhibitors are shown on the bottom of panels B and C. Note: This color scheme is preserved in all the other figures depicting crystal structures.
bound in the aminoglycoside binding pocket (Figure S224).
Strikingly, despite their similar shapes, the tricyclic moieties of
2i and 39b lie in the same plane, sandwiched between Trp36
and Phe84 but in different orientations that are related by a
180° rotation around an axis lying in the plane of these
moieties and passing through the three rings. This difference,
nevertheless, preserves the common binding characteristic,
where the entirely hydrophobic ring A that is unsubstituted (in
2i) or modified by small nonpolar or weakly polar substitutions
(a fluorine in 39b) abuts the hydrophobic wall of the binding
pocket (lined with Trp13, the aliphatic portion of Arg37,
Val40, Leu63, and Met65), whereas the large (R1) substituent
on the opposite ring C is extended along the substrate binding
cleft toward the solvent, a general direction of the thioether
side chain. We used these properties of rings A and C and their
substituents in our analogue design (Figure 3).
C
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backbone and the side chain of Glu401. For both 39b and 2i,
the tertiary amines of R1 formed a salt bridge with Asp26,
which for 2i is enabled by its longer linker assuming a U-turn
conformation. The position and the overall dimensions of the
substituent on ring C, as a thin linker fitting the narrow Eis
cleft, were also preserved in the analogues, whereas the nature
of the substituents was different. An Eis loop spanning residues
25−30 is in two somewhat different conformations in the two
structures, apparently induced by binding of the two different
inhibitors. Most significantly, rotamers of Ile28 and Asp26
differ considerably in the two structures to optimally interact
with the R1 linker and the terminal R1 group, respectively. The
side chain of Arg37 is also in two different conformations,
interacting with either the unmodified ring A of 2i or with the
fluorinated ring A of 39b.
The drastic differences in core orientations, locations of
bound water molecules, and consequently the inhibitor−
protein interactions, which all arise as a result of seemingly
minor chemical differences of the core structures, illustrate
potential serious pitfalls of modeling and computational
docking based on a crystal structure with a similar
pharmacophore. We used the interactions observed in the
crystal structure of Eis in a complex with 2i to design analogues
with the thieno[2,3-d]pyrimidine-containing core.
Figure 3. Chemical structures of all compounds synthesized and
investigated in this study. The three rings of the tricyclic inhibitors in
the library are labeled as A, B, and C for simplicity. The library is
divided into three families (I−III) based on the size of ring A. Further
subdivision of the families is based on R1, R2, and R3 substituents.
Design and Chemical Synthesis of Eis Inhibitors. By
using the guidelines derived from the crystal structure of the
Eis−2i complex and its comparison with the structure of Eis−
39b interactions, as described in the previous section, we made
substitutions on the A−B−C core of 2i (Figure 3; family II) as
well on the same thieno[2,3-d]pyrimidine B−C core but with a
five-membered ring A (Figure 3; family I) as well as a seven-
membered ring A (Figure 3; family III). To explore optimal
filling in the binding pocket for ring A, we modified ring A with
various hydrophobic moieties in family II. In this family, the R3
position in ring A was unsubstituted (H) or substituted with a
methyl, an ethyl, a tert-butyl, or a phenyl group (Figure 3). To
optimize hydrophobic interactions with residues Met65 and
Leu63, we generated molecules in which the R2 position in ring
A was either unsubstituted (H) or substituted with a methyl
group. The analogues were divided into eight series (1−8)
based on the ring A structure, with family I defining series 1,
family II defining series 2−7 with different R2 and R3
substituents on ring A, and family III defining series 8.
Furthermore, within all eight major series, we also installed
side chains R1 on the sulfur of ring C, in which tertiary amino
groups were connected to the sulfur of ring C by an aliphatic
linker (with or without an amide), consistent with the
restrictions of the narrow binding cleft. These tertiary amines
mimic the amino groups of aminoglycoside substrates in the
active site, located in the negatively charged environment of
the Eis active site (Asp26, Glu401, and the C-terminal
carboxyl; Figure 2). We used our previous experience in
diversifying substituents at this binding site to install a
pyrrolidine or a piperidine, which were observed to improve
inhibitor potency in other inhibitors, whereas substituents like
carbonyl, hydroxyl, ether, morpholine, or cyano group were
not well tolerated.20 Here, we varied the size of the terminal
group on R1 (for example, a pyrrolidine vs a piperidine ring),
the position of the nitrogen, for a constant chain length, the
linker length, the presence of an amide bond (imposing
constraints in the R1 side chain and introducing hydrogen
bonding potential). A total of nine R1 variants were generated
Since relatively small hydrophobic substitutions on ring A
were allowed, consistent with its environment, we varied the
size of this ring and the presence and the nature of small
hydrophobic substituents R2 and R3 at two positions on this
ring. The difference in the orientations indicates that the
specific chemical structures of the rings and their substitutions
specify how the inhibitors are bound to the enzyme. Indeed,
ring C in the thieno[2,3-d]pyrimidine core of 2i is decorated
with a primary amino group, absent in 39b, which forms a salt
bridge with the C-terminal carboxyl group of Eis. The primary
amine on ring C is also preserved in all analogues, as it defines
the overall disposition of the core of these the thieno[2,3-
d]pyrimidines in the Eis binding pocket. There is not enough
room to accommodate substitutions on this amino group,
which explains inactivity of library compounds with such
substitutions (Figure S223). The nonpolar sulfur of the central
ring B of 2i is in favorable hydrophobic contact with the side
chain of Ala33 (the Cβ−S distance is 3.6 Å), whereas the
orientation of 39b places its nitrogen-containing ring C
approximately in the same site as the sulfur of 2i but
somewhat closer to the protein surface, enabling water-
mediated hydrogen bonding of this ring with the amide
nitrogens of Ile28 and Gly29. The sulfur-containing ring B is
preserved in the analogues to maintain the interactions of this
ring with Eis. For 2i, a water mediates hydrogen bonding of a
nitrogen of ring C with the side chain of Asp26. In both
structures, the R1 is a thioether-linked side chain on ring C. In
2i, R1 contains an additional amide ultimately connecting to a
terminal morpholine ring, whereas in 39b R1 is shorter and is
terminated by a piperidine ring on an entirely hydrophobic
linker. As a result of the different core orientations, the
directions and interactions with the protein of R1 linkers (i.e.,
what links the sulfur atom and the tertiary amine of the
thioether side chain) are somewhat different for 2i and 39b.
While in 39b the R1 linker is directed to sterically contact the
backbone of Phe27-Asp26, the R1 of 2i is directed along the
other side of the binding cleft, in steric contact with the
D
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as indicated by the letters a−i after the series number. The
entire library contained 72 molecules (Figure 3).
were not commercially available and were prepared by reacting
commercially available primary amines with bromoacetyl
chloride (Scheme 1B). The alkyl halides 41−44 were used
in alkylation reactions with intermediates 33−40 to afford final
products with side chains f, g, h, and i (R1 groups in Figure 3).
SAR Deduced from Structural and Functional
Studies. We next sought to identify SAR for the 72 rationally
designed Eis inhibitor analogues. The inhibitory potency of the
analogues was determined by measuring their effect (IC50) on
the acetylation of the clinically relevant KAN by Eis (Figure 1;
step 5; Table 1 and Figures S225−S228). In order to interpret
these data, in addition to the crystal structure of the Eis−2i
complex, we determined crystal structures of Eis in complexes
with 11 other inhibitors (Tables S1−S3) to avoid making
potentially incorrect assumptions based on the structure of Eis
in a complex with the parent compound.
To access the thieno[2,3-d]pyrimidine core, we initially
prepared a series of 2-aminothiophenes (17−24) as building
blocks via the classic Gewald aminothiophene synthesis
starting from the commercially available malononitrile, sulfur,
and various cyclic ketones (9−16) (Scheme 1A). The building
Scheme 1. Synthetic Scheme for the Preparation of (A)
Compounds 1−8(a−i) and (B) Intermediates 41−44
Effect of the Size of Ring A. The inhibitors with the six-
membered ring A were nearly always much more potent Eis
inhibitors than those with the five- or the seven-membered ring
A and the same R1 side chain, regardless of the identity of R1,
whereas the molecules with the seven-membered ring A were
generally the least potent of the three (Table 1). This
observation explains why most substitutions were made on the
six-membered ring (Figure 3; family II). For example, the IC50
values for compounds 1g, 2g, and 8g were 1.9 0.6 μM, 0.25
0.02 μM, and 3.1 0.3 μM, respectively. To interpret this
effect, we determined crystal structures of Eis−1g and Eis−2g
complexes (Figure 4). The six-membered ring A of 2g appears
to fill the hydrophobic pocket of Eis more efficiently than the
five-membered ring of 1g. A Cγ atom of Val40 is 3.8 Å away
from the closest C atom of 2g, whereas it is 4.0 Å away in the
case of 1g. Analogously, the Sδ of Met65 is at 4.2 and 4.3 Å
from the closest C atoms in 2g and 1g, respectively. These C−
C distances are likely optimal for the six-membered ring, and
the seven-membered ring of 8g is not accommodated as well.
While this straightforward structural interpretation of the effect
of the ring A size on inhibitor potency explains the general
trend, there are other consequences to ring A size changes that
propagate to the rest of the inhibitor structure and that are not
readily interpretable. A subtle (0.5 Å) positional shift of the
tricyclic cores apparently occurred due to ring A differences,
which resulted in different conformations of the R1 side chain
and the side chain of Glu401, as well as differences in water-
mediated inhibitor−Eis interactions involving these moieties.
The differences in the R1 conformations resulting from the
positional shifts of the cores with different A rings must be
responsible in rare cases for the exceptions of the general rank
order of potencies for A ring sizes (6 > 5 > 7). Such a rare
exception was observed for inhibitor 8h with the seven-
membered ring, which exhibited the highest potency among
the compounds with this R1 side chain. This potency was
similar to that of 2h with the six-membered ring: the IC50
values for 1h, 2h, and 8h were 2.5 0.4 μM, 0.46 0.06 μM,
and 0.30 0.06 μM, respectively.
To investigate this effect in more detail, we determined
crystal structures of Eis−1h and Eis−8h complexes (Figure 5).
As anticipated, the seven-membered ring A of 8h is bent
against the hydrophobic pocket of Eis, and its large size caused
a 0.5 Å shift of the thieno[2,3-d]pyrimide core with respect to
that of 1h, resulting in a different conformation of R1, where it
makes additional hydrophobic contacts with the indole ring of
Trp36 and positions the tertiary amine of R1 closer to an Oδ of
Asp26 (N−O distance of 3.6 Å) than in 1h (N−O distance of
blocks 17−24 were reacted with benzoyl isothiocyanate in 1,4-
dioxane to afford the thioureas (25−32). The intermediates
25−32 were cyclized in 2 N NaOH and EtOH to afford the
thieno[2,3-d]pyrimide core scaffolds (33−40). Side chains
were introduced to compounds 33−40 via thiol alkylations to
afford the final products 1−8(a−i). Cesium carbonate was
found to be superior to potassium carbonate in terms of
reaction time and yields. Please note that alkyl halides 41−44
E
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a
Table 1. IC50 and MIC Values
K204 MICKAN
H37Rv mc2
6230 (6206)
MIC (μg/mL)
H37Rv mc2
K204 MICKAN
H37Rv mc2
6230 (6206)
MIC (μg/mL)
H37Rv mc2
6230 (6206)
MIC (μM)
(μg/mL) with
6230 (6206)
(μg/mL) with
b
b
cmpd
IC50 (μM)
100 μM cmpd
MIC (μM)
cmpd
IC50 (μM)
100 μM cmpd
d
KAN
1a
2a
3a
4a
5a
6a
7a
8a
N/A
>10, >10
10, 10
5, 5
<1
8
4
2−4
4
>128
4
4
4
64
32
32
8
4 (8)
4 (4−8)
16
32
32−64
64
<1.72
25
12
5.7−12
12
>353
10
9.7
11
191
92
88
22
11 (21)
9.9 (9.9−20)
38
88
92−183
176
42
42
41
76
9.1 (18)
5e
6e
7e
8e
1f
2f
3f
4f
5f
6f
7f
8f
7.0 0.7
4.6 0.6
0.46 0.17
2.5 0.1
10, 10
toxic
toxic
5, toxic
10, 10
5, 5
>10, >10
10, 10
NT
NT
toxic
10, 10
NT
2.5, 2.5
NT
>128
4 (8)
8
>340
9.9 (20)
19
2.2 0.3
0.99 0.06
6.7 0.7
2.4 0.2
>200
10, 10
5, 2.5, toxic
NT
32
88
11
1.5 0.1
52
1
>128
>128
>128
128
64
>363
>350
>337
337
162
>303
36
>263
>337
>325
314
157
38
>284
68
20
>327
>315
>305
152
23
5
toxic
toxic
10, >10
5, 5
5, 5
10, 10
5, 10
toxic
toxic
10, 10
toxic
2.5, 2.5
2.5, 2.5
5, 5, 2.5
toxic
toxic
toxic
7
4.0 1.1
6.6 0.6
1.9 0.2
1.1 0.1
14
2.0 0.3
5.4 0.5
16
16
7.6 1.1
0.75 0.06
0.17 0.04
3.8 0.5
0.61 0.08
0.91 0.13
6.6 1.2
3.6 0.4
1.1 0.1
∼200
3.0 0.6
8.7 1.2
1.9 0.6
0.25 0.02
∼2.4
0.20 0.03
1.1 0.2
>200
0.48 0.06
3.1 0.3
2.5 0.4
0.46 0.06
6.8 0.6
3.7 0.6
0.68 0.09
1b
2b
3b
4b
5b
6b
7b
8b
>128
16
1
>100
>128
>128
128
64
16
>128
32
c
1g
2g
c
2
2
3g
4g
5g
6g
7g
8g
5, 2.5
toxic
NT
toxic
5, 5
c
1c
2c
3c
4c
16
16
16
32
4 (8)
16
8
c
c
1h
5, 5
>128
>128
>128
64
5c
6c
2h
3h
4h
2.5, 2.5
10, 10
2.5, 2.5
toxic
c
7c
c
0.23 0.08
1.2 0.1
toxic
toxic
c
8c
42
5h
32
74
1d
2d
3d
4d
5d
6d
7d
8d
1e
1.8 0.1
0.45 0.03
3.6 0.2
0.81 0.13
>200
6.3 1.5
0.53 0.04
2.3 0.2
0.80 0.10
0.15 0.02
2.0 0.1
5, 5
5, 5
2.5, 5
toxic
NT
16
16
16
8
>128
4 (8−16)
16
16
128
64
48
46
44
22
6h
7h
8h
10
4
toxic
toxic
NT
5, 5
2.5, 2.5
10, 10
1.25, 2.5, 2.5
5, 5
toxic
5, toxic, toxic
5, 5
16
8
35
17
0.93 0.29
0.30 0.06
17
1.6 0.2
56
0.47 0.08
19
>200
16
c
>128
>128
>128
>128
>100
128
32
>305
>325
>314
>303
>237
294
69
66
>303
1i
2i
1
c
>340
9.9 (20−40)
38
44
382
183
176
21
toxic
NT
3i
4i
5i
6i
7i
8i
6
5, 10
10, 5
2.5, 2.5
5, 2.5, 5, 5
toxic
3
c
2e
3
32
>128
3e
4e
64
8
8.5 1.3
c
0.36 0.05
a
b
c
N/A = not applicable; NT = not tested. Multiple MIC values are the results of independent experiments. X-ray structures of Eis in complex with
d
these inhibitors are presented in this study. Note that the MIC of KAN in the parent H37Rv from which the K204 strain is derived is 1.25 μg/mL.
4.2 Å). Shifts in positions of bound waters in the vicinity of this
interface were also observed. The significant conformational
changes that propagate through the inhibitor and the
surrounding solvent, ultimately accounting for the observed
effects on inhibitor potency, would not have been possible to
model based on the single crystal structure of Eis in a complex
with the parent compound or based on molecular docking.
Effect of Substituents R2 and R3 in Family II. Next, we
planned to verify the effect of various substitutions on ring A of
the core, by considering the effect of R2 and R3 substituents
(series 2−7; Figure 3 while keeping the R1 side chain (a−i)
constant. We found that for each R1, regardless of its identity,
the compounds with no substituents at R2 and R3 (series 2)
were nearly always the most potent. Even the small methyl
group at R2 (series 3) was not tolerated, generally resulting in
at least a 10-fold higher IC50 value than that of its
unsubstituted counterpart from series 2. Indeed, the methyl
group at R2, depending on the stereoconfiguration, would
sterically clash with the Sγ of Met65 or with the Cβ atom of
Phe84, because the distances from the C atom of the core and
the respective Sγ and Cβ are 4.2 and 4.1 Å along the potential
C−C bond between the core and the R2 methyl group (Figure
4B). Nonpolar substituents, such as a methyl, an ethyl, and a
phenyl group at R3, were well tolerated while a tert-butyl
substituent was the least tolerated, regardless of R1 (Table 1).
The relatively small methyl and ethyl substituents at R3 were
predicted to be less hindered than at R2, because in either
stereoconfiguration they would not clash with the protein
(Figure 4B). Furthermore, the flexible side chain of Arg37
could change its conformation to enlarge the binding pocket.
On the other hand, a tert-butyl group (series 6) or a phenyl
ring (series 7) would not be readily accommodated. Indeed,
series 6 nearly always contained the least potent compounds.
Unexpectedly, some of compounds in series 7 were highly
potent Eis inhibitors. To learn about the structural
consequences of a methyl substitution at R3, we determined
crystal structures of Eis in complexes with highly potent
inhibitors 2e (IC50 = 0.15 0.02 μM; PDB ID 6VUR) and 4e
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Figure 4. Zoomed-in views of the Eis−inhibitor interfaces from crystal structures of Eis−1g and Eis−2g complexes, and the inhibitor structures:
(A) Eis−1g interface and (B) Eis−2g interface.
Figure 5. Zoomed-in views of the Eis−inhibitor interfaces from crystal structures of Eis−1h and Eis−8h complexes and the inhibitor structures.
(A) Eis−1h interface and (B) Eis−2h interface.
(IC50 = 0.36 0.05 μM; PDB ID 6VUW), which differ from
each other only by the presence of a methyl group at R3 in 4e
(Figure 6). As anticipated, the methyl group was accom-
modated largely owing to a change of the conformation of the
side chain of Arg37 and a minor shift by 0.4 Å of the inhibitor.
The methyl group interacts favorably with Arg37 and Trp13,
with the closest C−C distances of 3.8 and 3.6 Å, respectively.
To investigate how a bulky phenyl group was accommodated
at R3, we determined a crystal structure of Eis in a complex
with the most potent inhibitor in series 7, 7c (IC50 = 0.23
0.08 μM; PDB ID 6VUY; Figure 7A). As expected, large
conformational changes had to occur to fit the phenyl group
into the binding pocket. The indole ring of Trp13 rotated by
90° around the Cβ-Cγ bond and stacked orthogonally against
the R3 phenyl group of 7c. In turn, the side chain of Arg37
changed conformation to stack orthogonally with the indole of
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Figure 6. Zoomed-in views of the Eis−inhibitor interfaces from crystal structures of Eis−2e and Eis−4e complexes and the inhibitor structures: (A)
Eis−2e interface and (B) Eis−4e interface.
Trp13 against its edge. Notably, computational molecular
docking, which relies heavily on rigid-body assumptions, would
not predict that 7c is a potent inhibitor of Eis.
Effects of R1 Substituents for Each of the Three Inhibitor
Families. The effect of different R1 substituents is complex,
because the R1 side chain is extended into a large hydrophilic
cleft lined with polar and charged residues. SAR have emerged,
as follows.
conformations, all related by rotations along the C−N bond
connecting the ring to the R1 linker.
c. Effect of the Position of the Tertiary Amino Group in R1.
Since all R1 side chains contain a tertiary amino group in them,
we examined the impact of the position of the tertiary amino
group in the pyrrolidine ring on the potency of the compounds
(R1 = a vs d). We observed that the ring nitrogen that is not
directly attached to the 2-carbon linker (R1 = d) is preferred
(Table 1). Having noted this, we then asked if we could further
improve potency by keeping four bonds between the ring
nitrogen and the core but enlarging the R1 ring to the
piperidine (R1 = d vs e). In all but two series, 7 and 8, the
inhibitors with the piperidine ring (e) were more potent than
those with the pyrrolidone ring (d), and for series 7 and 8 the
potency was not affected. The crystal structures of Eis−2e and
Eis−4e complexes (Figure 6) showed that in series e, the
tertiary amino group is placed directly between the carboxyl
groups of Asp26 and the C-terminus for salt bridge formation,
whereas the aliphatic side of the appropriately sized piperidine
ring interacts with the aliphatic portion of the side chain of
Glu401, explaining the observed trend of potencies.
d. Substituents with an Amide in R1. We explored a
potential benefit from further lengthening of an R1 linker and
an introduction of an amide into it, to add polar character. In
addition, we assessed the variability of the terminal R1 groups,
which included a dimethyl amine (f), a diethyl amine (g), a
piperidine (h), and a morpholine (i) groups. The compounds
with a diethyl amino group (g) and a piperidine ring (h) were
more potent that those with the other two choices of the R1
termini (Table 1). To examine the interactions of side chains g
and h with Eis, in addition to the structures of Eis in complex
with the potent inhibitors 2g (Figure 4B) and 8h (Figure 5B)
described above, we determined the crystal structure of Eis in
complex with inhibitor 5h (IC50 = 0.68 0.09 μM; PDB ID
6VUX; Figure 8), the most potent inhibitor in series 5.
Apparently, the side chain R1 = h overcame the unfavorable
effect of the ethyl group at R3 observed with other choices of
a. Effect of the Ring Size at the Terminus of R1. First, we
investigated an effect of a terminal pyrrolidine (R1 = a) vs a
piperidine ring (R1 = b) for the same 2-carbon linker. These
substituents generally did not lead to significant improvement
in potency from that of the parent compound 2i. In families I
and III and in series 2 and 4 of family II, the R1 ring size had
no effect. In the rest of the series of family II the effect was
generally 2-fold or greater, favoring either the five- or the six-
membered ring. In all of these last cases, the inhibitors had
relatively low potencies (in the IC50 range from 4.0 1.1 μM
to >200 μM).
b. Effect of the Length of the Linker in R1. To test the
impact of the length of the R1 linker, we compared compounds
with a 2-carbon linker to those with a 3-carbon R1 linker, both
containing a terminal piperidine ring (series b vs c). The
longer, 3-carbon linker (R1 = c), was strongly favored over the
2-carbon linker (R1 = b) in all three families (Table 1). In
order to visualize the structural basis for this significant
improvement, in addition to Eis−7c, we determined crystal
structures of Eis in complexes with three additional potent
inhibitors: 1c (IC50 = 0.75 0.06 μM; PDB ID 6VUZ), 4c
(IC50 = 0.61 0.08 μM; PDB ID 6VUU), and 8c (IC50 = 1.2
0.1 μM; PDB ID 6VV2) (Figure 7). These structures
revealed that this longer R1 linker places the positively charged
amino group of the piperidine so that it is equidistant from the
carboxyl groups of Asp26, Glu401, and the C-terminal carboxyl
group, all three at appropriate distances for strong salt bridge
formation. The piperidine ring was found in different
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Figure 7. Zoomed-in views of the Eis−inhibitor interfaces from crystal structures of Eis−7c, Eis−1c, Eis−4c, and Eis−8c complexes and the
inhibitor structures: (A) Eis−7c interface, (B) Eis−1c interface, (C) Eis−4c interface, and (D) Eis−8c interface.
R1 in this series. Analogously, the side chain R1 = h was the
most favored in family 8. In all these three structures, the R1
side chain is in the same U-turn conformation, where the
thioether moiety is coplanar with the tricyclic core and the
amide is solvent exposed. The tertiary amino group formed a
salt bridge with Asp26. The diethyl group of 2g and the
piperidine rings of 5h and 8h abutted the phenyl ring of Phe24,
explaining why the morpholine ring in series i containing a
polar oxygen was less preferred. The rigidity of the amide-
containing linker and the restraint imposed by Phe24 likely
accounted for the high potency of these molecules. Other
structural factors seemed to modulate the potency. For
example, the ethyl group at R3 of 5h wedged against Trp13
and Arg37, pushing the latter off and compromising its
stacking against Trp13, likely caused a 2-fold weaker potency
of this analogue than those of the equipotent 2g and 8h, in
which ring A was unsubstituted.
Buried Surface Area of Eis−Inhibitor Complexes.
Binding of macromolecules to each other and to druglike
molecules commonly depends on geometrical and physical
complementarity of the two interacting interfaces. Upon such
binding, water is excluded from the interface between the
nonpolar surfaces, providing a favorable entropic contribution
in addition to the favorable van der Waals energy gain by the
I
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conformational freedom of the polar and charged side chains
upon inhibitor binding may be another contributor. These
calculations are consistent with the notion that the interactions
between complementary nonpolar surfaces are drivers of
inhibitor potency and they advocate for frugal and rational
incorporation of polar and charge groups.
Competitive Inhibition Mode. All 12 crystal structures of
Eis−inhibitor complexes presented here show that the
inhibitors are bound at the Eis active site in the pocket that
overlaps with the aminoglycoside substrate binding pocket
(Figure S224), strongly suggesting that these molecules inhibit
the acetyl transfer by competing with the aminoglycosides for
binding Eis. We tested this directly by monitoring kinetics of
acetylation of KAN by Eis in the steady-state, as a function of
KAN in the absence of the inhibitor and at several
concentrations of each of three potent inhibitors, 4c, 7c, and
4g (Figure 9). These data show that, within experimental
uncertainty, these inhibitors display a competitive (with
respect to KAN) inhibition mode, in agreement with the
crystal structures. The global fit of the competitive mode of
inhibition to these data yielded the Ki values for these three
inhibitors (Table 2), all in the 0.15−0.34 μM range.
Effect of Inhibitors on the MICKAN against Mtb in
Vitro. In addition to the parent inhibitor 2i, we measured the
effect of other Eis inhibitors at 100 μM on KAN resistance
caused by Eis overexpression (Table 1). Inhibitors that had
relatively low potencies (IC50 > 10 μM) generally had no effect
on the MICKAN of the KAN-resistant Mtb strain K204, whereas
compounds that were highly potent generally lowered the
MICKAN as much as 4-fold, to 2.5 μg/mL. Intriguingly, while
many of the compounds did not affect the growth of Mtb K204
in the absence of KAN, several compounds from families II
and III were toxic (at MIC < 100 μM) to this strain even in the
absence of KAN, which means that the compounds act via an
additional mechanism besides Eis inhibition. This unknown
mechanism merits investigation in the future. As a control, we
also determined MIC values of all the inhibitors in the absence
of KAN against another Mtb strain that contained the wild-
type eis promoter, H37Rv mc2 6230 (Table 1). In agreement
with the inhibitory effect on growth of Mtb K204, these
compounds displayed MIC < 100 μM, some inhibitors as low
as 9 μM. Five of the molecules were also tested against the
culture of another H37Rv variant, mc2 6206. These MIC
values were comparable to those for Mtb H37Rv mc2 6230
(Table 1).
Mammalian Cytotoxicity. The cytotoxic effect of three
Eis inhibitors and KAN was evaluated against three
mammalian cell lines: human adenocarcinoma (A549),
human embryonic kidney (HEK-293), and mouse macrophage
(J774A.1) (Figure 10 and Figure S230). Inhibitors 2e and 2i
were chosen as at 100 μM they restore the activity of KAN in
Mtb K204 (Table 1). Compound 7c was chosen as it is toxic to
Mtb strains with MIC values of 4 and 8 μM for strains H37Rv
mc2 6230 and mc2 6206, respectively. We observed that 7c
displayed toxicity against all three cell lines at 25 μM. Against
A549 cells, 7c displayed no toxicity at 6.3 μM and
approximately 20% cell survival at 12.5 μM. Similarly, against
the J774A.1 cell line, no toxicity was observed up to 3.1 μM,
with 30% cell survival at 6.3 μM. These values are very similar
to the concentrations that are toxic to both strains of Mtb
tested. In contrast, inhibitors 2e and 2i displayed no toxic
effect at the highest concentration tested, 50 μM, which is
highly promising.
Figure 8. Zoomed-in view of the Eis−inhibitor interface from crystal
structure of Eis−5h complex and the structure of 5h.
contact of the two properly spaced surfaces. Formation of the
interface between the two polar or charged surfaces, in
addition to the geometrical complementarity similarly needed
to ensure favorable van der Waals interactions, requires that
the energy stored in hydrogen bonds and salt bridges involved
in hydration and solvation of such surfaces is not lost upon
binding. Therefore, one can observe that in the interfaces
between polar surfaces buried from bulk solvent, hydrogen
bonds, and salt bridges are formed across the interface (when
solvent is also excluded with a favorable gain of entropy) or
with water molecules or ions trapped in the interface, without
losing these energetically costly interactions. With the benefit
of obtaining a set of crystal structures of Eis in complexes with
12 inhibitors, we calculated23 the total solvent accessible
surface area buried in the interface between the protein and the
inhibitors (ASAb,t) and examined whether there is any trend in
the inhibitor potency with respect to ASAb,t. We also
considered separately the buried nonpolar (ASAb,np) and
polar (ASAb,p) surface areas (Figure S229). ASAb,np comprised
approximately two-thirds of ASAb,t and, as expected based on
the complementarity principles discussed above, was the
dominant contribution to the trend in ASAb,t with respect to
IC50. The larger ASAb,t and ASAb,np were associated with higher
potency (smaller IC50 values), underscoring the importance of
hydrophobic interactions, although the correlation was
relatively weak: R = 0.040 and −0.259, respectively. To our
surprise, ASAb,p had a stronger correlation with the opposite
trend (R = 0.691), where the larger ASAb,p were associated
with lower potency. We interpreted this to mean that in
burying polar surfaces, the resulting hydrogen bonds and salt
bridges in the interface were not as favorable as those with bulk
solvent in the unbound state. Additionally, the loss of the
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Figure 9. Analysis of inhibition kinetics for inhibitors 4c, 7c, and 4g. Dose−response curves are shown in panels A, D, and G; Michaelis−Menten
analysis is in panels B, E, and H; and the Lineweaver−Burk representation of this analysis is in panels C, F, and I. These data indicate that the
molecules are competitive with KAN. These data are used in obtaining Ki by global nonlinear regression (Table 2).
Table 2. Kinetic Parameters of Eis and Ki Values for Inhibitors 4c, 4g, and 7c
cmpd
[inhibitor] (μM)
Km,KAN (μM)
kcat (s−1
)
k
cat/Km,KAN (s−1 μM−1
)
Ki (μM)
4c
0
460 150
460 120
530 120
650 150
310 74
370 59
440 82
390 64
510 160
500 140
490 140
1200 400
14
2
0.031 0.01
0.021 0.006
0.017 0.005
0.013 0.003
0.036 0.009
0.027 0.004
0.020 0.004
0.016 0.003
0.029 0.01
0.027 0.008
0.022 0.007
0.011 0.004
0.34 0.02
0.4
0.6
0.8
0
0.1
0.2
0.4
0
9.8 1.2
8.9 1.0
8.3 1.0
4g
7c
11
1
0.15 0.01
0.31 0.03
9.6 0.6
8.8 0.7
6.4 0.4
15
14
11
13
2
2
1
3
0.125
0.25
0.5
Microsomal Stability. In any drug discovery process,
preclinical drug metabolism assessment plays a vital role in
drug optimization. We tested the metabolic stability in human
and mouse microsomes of several compounds in family II that
were selected to represent the structural diversity of the family
and predicted as stable by using StarDrop software
(Optibrium) (Table 3). As expected, stabilities in human
microsomes were grouped according to the nature of the R1
side chain, whereas in mouse microsomes most compounds
were quite labile. Two compounds (6i and 7i) were completely
metabolized by both human and mouse microsomes, while
most of the other tested compounds were modestly
metabolized by human microsomes. For these and several
other molecules, the metabolic liability can be predicted. The
instability of 7i may be due to the presence of an unsubstituted
phenyl ring with oxidation at the para position of the phenyl
ring or on the morpholine ring as well as hydrolysis of the
amide bond. Similarly, 6i could be metabolized by oxidation at
three major sites, namely, the tert-butyl group, the amide
linker, and the sulfur linkage. Several compounds showed
moderate stability (25−40% metabolized) with human micro-
somes (7a, 4c, 4d, 7e). Metabolism of 4d, 6d, and 7e is likely
to be demethylation. Several compounds had improved
stability with <25% metabolized in human microsomes (6a,
K
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Table 3. Metabolic Stability of Eis Inhibitors
inhibitor
human
mouse
53
38 16
99
52 21
47 25
a
6a
7a
4c
5c
7c
4d
6d
7e
7h
6i
23 21
36 24
5
31
7
4
5
0
22
38
20
38
47
100
94
2
6
2
4
5
99
0
62 16
75
58
3
4
0
7
100
100
0
0
7i
a
The values in the table are % metabolized compound after a 30 min
incubation with microsomes. The data are the mean standard
deviation for a minimum of three samples (except 4d, which was
tested in duplicates).
CONCLUSION
■
A new class of Eis inhibitors having thieno[2,3-d]pyrimidine
core was discovered, with various analogues synthesized and
rationally optimized guided by structural studies, followed by
their extensive characterization using chemical, biochemical,
structural, and biological studies. Several analogues with potent
inhibitory activity against purified Eis were able to restore the
MICKAN in cellular assays, while displaying no toxicity against
either Mtb or mammalian cells on their own. For compounds
that displayed toxicity against Mtb, it remains to be
investigated whether all these compounds are also toxic to
mammalian cells (inhibitor 7c is toxic to both). In vitro
metabolism assays show that several side chains were tolerated
well metabolically, with R1 = c being especially promising.
Pending further toxicity testing, compounds 1c and 2c, which
are nontoxic to Mtb on their own, but potent in Eis inhibition
and synergistic with KAN, emerged as promising compounds
for further testing.
EXPERIMENTAL SECTION
■
Details of all experimental procedures for synthesis and character-
ization of all compounds and intermediates, HTS, hit validation,
selectivity of inhibitors toward Eis, Mtb MIC value determination,
dose-dependent Mtb MIC values determination, crystallization, and
crystal structure determination of Eis−inhibitor complexes, solvent
accessible surface area calculations, and metabolic stability assays are
included in the Supporting Information. The Supporting Information
also includes all 1H and 13C NMR spectra as well as HPLC traces for
the molecules generated (Figures S1−S222).
Figure 10. Evaluation of cytotoxicity for compounds 2e (red), 2i
(yellow), 7c (purple), and KAN (gray) against three cell lines: (A)
A549, (B) HEK-293, and (C) J774A.1. Controls include treatment
with Triton-X (TX, 1% v/v, positive control) and 0.5% DMSO
(negative control). It is important to note that testing xenobiotics at
sub-IC50 concentrations can result in increase in cell growth, resulting
in >100% cell survival in the treatment groups. In instances where
>100% cell survival was observed, we displayed the data as 100% cell
survival. Note: The raw data are presented in Figure S230.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschembio.0c00184.
■
sı
Additional details of the chemistry and the biochemical,
biological, and biophysical assays (PDF)
Accession Codes
5c, 7c, 6d). The molecules that were most labile in the human
microsomes were also highly labile in the mouse microsomes,
but the converse was not true. For example, 6d and 5c were
stable with human microsomes (20% and 7%, respectively),
but not with mouse microsomes (62% and 52%, respectively).
PDB accession codes: Eis−1c (6VUZ), Eis−1g (6VV0), Eis−
1h (6VV1), Eis−2e (6VUR), Eis−2g (6VUS), Eis−2i
(6VUT), Eis−4c (6VUU), Eis−4e (6VUW), Eis−5h
(6VUX), Eis−7c (6VUY), Eis−8c (6VV2), Eis−8h (6VV3).
L
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UK PharmNMR Center (in the College of Pharmacy) for
NMR support and S. Van Lanen for managing the LCMS
system used in this study. We thank S. Vander Roest, M.
Larsen, and P. Kirchhoff from the CCG at the University of
Michigan for their help with HTS. We thank J. J. Johnson and
R. M. Holmes for synthesizing and characterizing a few of the
molecules presented in this manuscript. We thank C. Hou for
assistance with the protein purification and crystallization. We
also thank S. Chowdhury and B. Berube for discussion and
technical assistance with metabolic stability assays. Use of trade
names is for identification only and does not constitute
endorsement by the U.S. Department of Health and Human
Services, the U.S. Public Health Service, or the CDC. The
findings and conclusions in this report are those of the authors
and do not necessarily represent the views of the funding
agency.
AUTHOR INFORMATION
Corresponding Authors
■
Sylvie Garneau-Tsodikova − Department of Pharmaceutical
Sciences, University of Kentucky, Lexington, Kentucky 40536-
0596, United States; orcid.org/0000-0002-7961-5555;
Phone: 859-218-1686; Email: sylviegtsodikova@uky.edu
Oleg V. Tsodikov − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40536-0596,
United States; orcid.org/0000-0001-7094-4216;
Phone: 859-218-1687; Email: oleg.tsodikov@uky.edu
Authors
Ankita Punetha − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40536-0596,
United States
Huy X. Ngo − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40536-0596,
United States
Selina Y. L. Holbrook − Department of Pharmaceutical
Sciences, University of Kentucky, Lexington, Kentucky 40536-
0596, United States
Keith D. Green − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40536-0596,
United States
Melisa J. Willby − Division of Tuberculosis Elimination,
National Center for HIV/AIDS, Viral Hepatitis, STD, and TB
Prevention, Centers for Disease Control and Prevention, Atlanta,
Georgia 30333, United States
Shilah A. Bonnett − TB Discovery Research, Infectious Disease
Research Institute, Seattle, Washington 98102, United States
Kyle Krieger − TB Discovery Research, Infectious Disease
Research Institute, Seattle, Washington 98102, United States;
Center for Global Infectious Disease, Seattle Children’s Research
Institute, Seattle Children’s Hospital, Seattle, Washington
98145, United States
Emily K. Dennis − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40536-0596,
United States
James E. Posey − Division of Tuberculosis Elimination, National
Center for HIV/AIDS, Viral Hepatitis, STD, and TB
Prevention, Centers for Disease Control and Prevention, Atlanta,
Georgia 30333, United States
ABBREVIATIONS
■
AcCoA, acetyl coenzyme A; ASA, accessible surface area; Eis,
enhanced intracellular survival; HPLC, high-performance
liquid chromatography; HTS, high-throughput screening;;
IC50, inhibitory concentration at half-maximum inhibition;
KAN, kanamycin; MDR, multidrug-resistant; MIC, minimum
inhibitory concentration; Mtb, Mycobacterium tuberculosis;
PDB, Protein Data Bank; SAR, structure−activity relationship;
TB, tuberculosis; XDR, extensively drug-resistant
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■
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Tanya Parish − TB Discovery Research, Infectious Disease
Research Institute, Seattle, Washington 98102, United States;
Center for Global Infectious Disease, Seattle Children’s Research
Institute, Seattle Children’s Hospital, Seattle, Washington
98145, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschembio.0c00184
Author Contributions
⊥A.P. and H.X.N. contributed equally to this work.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This study was funded by a National Institutes of Health
(NIH) Grant AI090048 (to S.G.-T.), a grant from the Firland
Foundation (to S.G.-T.), a grant from the Center for Chemical
Genomics (CCG) at the University of Michigan (to S.G.-T.),
as well as by startup funds from the College of Pharmacy at the
University of Kentucky (to S.G.-T. and O.V.T). We thank the
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