Selective inhibitors of bacterial t-RNA-(N1G37) methyltransferase (TrmD) that demonstrate novel ordering of the lid domain

Article abstract of DOI:10.1021/jm400718n

The tRNA-(N¹G37) methyltransferase (TrmD) is essential for growth and highly conserved in both Gram-positive and Gram-negative bacterial pathogens. Additionally, TrmD is very distinct from its human orthologue TRM5 and thus is a suitable target for the design of novel antibacterials. Screening of a collection of compound fragments using Haemophilus influenzae TrmD identified inhibitory, fused thieno-pyrimidones that were competitive with S-adenosylmethionine (SAM), the physiological methyl donor substrate. Guided by X-ray cocrystal structures, fragment 1 was elaborated into a nanomolar inhibitor of a broad range of Gram-negative TrmD isozymes. These compounds demonstrated no activity against representative human SAM utilizing enzymes, PRMT1 and SET7/9. This is the first report of selective, nanomolar inhibitors of TrmD with demonstrated ability to order the TrmD lid in the absence of tRNA.

Full text of DOI:10.1021/jm400718n

Article
pubs.acs.org/jmc
Selective Inhibitors of Bacterial t‑RNA-(N1G37) Methyltransferase
(TrmD) That Demonstrate Novel Ordering of the Lid Domain
Pamela J. Hill,*,† Ayome Abibi,‡ Robert Albert,† Beth Andrews,‡ Moriah M. Gagnon,† Ning Gao,‡
Tyler Grebe,† Laurel I. Hajec,‡ Jian Huang,‡ Stephania Livchak,‡ Sushmita D. Lahiri,‡
David C. McKinney,† Jason Thresher,‡ Hongming Wang,† Nelson Olivier,§ and Ed T. Buurman‡
‡
§
†Departments of Chemistry, Biosciences and Discovery Sciences, Infection Innovative Medicines Unit, AstraZeneca R&D Boston,
35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: The tRNA-(N1G37) methyltransferase (TrmD) is
essential for growth and highly conserved in both Gram-positive and
Gram-negative bacterial pathogens. Additionally, TrmD is very distinct
from its human orthologue TRM5 and thus is a suitable target for the
design of novel antibacterials. Screening of a collection of compound
fragments using Haemophilus influenzae TrmD identified inhibitory,
fused thieno-pyrimidones that were competitive with S-adenosylme-
thionine (SAM), the physiological methyl donor substrate. Guided by
X-ray cocrystal structures, fragment 1 was elaborated into a nanomolar
inhibitor of a broad range of Gram-negative TrmD isozymes. These
compounds demonstrated no activity against representative human
SAM utilizing enzymes, PRMT1 and SET7/9. This is the first report of selective, nanomolar inhibitors of TrmD with
demonstrated ability to order the TrmD lid in the absence of tRNA.
both a large pocket and the methionine moiety of the substrate,
further ordering of the linker would probably require binding of
the tRNA substrate. However, such structures have not yet been
published.
INTRODUCTION
■
Although all tRNAs are composed of nucleosides A, C, U, and G,
a total of 81 different modifications have been described.1
Methylation at the N1-position of guanosines immediately 3′ to
the anticodon of seven tRNAs (m1G37) is present in virtually all
species in the three kingdoms of life, suggesting an ancient and
important function in translation.2,3 The presence of the
modification prevents Watson−Crick base-pairing of this
guanosine with cytosine in mRNA and translational frame-
shifting that would result as a consequence.4
The methylation of G37 in bacteria is performed by tRNA-
(N1G37) methyltransferase (TrmD)5 and has been shown to be
essential for growth in a range of bacterial species.6−11 A TrmD
orthologue in Archaebacteria could not be identified by sequence
homology in genome databases. However, a functional, yet
structurally distinct, homologue of TrmD was found by selection
of a genome library of Methanococcus vannielii for complementa-
tion of a growth defect due to a temperature sensitive trmD allele
in Salmonella typhimurium.2 This led to the identification of
TrmD in Methanococcus jannaschii and subsequently the
Saccharomyces cerevisiae orthologue TRM5.2
Although TRM5 performs an identical function to TrmD, the
two seem to have evolved independently. The most striking
difference for drug discovery purposes is the conformation with
which SAM binds to the respective enzymes. In TrmD, the
trefoil-shaped binding site binds the substrate in a bent
conformation, whereas the substrate binds in an open
conformation with TRM5.15 This strongly indicates that TrmD
inhibitors that bind in the SAM binding site will be selective,
without showing mechanism-based toxicity toward mammalian
cells.
Therefore, we set out to identify chemical starting points that
inhibit TrmD by binding in the SAM binding pocket and thereby
preventing methylation of tRNA. Co-crystallization and
structure elucidation of one such fragment bound to H. influenzae
TrmD demonstrated binding in the adenosine portion of the
pocket. The ensuing structure-guided effort allowed extension of
the fragments and additional binding in the methionine portion
of the SAM pocket, thus yielding broad-spectrum nanomolar
inhibitors of TrmD.
X-ray crystal structures of TrmD of Escherichia coli12 and
Haemophilus influenzae13 showed the protein as a homodimer
with the S-adenosylmethionine (SAM) binding site at the
interface. This suggests that under physiological conditions
TrmD exists as a dimer, a fact that was corroborated in
Streptococcus pneumoniae.14 Each monomer consists of two
domains held together by an ill-defined linker that showed
improved ordering upon binding of SAM. Given the proximity to
Received: May 14, 2013
Published: August 27, 2013
© 2013 American Chemical Society
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fragment, as this position was occupied by carbon C2. A
hydrogen bond not present between SAM and H. influenzae
TrmD was observed between the Oγ of S132 and N7 of the
pyrimidone ring in compound 1.
RESULTS AND DISCUSSION
■
Fragment Screening. An AstraZeneca fragment collection
(14 000 compounds) was screened at a compound concentration
of 100 μM to identify inhibitors of H. influenzae TrmD using a
biochemical assay monitoring methylation of tRNA. The
resulting hits were then screened for activity using a similar
assay with Staphylococcus aureus as a representative Gram-
positive isozyme. This secondary screen selected for compounds
with the potential to be broad spectrum inhibitors. Sinefungin, a
natural product derivative of SAM, was used as a positive control.
All fragments that displayed IC50 < 50 μM in both assays were
cocrystallized with H. influenzae TrmD to determine the binding
location. X-ray cocrystal structures revealed that several
fragments bound in the SAM binding pocket (Figure 1). All
Lead Generation. The X-ray cocrystal structure of 1 with H.
influenzae TrmD shows a very tight binding pocket around the
pyrimidone core and suggests few options for substitution other
than at the 3-position. Maintaining this substitution trajectory, a
series of alternate heterocycles was fused to the pyrimidone core
to probe the H-bond donor−acceptor tolerability of the binding
site (Table 1). A hydrogen bond donor at position 1 (4, 5) was
not optimal for inhibition of Gram-negative activity, while the
Gram-positive isozyme appeared more forgiving. A five-
membered ring was preferred to a six-membered ring, likely
due to the resulting vector with which the phenyl substituent is
forced to adopt in the six-member example. Both 1 and 2
demonstrated good potency against all isozymes tested.
However, the isoxazole was not as amenable to varying the
substitution at the 3-position. The larger atomic radius of the
sulfur atom in compound 1 results in the substituent at the 3-
position, taking on a nonplanar orientation relative to the core.
This lowest energy conformation is identical to the binding
conformation. In the isoxazole ring, the 3-position substituent
takes on a planar geometry relative to the core. For the phenyl
substitution there was little impact; however, for most other
substitutions, this resulted in a significant entropic penalty and
resulting loss in potency to adopt the binding conformation.
Therefore, 1 was the lead compound chosen for SAR studies.
Synthesis and Biochemical Structure−Activity Relationship
(SAR) of Thienopyrimidones. The thienopyrimidones were
prepared in three steps (Scheme 1) beginning with the
condensation of disubstituted ketone 6 with ethylcyanoacetate
to form intermediate 7, which was then reacted with elemental
sulfur to form the desired amino thiophene ester 8. Cyclization
with formamidine acetate afforded the desired thienopyrimi-
dones 9−16. Utilizing this sequence, a variety of substituents at
the R1 and R2 positions of the thiophene ring were introduced
and assessed for biochemical activity to establish SAR around the
thiophene ring.
The cocrystal structure of 1 shows the phenyl ring at the 3-
position of the thiophene moiety extending into the ribose
portion of the binding pocket. To gather a better understanding
of the SAR associated with occupying the ribose pocket, a series
of analogues was prepared where the phenyl ring was substituted
or replaced (Table 2). The des-phenyl compound (10) showed
almost an order of magnitude loss in potency against E. coli with
an IC50 of 120 versus 17 μM for compound 1. This demonstrated
the importance of occupying the ribose pocket for activity.
Substituting off the para position of the phenyl ring with a small
lipophilic group improved potency 2-fold (11). The phenyl ring
was replaced with a variety of aromatic heterocycles, of which the
most potent was the pyrazole variant 12 at 2.1 μM. Saturated ring
systems (13−15) resulted in a significant loss in activity. A series
of acyl linked substituents was also prepared, of which the amide
16 appeared most promising with an IC50 of 6.5 μM. The phenyl
and pyrazole substituted compounds both displayed good
potency; however, they resulted in an undesirable vector to
further extend substitution into the methionine pocket.
Substitution off the meta or para position would require the
phenyl ring to rotate out of plane disrupting the binding
orientation. Therefore, compound 16 was the focus for further
analogue design.
Figure 1. Hits from fragment screening.
bound fragments were heteroaromatic systems which mimicked
the key acceptor−donor−acceptor hydrogen bonding motif
present in the adenine core of SAM. The low molecular weight
(MW < 300 Da) and good aqueous solubility (>500 μM) of
these fragments made them ideal starting points for SAR studies.
It was apparent that a fused pyrimidone motif was common to
several of the fragments. This structural motif bound in the SAM
pocket in an orientation that should permit chemical
modification to access the methionine portion of the pocket.
As such, this core structure was chosen as the starting point for a
structure-guided chemistry optimization effort.
The cocrystal structure of compound 1 (Figure 2A,B) in
complex with TrmD from H. influenzae was solved at 1.6 Å
resolution in the space group H32 with one protein molecule in
the asymmetric unit (see Supporting Information for data
collection and refinement statistics). By superimposing this
structure with the published H. influenzae structure in complex
with SAM (PDB: 1UAK13), it was found that 1 occupied the
same space as the adenine and ribose of SAM. It was apparent
that the thienopyrimidone ring system of 1 did not superimpose
with the purine ring system of SAM by aligning the adenine N6
with the O6 of the thienopyrimidone. Instead, the binding mode
showed the respective nine-member rings offset by 90°, placing
the phenyl group of 1 in the vicinity of the SAM ribose moiety.
The thienopyrimidone ring system formed key hydrogen bonds
to three of the four protein residues known to interact with the
adenosine base of SAM: I133, Y136, and L138. Additionally, the
hydrophobic contacts between adenine and P89 and P144
confirmed binding. In contrast, N6 from the adenine base of
SAM formed a hydrogen bond with the carbonyl oxygen of
G134. This interaction was not possible for the pyrimidone
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Figure 2. Inhibitor-bound co-crystal structures of H. influenzae TrmD. (A,C,E) Compounds 1, 27, and 38, respectively. Protein monomers of the
biologically relevant dimer are colored yellow and cyan. Dotted lines represent possible hydrogen bonds between protein atoms and inhibitors, labels
indicate residue code and H. influenzae TrmD sequence position of the amino acids. (B,D,F) Superposition of inhibitor-bound H. influenzae TrmD
cocrystal structures against SAM-bound H. influenzae TrmD cocrystal structure (1UAK13), inhibitors are colored yellow, SAM is colored in gray, and
protein atoms have been removed for clarity. (E) 2Fo − Fc electron density map contoured to 1.0σ of compound 38. Hydrogen bonds between inhibitor
and labeled amino acids from both protein monomers of the biologically relevant arrangement are indicated with dashed lines, and hydrogen bonds
between the thienopyrimidone core and protein are omitted for clarity. Amino acid D177 is modeled with its carboxylate in alternate conformations, and
only one conformation is within hydrogen bond distance to the imidazole ring on the compound.
Synthesis and Biochemical SAR of Amide-Linked
Thienopyrimidones. The amide-linked compounds (16,
21−29) were synthesized as shown in Scheme 2. Condensation
of ethylacetoacetate and ethylcyanoacetate followed by cycliza-
tion with elemental sulfur yielded the desired thiophene amino
diester 17. Subsequent cyclization with formamidine acetate
yielded the fused thienopyrimidone ethyl ester (18) which
resulted from transesterfication with ethanol during the
cyclization. Finally, amide 20 was arrived at by heating with the
desired amine or via a two-step procedure of saponification
followed by T3P mediated amide coupling.
Replacement of the phenyl ring of 16 with a pyridyl ring
caused a loss in potency, 20-fold for the ortho isomer (21, Table
3) and 2-fold for the meta and para isomers (22 and 23).
Methylation of the amide N−H (24) caused a significant loss of
activity as did alkylation of the α-methylene (25 and 26). The
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a
Table 1. Biochemical Potency, in IC50s (μM), of Fused Core Variations against Various TrmD Isozymes
a
ND = not determined.
a
Scheme 1. Synthesis of Fused Thienopyrimidones
a
Conditions: (a) NH4OAc, acetic acid, toluene, reflux; (b) sulfur, Et2NH, EtOH, reflux; (c) formamidine acetate, EtOH, reflux.
cocrystal structure suggests that the para position of the phenyl
ring offers the best vector for substitution to occupy the space of
the physiological substrate. To this end, compound 27 bearing a
p-methylamine substituent was made. While both 27 and 28
showed a significant boost in potency, compound 28 was slightly
less active, perhaps indicating some value in hydrogen bond
donor capacity at this position. Saturation of the phenyl ring in
27 led to the cyclohexyl variant (29) which, while still active, was
6-fold less potent. The decrease in activity may be attributed to a
steric clash between the ring system of P89 and the nonplanar
geometry of a saturated cyclohexyl group.
The analysis of the SAM-bound X-ray crystal structure (PDB:
1UAK13) suggests the methionine portion of the binding pocket
would allow binding of the methylene groups of SAM in an
extended conformation. Comparison of the apo- and SAM-
bound X-ray crystal structures suggests little change in
conformation upon substrate binding and, thus, the potential
exists for an inhibitor exploiting the spatial electrostatic
characteristics of SAM. The X-ray cocrystal structure of the
TrmD with compound 27 (Figure 2C,D) was solved to
determine the binding interaction of the phenyl methylamine
moiety. The amide link extends out of the adenine pocket,
forming an intramolecular hydrogen bond between the cognate
nitrogen and the pyrimidone oxygen. The trajectory of the
phenyl ring superimposed with that of the methylene groups
from the ribose of SAM and the methylamine group terminates
in a region similar to where the amino end of the substrate binds
(Figure 2D). Although S170 from the opposing monomer has
been shown to interact with both SAM and S-adenosylhomo-
cysteine (AdoHcy),13 this residue was disordered in the cocrystal
structure of 27. A hydrogen bond between the terminal amine of
27 with the carboxylate side chain of E116 was evident. This
specific interaction was not observed between SAM and TrmD.
E116 is strictly conserved among TrmD orthologues and has
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Table 2. Biochemical Potency, in IC50s (μM), of Variations of the Ribose Pocket Group against Various TrmD Isozymes
a
Scheme 2. Synthesis of Amide Substituted Thienopyrimidones
a
Conditions: (a) (i) NH4OAc, acetic acid, toluene, reflux, (ii) sulfur, Et2NH, EtOH, reflux; (b) formamidine acetate, EtOH, reflux; (c) RNH2,
EtOH, 70 °C; (d) LiOH, MeOH/water, 80 °C; (e) Et3N, T3P, RNH2, DCM.
been proposed to be involved in the recognition of G37 of the
tRNA substrate.13
Our hypothesis was that engaging with D169 could facilitate
stabilization of the residue and concomitantly improve potency.
Replacing the aminomethyl of 27 with a hydroxymethyl (30)
or phenyl (31) resulted in a significant decrease in potency
(Table 4). Changing the electron donating character of the
amine by converting to the amide (32) also showed a decrease in
potency while the amidine (33) was equipotent with 27. To build
into the methionine pocket, substituted amines were inves-
tigated. Propylamine (34) was equipotent to the primary amine
as was methoxyethylamine (35). Incorporating an additional
hydrogen bond donor, like aminoethyl (37) or imidazole (38),
improved potency approximately 2-fold. Following up on the
potent amidine 33, substituted guanidine (39) was prepared and
significantly improved potency to an IC50 of 0.19 μM.
Compounds 40−51 represented a series of cyclic amines
designed to explore the effect of removing the hydrogen bond
donor proximal to the core and extending this group deeper into
the pocket to target the D169 interaction. Compared to 27,
Accessing Beyond the Methionine Pocket: Synthesis
and Biochemical SAR of the Substituted Amines. A series
of analogues was prepared substituting on the para position of
the phenyl ring of 27 to build into the methionine portion of the
binding pocket (Scheme 3, Table 4). Ahn et al.13 reported the
ternary complex of H. influenzae TrmD with AdoHcy and a
phosphate ion. In this X-ray crystal structure, an ordering of
residues G161 to D169 from an opposing monomer was
observed, forming a so-called “lid,” which covered the substrate
binding cleft. Although the lid contains the acidic and
catalytically relevant residue D169, none of our cocrystal X-ray
structures indicated an interaction between the inhibitor and
D169. As such, compound 27 was extended beyond the confines
of the SAM pocket to probe the disordered region of the protein
and hopefully establish a hydrogen bond interaction with D169.
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Table 3. Biochemical Potency, in IC50s (μM), of Variations of the Amide Substituents against Various TrmD Isozymes
a
Scheme 3. Synthesis of the Substituted Phenyl Amides
a
Conditions: (a) ethylene glycol, pyridinium-p-toluenesulfonic acid, toluene 130 °C, Dean−Stark; (b) LAH, THF, 0 °C to room temperature; (c)
Et3N, EtOH, reflux; (d) 6 M HCl, 1,4-dioxane; (e) R1R2NH, Ti(OPri)4, NaBH(OAc)3, THF, 45 °C.
amino-azetidine (40) showed a 3-fold improvement in potency.
An exploration of ring size demonstrated that the pyrrolidine as
well as the piperidines and piperazines were well tolerated.
Incorporation of hydrogen bond donating groups onto the
pyrrolidine resulted in compounds with IC50s of 0.2−0.4 μM
(42−44). The unsubstituted piperazine was equipotent to
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Table 4. Biochemical Potency, in IC50s (μM), of Substituted Phenyl Amides
compound 27, while altering the basic character of the free N−H
with the piperizinone 46 showed more than a 3-fold loss of
activity. Building off the piperazine nitrogen to explore the
methionine pocket resulted in a modest improvement in potency
(47−48). Switching to the piperidine and moving the donor
outside of the ring resulted in a significant improvement in
potency (51) to 0.18 μM.
Thienopyrimidone TrmD Inhibitors Have Broad Spec-
trum Enzymatic Activity. A representative set of the most
potent compounds were tested for broad spectrum TrmD
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activity employing a variety of Gram-positive and Gram-negative
isozymes (Table 5). Inhibitory activity against E. coli,
confirm or refute mode of action via TrmD were not successful
due to a low resistance frequency of less than 10−9. While the
inability to isolate resistant mutants did not enable the
confirmation of the mode of action, it is an indication of a low
likelihood of developing resistance.
Table 5. Biochemical Spectrum, in IC50s (μM), of Selected
Inhibitors against a Wide Range of TrmD Isozymes
Given their potent enzymatic activity but weak antibacterial
activity, including against the efflux mutant strains, these
compounds presumably penetrate slowly into the cytosol. This
is likely due to the complex nature of the bacterial cell wall and
membrane that can act as a barrier to charged compounds such as
these. This may indicate a dichotomy for this series between what
is required for binding to the target and what is desirable to
penetrating bacterial cells. Given the nature of the binding
pocket, the most potent compounds were those that contained
dibasic groups to extend into the methionine pocket. However,
these same dibasic groups make penetrating the bacterial cell
more difficult.17 Modulation of compound physical properties
would be necessary to enhance cell penetration.
TrmD Inhibitors Demonstrate Novel Ordering of the
“Lid” Region. To further analyze the structural implications of
substituted amines binding to TrmD, the cocrystal structure of
TrmD with 38 (Figure 2E,F) was solved at a resolution of 1.9 Å.
The position of the thienopyrimidone and phenyl ring of 38 in
the SAM binding pocket is virtually identical to the binding mode
for 27. Consistent with our hypothesis, the hydrogen bond
donating nitrogen (N1, pKa = 14.4) of the imidazole ring was
within binding distance to D169 carboxylate (2.9 Å). Addition-
ally, the resulting electron density map supports an alternate
conformation of the D177 side chain on the opposing monomer.
The refined occupancy state for the carboxylate orientation
pointing toward or away from the main chain amide was 0.56/
0.44, respectively. The conformer with the carboxylate pointing
toward the main chain amide was 2.8 Å from the second
imidazole nitrogen (N3, pKa = 6.5) of 38, a reasonable distance
for a hydrogen bond contact to be established. The conformer
that placed the carboxylate away was beyond hydrogen bond
distance to the inhibitor. Given that crystallization was
A.
H.
K.
P.
S.
E. coli baumannii influenzae pneumoniae aeruginosa aureus
27
37
38
40
43
44
51
0.91
0.44
0.52
0.33
0.29
0.40
0.18
0.61
0.74
0.23
1.3
0.56
0.33
1.5
0.83
0.61
0.31
1.3
0.098
0.044
0.067
0.25
4.3
7.4
8.2
1.8
2.7
6.3
1.2
0.33
0.42
0.60
0.33
0.24
0.63
0.58
0.51
0.63
0.35
0.063
0.12
<0.039
Acinetobacter baumannii, and Klebsiella pneumoniae isozymes
was equipotent to that observed for H. influenzae, suggesting a
high degree of binding site homology across these isozymes.
Gram-positive activity was much weaker, as evidenced by the
IC50s against S. aureus TrmD generally about 5-fold less potent
than that from E. coli. Surprisingly, all compounds tested
demonstrated potent activity against Pseudomonas aeruginosa
TrmD, about 10-fold more potent than E. coli TrmD. Most
notable was 51 with a P. aeruginosa IC50 below the limit of the
assay detection at <0.039 μM.
Antibacterial Activity. Synthesized inhibitors with potent
biochemical activity (IC50 < 10 μM) were further profiled for
antibacterial activity against a range of Gram-positive and Gram-
negative pathogens. The bacterial cell wall and membrane
contain efflux pumps which protect the cell from xenobiotics, and
so we also tested compounds against a series of recombinant
strains of E. coli and H. influenzae debilitated in the AcrAB TolC
efflux pumps.16 In general, only weak activity at the maximum
test concentration of 200 μM was observed against H. influenzae
(data not shown). Compound 42, however, displayed some
activity with a minimum inhibitory concentration (MIC) of 3.1
μM against H. influenzae. Attempts to isolate resistant mutants to
Figure 3. Stereo representation of the different binding conformations of SAM or AdoHcy. Stereo representation of S-adenosylmethionine (SAM) or S-
adenosylhomocysteine (AdoHcy) conformation when bound to distinct methyltransferases. Compound coordinates were extracted from the following
crystal structures deposited in the Protein Data Bank (www.rcsb.org): TrmD-SAM tRNA methyltransferase from H. influenzae in gray (1UAK),13
SET7/9-SAM histone lysine methyltransferase in green (1N6A),20 PRMT1-AdoHcy histone arginine methyltransferase in pink (3QOW),19 and Trm5-
SAM tRNA methyltransferase from M. jannaschii in yellow (2ZZM).15 Compounds were spatially aligned according to the adenosine ribose ring only;
respective protein atoms omitted for clarity.
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mM DTT, and 0.005% Tween 20. When testing for compound
inhibition of isozymes of other species, the TrmD concentration in
reagent 1 was adjusted to 2 nM for A. baumannii, 5 nM for P. aeruginosa,
4 nM for H. influenzae, 10 nM for K. pneumoniae, and 10 nM for S. aureus.
Reagent 2, also made in assay buffer, consisted of 0.05 μM 80.7 Ci/mmol
S-[methyl-3H] adenosyl-L-methionine (GE Healthcare), 2 μM non-
radioactive S-adenosyl-L-methionine (VWR), and 2 μM synthetic E. coli
leucine-tRNA (Trilink BioTechnologies, San Diego, CA).
Stock solutions of compounds were prepared at 10 mM, and 2-fold
serially diluted, in DMSO. Diluted compound (0.6 μL) was added to 96-
well white polystyrene plates (Corning, Corning NY). Equal volumes of
reagents 1 and 2 (15 μL of each) were added to the compound plates.
The plates were covered with lids and incubated at room temperature
for 1 h. Subsequently, 30 μL of 8 mg/mL WGA beads (Perkin-Elmer
RPNQ0003) resuspended in 0.15 M sodium citrate (pH 2.0) were
added. Plates were sealed with foil and incubated at room temperature
for 3 h, and radioactivity was measured by scintillation counting on the
TopCount microplate scintillation counter (Perkin-Elmer). For 100%
inhibition, the reactions contained 100 μM sinefungin (Sigma, IC50 = 5
μM).
performed at pH 7.0, the inhibitor imidazole N3 should be
partially protonated, consistent with the conformer dependent
hydrogen bond state of D177.
Ahn et al., identified residues G161-D169 as playing the role of
a lid that covers the active site cleft as well as interacting with the
tRNA substrate.13 Although this segment of protein was largely
ordered in the presence of phosphate and AdoHcy in their
reported cocrystal structure, they also reported the lid residues
were disordered in the presence of SAM or AdoHcy alone.
Presuming the lid region harbors an inherent flexibility, we
speculate that compound 38 is able to exploit this property by
presenting a hydrogen bond donor in the vicinity of D169 and
D177, thus promoting positional stability of the amino acids in
the lid domain. Because the inhibitors were able to order the lid
without the presence of phosphate, this established a new
binding mode for this protein. This ordering likely reflects
structural changes upon binding of tRNA when the SAM methyl
group is transferred to N1G37.
Susceptibility Testing. Minimum inhibitory concentrations
(MIC) were determined using broth microdilution according to the
guidelines of the Clinical and Laboratory Standards Institute.21
Chemistry. All solvents used were commercially available and were
used without further purification. Reactions were typically run with
anhydrous solvents under an atmosphere of nitrogen unless otherwise
noted. Starting materials were obtained from commercial sources or
prepared as described in Supporting Information. Microwave heating
was performed in a CEM Discover unit at the temperatures noted.
LC-MS analysis was performed by reversed phase LC-MS using
Varian Polaris C18A, 2 mm × 50 mm, 3 μm particle size columns. An
Agilent HP1100 (Wimington, DE, USA) LC system was used with a
gradient elusion profile of 5−95% B over 4.5 min at 1 mL/min, then re-
equilibration at initial conditions to 6 min. Injection volume was 2 μL
and column temperature 30 °C. Mobile phase A was 0.1% formic acid in
water and mobile phase B 0.1% formic acid in acetonitrile. Detection was
based on electrospray ionization (ESI) in positive and negative polarity
using Waters ZQ mass spectrometer (Milford, MA, USA), diode-array
UV detector from 210 to 400 nm, and evaporative light-scattering
detector (Sedex 75, Sedere, Alfortville Cedex, France). Alternatively, the
same method as above was used, except A was 10 mM ammonium
acetate in (5/95 acetonitrile/water) and B was acetonitrile.
Accurate mass was done using a hybrid quadrupole time-of-flight
mass spectrometer (microTOFq II, Bruker Daltonics) in ESI+ mode
connected to Waters Acquity UPLC. Method: 0−95% B from 0.0−1.5
min, hold at 95% B to 1.75 min, then equilibrate to 2 min at 5% B.
Mobile phase A, 0.1% formic acid in water; B, 0.1% formic acid in
acetonitrile. Flow rate was 1.0 mL/min. Waters Acquity HSS T3, 2.1
mm × 3.0 mm, 1.8 μm particle column was used. Then 2 μL of sample
were injected using an externally calibrated instrument for accurate mass
measurement. Sample temperature was set to 10 °C and column
temperature was 30 °C.
Inhibitors Are Selective for Bacterial TrmD. In the course
of our investigations, an assay for the functional human
homologue of TrmD, TRM5, was not available due to challenges
encountered in generating the protein reagent. Although
expression of the human TRM5 was achieved, the material was
insoluble and deemed unsuitable for assay development. Seeking
an alternative, functional homologues were used as a tool. As
argued previously by Copeland and colleagues, a relevant
exercise toward understanding the chemical biology relatedness
of such functional homologues is to analyze the binding
conformation of SAM even though the primary or quaternary
structure of respective proteins demonstrate low homology.18
The bound forms of SAM or AdoHcy from representative
methyltransferases were superimposed along with the SAM
bound form of TRM5. (Figure 3). The TRM5 SAM and PRMT1
AdoHcy, a histone arginine methyltransferase, both show an
extended conformation for the methionine and homocysteine
moieties, respectively.19 Thus we selected PRMT1 as the
surrogate to determine selectivity against TRM5. The histone
lysine methyltransferase SET7/9 was also analyzed for selectivity
because SAM binds SET7/9 in a compact conformation as seen
with TrmD, although the methionine moieties are positioned
opposite each other due to a 118° difference in the respective
C3′−C4′−C5′−Sδ torsion angle.20 Compounds 27, 37−39, 42,
45, and 48−51 showed <10% inhibition at 20 μM concentration,
whereas Singefungin was equipotent against E. coli TrmD and
SET7/9. This reflects 50−100-fold selectivity.
CONCLUSION
■
1H NMR spectra (δ, ppm) were recorded using Bruker Advance
Ultrashield 300 MHz or Bruker DPX 400 MHz instruments. 13C NMR
spectra (δ, ppm) were recorded using Bruker Shielded DRX-500 MHz
fitted with QNP cryoprobe.
Column chromatography was performed using Silcycle FLH-
R10030B Silisep cartridges (12−330 g). Preparative reversed phase
HPLC chromatography was carried out using a Waters Atlantis T3-C18
column, 19 mm × 100 mm, 5 μm, a linear gradient from 10% to 90%
CH3CN in H2O over 12 min (0.1% trifluoroacetic acid), and a flow rate
of 20 mL/min.
A fragment screening approach to identify inhibitors of H.
influenzae TrmD yielded multiple hits of which binding to the
SAM binding pocket of TrmD was confirmed by X-ray
crystallography. Using structure-guided design and by making
additional interactions with the protein, these fragments were
elaborated into the first known series of highly potent, selective
inhibitors of TrmD. The most potent inhibitors which extend
into the methionine portion of the binding pocket were found to
order the TrmD lid, thus mimicking physiologically changes
presumed to occur upon binding of tRNA.
The purity of the final compounds was assessed on the basis of
analytical LC-MS, and the results were greater than 95%.
General Procedure A for Reductive Amination. To a solution of
57 (0.32 mmol) in THF (5.0 mL), amine (0.64 mmol) and Ti(OPri)4
(270 mg, 0.96 mmol) were added and stirred at 45 °C for 12 h. Sodium
triacetoxyborohydride (200 mg, 0.96 mmol) was added portionwise and
stirred at the same temperature for 24 h. The reaction mixture was
diluted with 10% aq sodium hydrogen carbonate (5 mL) and extracted
EXPERIMENTAL SECTION
■
Scintillation Proximity Assay for Compound Screening. The
assay for compound inhibition required two reagents. Reagent 1
contained purified E. coli TrmD protein diluted to 10 nM in assay buffer
consisting of 50 mM HEPES (pH 7.5), 5 mM magnesium chloride, 1
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dx.doi.org/10.1021/jm400718n | J. Med. Chem. 2013, 56, 7278−7288

Journal of Medicinal Chemistry
Article
with THF (3 × 5 mL). Combined organic layer was washed with brine
(5 mL), dried over anhydrous Na2SO4, and evaporated under reduced
pressure. Residue was purified by CombiFlash using 10% methanol in
chloroform as eluent to yield the title compound as a solid.
General Procedure B for trans Amidation. A solution of ethyl 4-
oxo-3,4-dihydrothieno[2,3-d]pyrimidine-5-carboxylate (18) (0.50
mmol) and amine (1.0 mmol) in ethanol (10 mL) was stirred at 80
°C for 18 h. Volatiles were evaporated under reduced pressure. Residue
was purified by flash chormatography on silica gel using a gradient of
methanol in dichloromethane to give the title compound as a semisolid
which was precipitated from hot ethyl acetate and hexanes to yield the
title compound as a white powder.
of 6 M aq HCl was added at 0 °C and then stirred at room temperature
for 2 h. Reaction mixture was evaporated under reduced pressure, and
the residue was triturated with diethyl ether (5 mL) and the solids were
filtered and dried under vacuum to yield the title compound as an off-
white solid; yield, 81 mg (92%). LCMS (m/z): 398 [M + 1]. HRMS
1
calculated for C20H23N5O2S [M + 1], 398.1645; found, 398.1631. H
NMR (400 MHz, DMSO-d6) δ ppm 1.94−2.02 (m, 2H), 2.04−2.14 (m,
2H), 2.92−3.02 (m, 2H), 3.16−3.26 (m, 1H), 3.32−3.38 (m, 2H), 4.21
(d, J = 4.76 Hz, 2H), 4.58 (d, J = 5.52 Hz, 2H), 7.42 (d, J = 8.00 Hz, 2H),
7.57 (d, J = 8.08 Hz, 2H), 8.29 (s, 1H), 8.34−8.42 (m, 3H), 11.02−11.08
(m, 1H), 11.38 (t, J = 5.52 Hz, 1H), 13.21 (s, 1H). 13C NMR (500 MHz,
DMSO-d6) δ ppm 26.78, 42.37, 45.18, 49.37, 58.47, 119.36, 127.39,
128.12, 131.33, 131.62, 132.26, 140.38, 145.98, 159.62, 160.33, 167.11
N-Benzyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-5-car-
boxamide (16). Prepared according to general procedure B using
phenylmethanamine, yield 78 mg (61%). LCMS (m/z): 286 [M + 1].
HRMS calculated for C14H11N3O2S [M + 1]: 286.0572 found:
ASSOCIATED CONTENT
* Supporting Information
■
S
1
286.0644. H NMR (300 MHz, DMSO-d6) δ ppm 13.07 (br s, 1 H),
Experimental procedures for all compounds, cloning and
purification of TrmD isozymes, selectivity data as well as crystal
structure determination and refinement procedure. This material
is available free of charge via the Internet at http://pubs.acs.org.
11.32 (t, J = 5.37 Hz, 1 H), 8.39 (s, 1 H), 8.28 (s, 1 H), 7.29−7.42 (m, 4
H), 7.18−7.29 (m, 1 H), 4.55 (d, J = 5.65 Hz, 2 H)
N-[4-(Aminomethyl)benzyl]-4-oxo-3,4-dihydrothieno[2,3-d]-
pyrimidine-5-carboxamide (27). tert-Butyl 4-[(4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-5-carboxamido) methyl] benzyl carba-
mate: A solution of ethyl 4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-5-
carboxylate (18) (150 mg, 0.67 mmol) and tert-butyl 4-(aminomethyl)-
benzylcarbamate (160 mg, 0.67 mmol) in ethanol (5 mL) was heated to
80 °C for 18 h. The volatiles were removed and the residue purified by
flash chromatography on silica gel eluting with a gradient of ethyl acetate
in hexanes to yield the title compound as a white powder. Yield: 160 mg
(57.7%). LCMS (m/z): 415 [M + 1]. N-[4-(Aminomethyl)benzyl]-4-
oxo-3,4-dihydrothieno[2,3-d]pyrimidine-5-carboxamide: A solution of
tert-butyl 4-((4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-5-carboxami-
do) methyl) benzyl carbamate (100 mg, 0.24 mmol) in methanol (10
mL) was treated with HCl (4 M in 1,4-dioxane, 1.0 mL, 4.0 mmol),
heated to 60 °C for 1 h. Upon cooling, the volatiles were removed and
the residue dissolved in methanol (3 mL). The title compound was
precipitated by the slow addition of ethyl acetate (30 mL) to give a tan
powder as the HCl salt. Yield: 30 mg (35.4%). LCMS (m/z): 315 [M +
1]. HRMS calculated for C15H14N4O2S [M + 1], 315.0910; found,
315.0909. 1H NMR (300 MHz, DMSO-d6) δ ppm 11.18−11.43 (m, 1
H), 8.39 (s, 1 H), 8.29 (s, 1 H), 7.42 (s, 4 H), 4.56 (d, J = 5.46 Hz, 2 H),
4.01 (s, 2 H). 13C NMR (500 MHz, DMSO-d6) δ ppm 42.10, 42.45,
118.60, 127.42, 128.10, 128.50, 132.31, 134.18, 139.25, 150.47, 160.90,
164.32, 167.41
Accession Codes
Co-crystal structure coordinates with corresponding structure
factor files have been deposited in the RCSB Protein Data Bank
under the following accession codes: 1, 4MCD; 27, 4MCC; 38,
4MCB.
AUTHOR INFORMATION
Corresponding Author
*Phone: (781) 839-4291. E-mail: pam.hill@astrazeneca.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Analytical Chemistry Department of AstraZeneca
R&D Boston for performing reverse phase HPLC purifications
and accurate mass determinations and the AstraZeneca R&D
Susceptibility Testing Group for MIC testing. We also thank Dr.
Henry Zhu of BPS Bioscience (San Diego, CA) for PRMT1 and
SET7/9 testing.
N-(4-{[(1H-Imidazol-2-ylmethyl)amino]methyl}benzyl)-4-
oxo-3,4-dihydrothieno[2,3-d]pyrimidine-5-carboxamide (38).
Prepared according to general procedure A using 1-(1H-imidazol-2-
yl)methanamine; yield, 25 mg (4%). LCMS (m/z): 395 [M + 1]. HRMS
ABBREVIATIONS USED
TrmD, tRNA methyltransferase; SAM, S-adenosylmethionine;
AdoHcy, S-adenosylhomocysteine
■
1
calculated for C19H18N6O2S [M + 1], 395.1285; found, 395.1278. H
NMR (400 MHz, DMSO-d6) δ ppm: 3.68 (s, 4H), 4.53 (d, J = 5.16 Hz,
2H), 6.91 (s, 2H), 7.30 (s, 4H), 8.27 (s, 1H), 8.37 (d, J = 2.28 Hz, 1H),
11.45 (br s, 1H). 13C NMR (500 MHz, DMSO-d6) δ ppm: 42.53, 45.45,
51.84, 119.26, 127.15, 128.22, 128.64, 130.82, 131.50, 132.40, 137.29,
138.58, 146.34, 146.42, 160.14, 160.18, 167.15
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