Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37- N1)-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism

Article abstract of DOI:10.1021/acs.jmedchem.9b00582

Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(N¹G37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from Pseudomonas aeruginosa and Mycobacterium tuberculosis, we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding. This tyrosine-flipping mechanism is uniquely found in P. aeruginosa TrmD and renders the enzyme inaccessible to the cofactor S-adenosyl-l-methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure-activity relationship studies provided insights into the mechanisms underlying the potency of thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against Gram-positive and mycobacterial pathogens. These results lay a foundation for further development of TrmD inhibitors as antimicrobial agents.

Full text of DOI:10.1021/acs.jmedchem.9b00582

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Article
Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA
1
(Guanine37-N)-Methyltransferase (TrmD) by Restructuring
the Active Site with a Tyrosine-Flipping Mechanism
Wenhe Zhong, Kalyan Pasunooti, Seetharamsing Balamkundu, Yee Hwa Wong, QianHui Nah,
Vinod Gadi, Shanmugavel Gnanakalai, Yok Hian Chionh, Megan E. McBee, Pooja Gopal, Siau Hoi
Lim, Nelson Olivier, Ed Buurman, Thomas Dick, Chuan-Fa Liu, Julien Lescar, and Peter C. Dedon
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00582 • Publication Date (Web): 23 Aug 2019
Downloaded from pubs.acs.org on August 24, 2019
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course of their duties.

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Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA
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(
Guanine37-N )-Methyltransferase (TrmD) by Restructuring
the Active Site with a Tyrosine-Flipping Mechanism
Wenhe Zhong1,3,‡, Kalyan Kumar Pasunooti , Seetharamsing Balamkundu , Yee Hwa
1, ‡
1, ‡
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,3
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1, †
Wong , Qianhui Nah , Vinod Gadi , Shanmugavel Gnanakalai , Yok Hian Chionh ,
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Megan E. McBee , Pooja Gopal , Siau Hoi Lim , Nelson Olivier , Ed T. Buurman , Thomas
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, †
2,*
2,3,*
& Peter C. Dedon1,7,*
Dick , Chuan Fa Liu , Julien Lescar
1Infectious Disease and Antimicrobial Resistance Interdisciplinary Research Groups,
Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, 138602, Singapore;
2School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,
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37551, Singapore;
3NTU Institute of Structural Biology, Nanyang Technological University, 636921, Singapore;
4Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore;
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Departments of Bioscience and Discovery Sciences , Infection Innovative Medicines Unit,
AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA;
7Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge,
MA 02139, USA.
‡
These authors contributed equally to this work.
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†Present address: Y.H.C., Tychan Pte. Ltd., 79 Ayer Rajah Crescent, #05-03, Singapore
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39955; E.T.B., Pfizer Vaccines Research and Development, Pearl River, NY 10965, USA;
T.D., Public Health Research Institute Center, New Jersey Medical School - Rutgers, The State
University of New Jersey, 225 Warren Street, Newark, NJ 07103 USA
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*Correspondence should be addressed to C.F.L. (CFLiu@ntu.edu.sg), J.L. (julien@ntu.edu.sg)
or P.C.D. (pcdedon@mit.edu)
ABSTRACT
Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many transfer
RNA (tRNA) modifications are critical to bacterial survival, which makes their synthetic
enzymes ideal targets for antibiotic development. Here we performed a structure-based design
1
of inhibitors of tRNA-(N G37) methyltransferase, TrmD, which is an essential enzyme in many
bacterial pathogens. Based on crystal structures of TrmDs from Pseudomonas aeruginosa and
Mycobacterium tuberculosis, we synthesized a series of thienopyrimidinone derivatives with
nanomolar potency against TrmD in vitro and discovered a novel active site conformational
change triggered by inhibitor binding. This tyrosine-flipping mechanism is unique found in P.
aeruginosa TrmD and renders the enzyme inaccessible to the co-factor S-adenosyl-L-
methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure-
activity relationship studies provided insights into the mechanisms underlying the potency of
thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against
Gram-positive and mycobacterial pathogens. These results lay a foundation for further
development of TrmD inhibitors as antimicrobial agents.
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INTRODUCTION
The emergence of antibiotic resistance in bacterial pathogens on a global scale and the lack of
new antibiotics represent a crisis with significant societal and economic impact. This is
illustrated by >2 million annual cases multi-drug resistant bacterial infections, which result in
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3,000 deaths . Mycobacterium tuberculosis (Mtb) and Pseudomonas aeruginosa (Pa) are
examples of major human pathogens for which drug resistance is emerging as a serious public
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health problem : multi-drug resistant Mtb and Pa cause >10,000 and ~6,700 deaths per year,
respectively. Pa is the cause of the most common hospital-acquired infection among the
immuno-compromised, the elderly, the chronically ill, and patients with in-dwelling medical
devices such as catheters, nasogastric tubes, and drains. These two pathogens illustrate the
point that new antibiotics, particularly those that avoid resistance mechanisms and are aimed
at novel targets, are urgently needed to alleviate the current antibiotic crisis.
Post-transcriptional ribonucleotide modifications of RNA, especially transfer RNA (tRNA),
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play critical roles in translation in all organisms . In addition to the essentiality of some of the
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3,5
enzymes catalyzing these modifications for growth, studies with bacteria , yeast , and
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parasites have demonstrated that many tRNA modifications are critical in the cell stress
response by facilitating selective translation of proteins critical to surviving the stress. Loss of
the ability to synthesize these tRNA modifications renders bacteria susceptible to killing by the
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immune response and other environmental stresses . Given their role in bacterial cell survival,
these critical tRNA modification synthesis enzymes constitute attractive targets for antibiotic
development.
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The bacterial tRNA (guanine37-N )-methyltransferase (EC 2.1.1.228; TrmD) catalyses methyl
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transfer from S-adenosyl-L-methionine (SAM) to the guanine N at position 37 in a subset of
bacterial tRNA isoacceptors (Supporting Information Figure S1) and has proven to be an
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essential enzyme in most bacterial species10-14. While of central importance to mammalian cell
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health , Trm5, the functional homologue of TrmD in eukaryotes, has dissimilar active sites
and different binding modes of SAM than TrmD15,16. These observations suggest that TrmD is
an attractive antibiotic target, a conclusion supported by the efforts of several groups to develop
TrmD inhibitors15,17,18. We recently developed a radioactivity-free bioluminescence-based
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high-throughput screening (HTS) assay that identified a series of novel TrmD inhibitors . In
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013, Hill et al. used X-ray crystallography-guided fragment screening to develop a series of
SAM-competitive TrmD inhibitors against a broad range of bacterial isozymes. These
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inhibitors showed minimal antibacterial activity presumably due to poor cell permeability .
Interestingly, one of these inhibitors, compound 51 (AZ51), showed remarkable inhibitory
potency toward all the tested TrmD isozymes and subnanomolar activity against P. aeruginosa
TrmD (PaTrmD). Despite this potent TrmD inhibitor activity, AZ51 lacked antibacterial
activity.
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Building on this work and the TrmD inhibitor scaffolds identified in a HTS , we sought here
to understand the structural basis for the potent TrmD inhibition caused by the
thienopyrimidinone compounds, with the goal of refining the structures for antibiotic activity.
Crystal structures of TrmD from Pa and Mtb in complex with AZ51 revealed conformational
changes unique to the Gram-negative bacterial TrmD. Based on these structures, we then used
the thienopyrimidinone scaffold (Figure 1) to design and synthesize a series of 33 derivatives
with the goal of improved potency and antibacterial activity. Structure-activity relationship
(SAR) studies defined critical features of the thienopyrimidinone that drive enzyme inhibition
potency as well as antibacterial activity.
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RESULTS
AZ51 has broad-spectrum TrmD inhibition activity. Previously, Hill et al. discovered an
interesting inhibition mechanism where one of the thienopyrimidinone derivatives (compound
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38) ordered the position of the lid domain of Haemophilus influenzae TrmD (HiTrmD) via
its terminal imidazole substituent. The lid domain covered the active site of TrmD and was
essential for tRNA binding19,20. AZ51 is another analogue derived from the thienopyrimidinone
scaffold (Fig. 1A; Table 1) and is generally more potent than other thienopyrimidinone
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inhibitors across a variety of Gram-negative and Gram-positive isozymes , in particular to
PaTrmD. Thus, it raised the question about the potential unique inhibition mechanism of AZ51.
We confirmed that it inhibited PaTrmD with nanomolar potency (IC 180 ± 20 nM; Fig. 2A,
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Table 1) using a different, bioluminescence-based assay . In addition, we determined the
binding affinities of AZ51 to three bacterial TrmDs by surface plasmon resonance (SPR; Table
2
). Interestingly, AZ51 has much stronger binding affinity with Gram-negative bacterial
TrmDs than mycobacterial and Gram-positive bacterial TrmDs (100- to 300-fold lower Kd;
Table 2). For PaTrmD and Staphylococus aureus TrmD (SaTrmD), this relationship paralleled
differences in TrmD inhibition potency by AZ51: PaTrmD K 91 nM and IC 180 nM here,
d
50
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39 nM Hill et al. ; and SaTrmD K 27,500 nM and IC 1200 nM . Since the basis for these
d
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striking differences in AZ51 binding energetics and inhibition potency for the different
bacterial classes may lie in different structures of the AZ51-enzyme complexes, we undertook
to solve the crystal structures of AZ51 with PaTrmD and MtbTrmD.
A
B
Figure 1. Structure of TrmD inhibitors based on the thienopyrimidinone scaffold (A) and their
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O -derivatives (B).
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a
Table 1. Structures and IC values for thienopyrimidinone PaTrmD inhibitors
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R2
N
R1
O
HN
O
NH
S
N
Compound
AZ51 b
R1
R2
IC (μM)
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N
0.18 ± 0.02
1.1 ± 0.2
H2N
N
9 b
N
10 b
3.0 ± 0.5
0.73 ± 0.13
0.49 ± 0.06
O
1
1
1
2
Et
Et
Et
1
1
3
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Et
2.2 ± 1.3
0.13 ± 0
H
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n-octyl
H
H
0.024 ± 0.004
0.79 ± 0.29
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H
0.72 ± 0.30
n-hexyl
n-octyl
n-octyl
2.2 ± 0.5
0.37 ± 0.01
~12
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Et
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0
2
22
2
2
1
n-butyl
n-pentyl
n-decyl
H
H
H
H
0.15 ± 0.02
0.085 ± 0.016
0.025 ± 0.001
0.38 ± 0.01
3
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n-dodecyl
H
N
2
2
5
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HO
H
H
H
0.11 ± 0.02
0.21 ± 0.03
O
O
H
O
N
H
31
32
O
O
N
0.45 ± 0.06
0.14 ± 0.01
0.11 ± 0.02
0.08 ± 0.02
ClH•N
ClH•N
H
H
H
N
H
3
3
3
4
ClH•N
ClH•N
N
N
H
H
O
HO
H
N
H
5
3
0.70 ± 0.01
N
OH
O
O
HO
O
H
H
5
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1.3 ± 0.4
N
N
N
H
OH
O
a
Biochemical potency is represented as IC value (μM); values are mean ± SD for at least two
independent experiments done in duplicate. The listed pharmacophores replace the group
N(R1)(R2).
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b
Table 2. Binding kinetics of TrmD ligands a
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-
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-1
Ligand
SAH
AZ51
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SAH
AZ51
K (μM)
k (M s )
k (s )
off
D
on
b
5.9
0.091
0.0016
10.1
10.4
0.19
3.3
55500 ± 479
128000 ± 318
13500000 ± 87800
7700 ± 14
0.326 ± 0.002
0.0117 ± 0.0000
0.0218 ± 0.0001
0.0775 ± 0. 0001
0.209 ± 0.0004
0.120 ± 0.0003
0.92 ± 0.03
PaTrmDc
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MtbTrmDd
20100 ± 80
1
5
645000 ± 3030
279000 ± 36000
59900 ± 566
SAH
AZ51
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SaTrmDe
27.5
3.1
1.65 ± 0.01
9.15 ± 0.73
2940000 ± 241000
a
Binding data determined by surface plasmon resonance (SPR) as described in
Experimental Procedures; S-adenosylhomocysteine; Gram-negative bacterial TrmD;
b
c
d
e
Mycobacterial TrmD; Gram-positive bacterial TrmD.
AZ51 induces a unique conformational change of a wall-loop residue in PaTrmD. To gain
insight into the molecular mechanism used by AZ51 for PaTrmD inhibition, we soaked a
PaTrmD-SAM cocrystal with AZ51 and determined the structure at a resolution of 2.21 Å
using a crystal form having a complete PaTrmD dimer in the asymmetric unit (Table 3). The
electron density of AZ51 is clearly visible at one active site of the PaTrmD, whereas the
copurifying SAM cofactor still occupies the other active site (Fig. 2B). The observation that
cocrystal soaking with the competitive inhibitor led to the displacement of SAM at only one of
the two active site suggests that either the binding affinity to SAM or the rigidity of binding
sites is different for the two active sites within a TrmD biological dimer. However, as the
difference in AZ51 binding of two active sites are found in the protein crystal by the soaking
method where the structural dynamics of the crystal form are restricted, we cannot exclude the
possibility that the difference in AZ51 binding is simply due to the nature of the method.
Similar to SAM binding (Supporting Information Fig. S2), inhibitor AZ51 is mainly
stabilized by three active-site loops in PaTrmD (named the “cover loop”, “bottom loop”, and
“wall loop”) (Fig. 2B): the thienopyrimidinone ring is tightly bound in the adenine pocket, is
hydrogen-bonded to residues Ile138, Tyr141 and Leu143, and also forms stacking interactions
with residues Pro94 and Leu143. The phenyl ring is locked in place by stacking interactions
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with residues Pro94 and Tyr120, while the piperidine ring forms stacking interactions with
residue Tyr120 and is potentially hydrogen-bonded to residue Asp182 by its protonated
nitrogen. The terminal amine (i.e., the -NH hydrogen bond donor incorporated onto the
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piperidine ring) interacts with the carboxylate of Glu121, a highly conserved residue among
TrmD isozymes (Supporting Information Fig. S3).
Figure 2. Crystal structures of AZ51 with PaTrmD and MtbTrmD. (A) The IC values
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determined for AZ51 at tRNA concentrations of 1.5 µM (), 4.5 µM () and 15 µM ()
respectively are: 0.18 ± 0.03 µM, 0.33 ± 0.04 µM and 0.84 ± 0.09 µM. Each data point
represents an average of duplicate experiments with error bars indicated as SD. (B, D) Close-
up views of catalytic sites of PaTrmD-AZ51 (B; PDB ID 6JOE) and MtbTrmD-AZ51 (D; PDB
ID 6JOF) showing the binding modes of inhibitor AZ51, respectively. Polypeptide chains are
shown as cartoons (chain A in grey and chain B in cyan), whereas key interacting residues are
shown as sticks. AZ51 are shown as sticks with an 2Fo-Fc electron density (grey) map
contoured at 1.0 σ. Potential interactions involved in AZ51 binding are indicated by broken
lines (black: hydrogen bonds; green: stacking interactions). (C) Inhibitor AZ51 binding at
catalytic site induces wall-loop conformational change and thereafter blocks substrate tRNA
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(
G ) binding. SAM-bound PaTrmD (PDB ID 5WYQ) and tRNA-bound H. influenzae TrmD
(PDB ID 4YVI) were superimposed onto AZ51-bound PaTrmD, respectively. Polypeptide
chains of PaTrmD-AZ51 are shown as surface while the bound AZ51 (yellow) and side-chain
flipped residue Tyr120 (grey) are shown as sticks. PaTrmD-SAM structure (light pink)
displays SAM and residue Tyr120 in sticks representation. G37of tRNA substrate in HiTrmD-
tRNA structure is shown as sticks in orange. Polypeptide chains of PaTrmD-SAM and
HiTrmD-tRNA are omitted for clarity. (E) The superposition of catalytic sites of PaTrmD-
AZ51 and MtbTrmD-AZ51. The inhibitor AZ51 and residues in different positions from
PaTrmD and MtbTrmD are indicated as sticks.
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Table 3. Data collection and refinement statistics for TrmD structures a
PaTrmD-AZ51
PaTrmD-11
PaTrmD-15
Apo MtbTrmD
MtbTrmD-SAH
MtbTrmD-AZ51
MtbTrmD-12
MtbTrmD-15
PDB ID
6JOE
5ZHM
5ZHN
5ZHI
5ZHJ
6JOF
5ZHK
5ZHL
Data collection
Space group
Cell dimensions
a, b, c (Å)
P3
2
21
P3
2
21
P3
2
21
P2
1
C121
C121
C121
C121
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
85.50,85.50,147.54
90.00,90.00,120.00
84.50,84.50,147.27
90.00,90.00,120.00
84.67,84.67,148.56
90.00,90.00,120.00
44.17,113.07,44.21
90.00,110.75,90.00
72.96,50.76,53.31
90.00,95.10,90.00
73.07,51.38,57.95
90.00,90.18,90.00
73.09,50.80,58.081
90.00,90.56,90.00
73.69,50.23,57.94
90.00,90.95,90.00
α, β, γ (°)
Solvent content (%)
Resolution (Å)
No. of reflections
No. of unique reflections
Wilson B-factor (Å )
Rmerge (%)
CC(1/2)
I/σI
Completeness (%)
Multiplicity
Refinement
Monomers in ASU
No. reflections
52
51
52
38
35
41
40
40
42.75-2.21
267240 (21374)
32130 (2724)
56.2
49.09-2.76
167650 (24471)
16240 (2335)
70.2
42.33-2.65
201645 (27032)
18516 (2392)
82.0
41.30-2.20
72052 (5534)
19717 (1588)
16.8
53.10-1.75
55961 (8132)
18952 (2704)
16.2
42.03-2.20
44682 (3655)
10831 (917)
20.5
58.08-2.30
23534 (3432)
8972 (1287)
19.7
11.5 (43.8)
0.989 (0.733)
6.6 (2.3)
41.50-2.25
32518 (4380)
9936 (1397)
24.3
5.8 (23.8)
0.997 (0.942)
13.9 (5.8)
97.8 (95.3)
3.3 (3.1)
2
5.1 (84.3)
0.999 (0.874)
21.1 (3.0)
99.8 (98.8)
8.3 (7.8)
8.8 (99.3)
0.998 (0.917)
15.2 (2.7)
99.9 (100.0)
10.3 (10.5)
5.7 (131.3)
0.999 (0.865)
22.0 (2.5)
100.0 (100.0)
11.3 (10.9)
5.5 (22.6)
0.997 (0.929)
16.1 (5.2)
96.1 (91.8)
3.7 (3.5)
9.0 (40.8)
0.994 (0.721)
6.9 (2.5)
96.7 (94.6)
3. 0 (3.0)
4.8 (17.0)
0.998 (0.945)
18.9 (6.7)
97.9 (94.8)
4.1 (4.0)
94.2 (92.8)
2.6 (2.7)
2
2
2
2
1
1
1
8972
0.1733/0.2265
1
9900
0.1583/0.2110
31929
16202
18474
18708
18930
10818
R
work / Rfree
0.2090/0.2455
0.2150/0.2751
0.2079/0.2543
0.1596/0.2001
0.1768/0.2021
0.2182/0.2619
No. of non-hydrogen atoms
Protein
Water
Ligands
Average B-factor (Å )
Protein
Water
Ligands
Inhibitor
RMS deviations
Bond lengths (Å)
Bond angles (°)
Ramachandran plots
Favored (%)
Allowed (%)
Number of outliers
3826
191
60
3786
123
53
3789
50
60
3253
87
N.A.
1634
145
32
1610
81
28
1629
122
30
1633
159
30
2
69.20
76.24
62.95
62.82
85.03
73.95
72.81
70.75
102.90
92.18
98.33
98.33
19.95
18.25
N.A.
N.A.
19.79
39.32
25.31
25.32
27.09
32.23
31.11
31.11
27.85
38.95
46.36
46.36
29.05
42.66
39.25
39.25
0.01
1.01
0.01
1.11
0.01
1.08
0.01
1.30
0.01
0.99
0.01
1.04
0.01
1.07
0.01
1.07
97.1
99.8
1
96.0
100
0
97.1
100
0
98.6
100
0
99.1
100
0
97.5
100
0
99.0
100
0
98.1
100
0
aValues in parentheses are for the highest resolution shell; N.A.: not applicable
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In contrast to the SAM-bound structure, AZ51 binding induces conformational changes of the
o
wall loop, whereupon the side-chain of aromatic ring of Tyr120 flips about 180 and forms
stacking interactions with both the phenyl and piperidine rings of AZ51 (Fig. 2C). This feature
appears unique to AZ51 and PaTrmD, and was not observed in other reported TrmD inhibitor
structures15,17. In addition, since residue Tyr120 is strictly conserved among TrmD orthologues
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(Supporting Information Fig. S3), AZ51 can presumably also trigger a comparable side-
chain flip in other TrmD isozymes. The conformational rearrangement of protein upon ligand
binding generally could link to complex binding kinetics21-23. Compared to SAH binding to
PaTrmD, SPR reveals a faster (~2-fold) on-rate (association rate) and slower (~30-fold) off-
rate (dissociation rate) for AZ51 binding (Table 2).
To further understand the biophysical parameters governing AZ51 binding, the thermal
stability of PaTrmD in the presence of AZ51 or the SAM byproduct S-adenosyl-L-
homocysteine (SAH) were determined (Fig. 3D; Supporting Information Fig. S4). Both
active-site ligands decrease the thermal vibration (ΔT = 4 - 29 °C), suggesting that the stability
m
of PaTrmD is enhanced by ligand binding. In particular, AZ51 is able to significantly increase
the T to 79 °C, which is 17 °C higher than thermostabilization conferred by SAH. Therefore,
m
formation of a highly stable complex of PaTrmD with AZ51 binding possibly causes a
conformational change at the active site, which locks the structure in a very stable state. We
also showed that AZ51 interacts with many other bacterial TrmDs to increase the enzymes’
thermal stability (Supporting Information Fig. S5). This further confirms that AZ51 has
broad-spectrum TrmD-binding activity.
The protein structural changes induced by AZ51 binding to PaTrmD also raised questions
about the effect of the changes on tRNA binding. To test this idea, we modelled the tRNA
substrate into the AZ51-bound structure by superimposing the structures of PaTrmD-AZ51
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(
this work) and HiTrmD bound to tRNA (PDB ID 4YVI) (Fig. 2C). Interestingly, we found
that the flipped side-chain of Tyr120 overlaps with G37 of the tRNA substrate, indicating that
the AZ51-bound structure becomes unsuitable for tRNA binding. This is consistent with the
results of the tRNA competition assay (Fig. 2A). Taken together, we conclude that, in the
PaTrmD structure, AZ51 not only mimics substrate SAM and competitively occupies the SAM
binding pocket, but also induces conformational changes in the active-site wall loop that
probably could prevent the binding of tRNA. This hypothesis of “dual functions” of AZ51 may
account for the high potency of this molecule in enzyme inhibitory activity for PaTrmD. Given
the striking differences in AZ51 binding energetics with PaTrmD and MtbTrmD, these results
with PaTrmD raised questions about AZ51-induced structural changes in MtbTrmD.
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9
0
Figure 3. Interactions of compound 15 with TrmDs. (A, B) The polypeptide chains are shown
as cartoons, whereas the residues involved in inhibitor binding are shown as sticks. Ligands
are shown as sticks with 2Fo-Fc electron density maps (grey) contoured at 1 σ. (A) Detailed
interactions between 15 and PaTrmD (chain A) (PDB ID 5ZHN). (B) The interactions between
1
5 and MtbTrmD (PDB ID 5ZHL). (C) In PaTrmD, compound 15 induces a conformational
3
7
change of wall-loop residues, which could block substrate tRNA (G ) binding. SAM-bound
PaTrmD and tRNA-bound H. influenzae TrmD (PDB ID 4YVI) were superimposed onto 15-
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bound PaTrmD, respectively. (D) The thermal shift of PaTrmD and MtbTrmD in the presence
of SAH (2 mM), AZ51 (0.1 mM) or 15 (0.1 mM) was analysed. Error bars represent mean ±
SD. (E) A superposition of PaTrmD-15 (subunit 1 and 2), MtbTrmD-15 and HiTrmD-tRNA
(PDB ID 4YVI) showing the close contacts between 15 and interdomain linker. The
interdomain linker of HiTrmD (residues 157-174) and tRNA are shown as cartons whereas
guanosine is shown as stick. (F) Dose-dependent inhibition of PaTrmD at varying tRNA
titrations. The IC values determined for compound 15 at tRNA concentrations of 1.5 µM (),
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.5 µM () and 15 µM (), respectively, are: 0.02 ± 0.01 µM, 0.06 ± 0.01 µM and 0.26 ±
.02 µM. Each data point represents an average of duplicate experiments with error bars
indicated as SD.
AZ51 binds to MtbTrmD without inducing conformational changes. As noted earlier,
compared with its binding activity with Gram-negative TrmDs, AZ51 binds with much lower
1
5
affinity to mycobacterial and Gram-positive bacterial TrmDs (Table 2). In addition, AZ51
increases the T of PaTrmD by 29 °C, while the corresponding ΔT values of mycobacterial
m
m
and Gram-positive bacterial TrmDs range from 1 to 13 °C (Fig. 3D; Supporting Information
Figs. S4, S5). These observations suggest that AZ51 may use a different mechanism for
binding to mycobacterial and Gram-positive bacterial TrmDs, in spite of the fact that the
overall protein sequence similarity is high among bacterial TrmDs (Supporting Information
Fig. S3, Table S1). To gain insight into the inhibitory mechanism on MtbTrmD, we crystallized
its free form and determined the structure at 2.20 Å resolution. We then optimized the crystal
soaking conditions and successfully soaked SAH and AZ51 into the MtbTrmD crystals, which
allowed determination of the structures at high resolution (Table 3).
As shown in Supporting Information Figure S2D, MtbTrmD forms a biological dimer with
the active site located at the dimer interface, as with PaTrmD. Indeed, MtbTrmD and PaTrmD
are highly similar in structure with RMS deviations of 1.3 Å for all C-α atoms after
superposition. The electron density of SAH is clearly identified at the active site in MtbTrmD-
SAH structure and SAH is positioned by three active-site loops (Supporting Information Fig.
S2D). The adenine base is stacked between Pro85 and Leu138, and forms hydrogen bonds with
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main-chains atoms of Ile133, Gly134, Tyr136 and Leu138. The 2’ hydroxyl group of the ribose
makes a hydrogen bond to the carbonyl oxygen of Pro83. The amino acid tail interacts with
side chain of Thr84 and carbonyl oxygen of Glu112 by forming hydrogen bonds. A protein
sequence alignment of multiple bacterial TrmDs shows that the residues involved in adenine
base interactions are strictly conserved but more diverse for those interacting with the ribose
and tail moiety of SAH (Supporting Information Fig. S3). Compared to MtbTrmD, SAM in
PaTrmD has four additional hydrogen bonds with side chains of residues Tyr91, Gln95,
Asp182 and His185, where the corresponding residues in MtbTrmD (Val82, Ala86, Glu180
and Ser183) do not interact with SAH (Supporting Information Fig. S2E). Thus, these
additional interactions observed at active site in PaTrmD could account for the higher binding
affinity for SAM and SAH, which is consistent with SPR data (Table 2).
1
1
1
1
1
1
1
1
1
1
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The crystal structure of MtbTrmD-AZ51 complex was determined at a resolution of 2.20 Å
(Table 3). Similar to the AZ51 binding mode in PaTrmD (Fig. 2B), AZ51 is positioned at the
SAH binding site by interacting with three active-site loops of MtbTrmD (Fig. 2D). Residues
involved in AZ51 interactions are strictly conserved in other bacterial TrmD including
PaTrmD (Supporting Information Fig. S3). However, the binding of AZ51 does not induce
the side-chain flip of Tyr111 in MtbTrmD, while the corresponding residue Tyr120 in PaTrmD
turned 180° to form stacking interactions with the phenyl ring of the inhibitor (Fig. 2E),
suggesting a more rigid active site in MtbTrmD compared to PaTrmD. In the absence of the
conformational changes at the wall loop, the piperidine ring of AZ51 in MtbTrmD is positioned
differently than in PaTrmD, resulting in the loss of interactions between the terminal amine
and the side chain of Glu112. Even though the binding mode of the thienopyrimidinone ring is
highly similar in MtbTrmD and PaTrmD, the conformational changes induced by AZ51 in
PaTrmD lead to additional interactions (hydrophobic and hydrogen-bonding) and eventually
higher binding affinity. Thus, the structural flexibility of the wall loop in Gram-negative
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bacterial TrmDs appears to play a unique role in the binding of AZ51. Exploiting this unique
mechanism of AZ51 inhibition of TrmD, we next designed and synthesized a series of
derivatives with the goal of improving enzyme inhibition and antibacterial activity.
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Design and synthesis of thienopyrimidinone derivatives. The PaTrmD crystal structure was
used to design a series of inhibitors based on the thienopyrimidinone molecular scaffold shown
in Figure 1. Inspired by the unique mechanism leading to a potential tRNA competition,
instead of exclusively designing substituents that make contacts with residues Glu121, Asp182
and Tyr120 as found with AZ51, our idea driving the inhibitor design process was that alkyl
and aryl substitutions at R and R would allow full access to the free space outside the SAM
1
2
binding site, with the hydrophobicity of the substituent and the charge distribution around the
centered ammonium ion enhancing enzyme inhibitory activity and thus antibacterial potency.
Based on these design principles, we synthesized a series of thienopyrimidinone analogues.
The key aldehyde 8 was synthesized using a reaction sequence and procedures similar to those
1
5
reported by Hill et al. : reaction of methyl 2-oxopropanoate (1) and ethyl 2-cyanoacetate (2)
in presence of sulfur in DMF afforded the thiophene amino diester (3), which upon
condensation with formamidine acetate in ethanol under reflux yielded thienopyrimidinone (4).
Trans amidation of 4 with benzylamine derivative (7), which as synthesized from 4-
formylbenzonitrile (5) followed by treatment with trifluoracetic acid, afforded the key aldehyde
1
5
8
(Scheme 1). We then modified the procedure of Hill et al. for reductive amination of
aldehyde 8 with various amines. We found that the reductive amination with Titanium
isopropoxide (Ti(OiPr) ), sodium triacetoxyborohydride (NaBH(OAc) ) was sluggish and
4
3
needed long reaction times. Thus, we optimized the same transformation using sodium
cyanoborohydride (NaCNBH ) in methanol with catalytic amount of acetic acid at ambient
3
temperature for 18 h, which afforded compounds 9-26. Various Boc-protected diamines were
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coupled using the above-mentioned procedure to afford compounds 27-30, which, on treating
with 4N HCl in dioxane, afforded compounds 31-34.
Scheme 1. Synthesis of TrmD inhibitors based on the thienopyrimidinone scaffold.
Synthesis of compounds 9-26 and 31-34 using reductive amination method
1
1
1
1
1
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1
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Conditions: (a) sulfur, triethylamine DMF, 50 °C; (b) formamidine acetate,ethanol, reflux; (c)
ethyleneglycol, pyridinium-p-toluenesulfonate, Deen-Stark; (d) LiAlH , THF, 0 °C to room
4
temparature; (e) Et N, EtOH, reflux; TFA , DCM, 0 °C to ambient temperature; (f) amine,
3
NaCNBH , cat. acetic acid, MeOH; (g) 4 N HCl in dioxane.
3
Biochemical structure-activity relationships (SAR) for TrmD inhibitors. To explore the
relationship between inhibitor structure and the potency of TrmD inhibition, we quantified the
concentrations of compounds producing 50% inhibition of TrmD methylation of a tRNA
substrate. Compared with AZ51, inhibitory activity was significantly reduced (~6- to 16-fold)
when the R and R positions are present in a cyclic structure (without the terminal amine)
1
2
shown in compounds 9 and 10 (Table 1). This observation is consistent with our structural data
explored next) where the terminal amine group as a hydrogen bond donor forms the interaction
(
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with PaTrmD. Furthermore, compounds 14, 15, 16 and 17 explore the optimal bulk structure
of a single substitution, with benzyl (14), cyclohexyl (16) and adamantyl (17) groups all
showing sub-micromolar activity. However, the n-octyl substitution in 15 confers 5- to 30-fold
more potent inhibitory activity (IC ~24 nM), which suggests that the long straight-chain alkyl
0
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group could play a role in PaTrmD binding. This observation prompted us to quantify the effect
of alkyl chain length on enzyme activity. Accordingly, we synthesized compounds 21-24 with
4
-, 6-, 10- and 12-methylene group alkyl chain lengths. This series of analogues of 15 shows a
clear optimal chain length of 8-10 carbons for the most potent TrmD inhibitory activity
Supporting Information Fig. S6). Substitutions at the R position, however, generally had a
(
2
detrimental effect on TrmD inhibitory activity (11-13, 18, 19 and 20) when the substituent at
R position is also bulky, which suggests steric limitations beyond the SAM binding pocket.
1
For example, when –H at R position of compound 15 is substituted with –Et where compound
2
19 is generated, the enzyme inhibitory activity is significantly decreased from 24 nM to 370
nM (Table 1). Take compound 20 as another example, when substituents at both R and R
1
2
positions are bulk structures (n-octyl and benzyl groups), its enzyme inhibitory activity is
nearly abolished.
Thienopyrimidinone inhibitors induce the same conformational change at active site as
AZ51 in PaTrmD. To further investigate the inhibitory mechanism of the thienopyrimidinone
compounds, we solved the crystal structures of PaTrmD in complex with 11 and 15 (Table 3).
The interactions of PaTrmD with 15 are illustrated in Figure 3A, and with 11 in Figure 4A.
Similar to the AZ51 binding mode (Figure 2A), these thienopyrimidinone inhibitors are mainly
stabilized by the three active-site loops (cover, bottom, and wall loops). At the cover loop, the
thienopyrimidinone ring remains tightly bound in the adenine pocket, with hydrogen bonds to
residues Ile138, Tyr141 and Leu143, and stacking interactions with residues Pro94 and
Leu143. The phenyl ring of 11 and 15 is locked in place by stacking interactions with residues
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Pro94 and Tyr120 in the bottom loop. Electron densities for the thienopyrimidinone and phenyl
rings are clearly visible in the structures of all thienopyrimidinone inhibitors. However, the
electron densities for the flexible tails (substituents) of the thienopyrimidinone inhibitors are
poorly visible and may possess multiple conformations. Similar to the AZ51 crystal structure,
the binding of thienopyrimidinone inhibitors to PaTrmD induces a conformational change of
1
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o
the wall loop, in which the side-chain of residue Tyr120 flips ~180 and forms stacking
interactions with the phenyl ring (Figure 3A; Figure 4A). Compound 15 also highly
thermostabilizes PaTrmD (ΔT = 12 °C) (Figure 3D; Supporting Information Fig. S4) and
m
other bacterial TrmDs (Supporting Information Fig. S5). It is noteworthy that the correlation
is not strong between enzyme inhibition IC50 values (Table 1) and thermal stability Tm
(Supporting Information Fig. S4), indicating that the substituents play additional roles in
enzyme inhibition other than forming a thermally stable complex with the protein, such as
tRNA competition. We will elaborate this point using compound 15 as an example next.
To investigate the potential role of thienopyrimidinone inhibitors in tRNA binding, we then
modelled the tRNA substrate with the PaTrmD-15 structure by superimposing the HiTrmD-
tRNA (PDB ID 4YVI) structure (Fig. 3C). Similar to the mechanism inferred for AZ51, we
found that the flipped side-chain of Tyr120 also overlapped with G37 of tRNA substrate
indicating that the 15-bound structure was unavailable for tRNA binding. The role of the
flexible alkyl chain of 15 was explored below.
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Figure 4. Crystal structures of compound 11 bound to PaTrmD (A; PDB ID 5ZHM) and
compound 12 bound to MtbTrmD (B; PDB ID 5ZHK), respectively. The polypeptide chains
are shown as cartoons, whereas the residues involved in inhibitor binding are shown as sticks.
Ligands are shown as sticks with 2Fo-Fc electron density maps (grey) contoured at 1.0 σ.
Potential interactions involved in inhibitor binding are indicated by broken lines (black:
hydrogen bonds; green: hydrophobic contacts including stacking interactions).
Crystal structures of MtbTrmD in complex with thienopyrimidinone inhibitors. To further
define the structural differences between PaTrmD and MtbTrmD, we soaked 12 (Fig. 4B) and
1
5 (Fig. 3B) into MtbTrmD crystals and solved the structures at high resolution (Table 3; 2.30
Å and 2.25 Å, respectively). Similar to the AZ51 binding mode in MtbTrmD, 12 (Fig. 4B) and
5 (Fig. 3B) are positioned at the SAH binding site by interacting with the three active-site
1
loops of MtbTrmD, but the inhibitors did not induce the side-chain flip of Tyr111 (Fig. 3A).
Inhibitor 15 shows over 100-fold lower affinity for MtbTrmD compared to the PaTrmD
binding affinity (Table 2), further indicating that the flip of the Tyr side-chain could play an
important role in binding to the TrmD protein. Furthermore, similar to the PaTrmD-15
structure, the location of the substituent of 15 is defined by relatively weak electron density in
MtbTrmD. Taken together, we found that the thienopyrimidinone-based inhibitors adopt
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similar binding modes in both MtbTrmD and PaTrmD by interacting with conserved residues,
except for the ability to induce a conformational change in the active-site wall loop.
Thienopyrimidinone substituent flexibility is critical for potent TrmD inhibition. The
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structure-activity studies noted earlier identified optimal R and R substituents on the
1
2
thienopyrimidinone scaffold, with a single alkyl chain of 8-10 carbons producing 8- to 50-fold
stronger affinity to TrmDs compared with AZ51 (Table 2). We next explored the mechanistic
basis for this potency. In crystal soaking experiments with PaTrmD, most thienopyrimidinone
compounds replaced SAM at only one of the two active sites in the dimer, presumably due to
the stronger affinity of SAM for binding to the second active site and/or different dynamics of
two active sites. Unexpectedly, we were able to identify 15 at both active sites of the dimer
(Supporting Information Fig. S7). We assume that the successful replacement of SAM at
both active sites resulted from a higher affinity of 15 relative to other compounds. The electron
densities of the alkyl substituent in 15 at the two active sites were poorly resolved, indicating
flexibility and multiple orientations. Interestingly, the unique side-chain flip of residue Tyr120
induced by 15 only occurs at one of the active sites, which indicates that, in the crystal soaking
condition, the two active sites have different structural environments that require
conformational flexibility in 15 for binding. To further investigate the role of the alkyl chain
of 15 in enzyme inhibition, we superimposed the published structure of tRNA-bound
2
0
HiTrmD onto the PaTrmD-15 structure and found a potentially close contact between the
flexible 15-alkyl chain and the interdomain linker, which was also observed in MtbTrmD (Fig.
3
E). Moreover, the hydrophobicity of the alkyl chain probably favors the wall-loop
conformational change leading to the formation of a hydrophobic core and facilitating ligand
binding. In Table 2, 15 shows much higher binding on-rate (k ) to PaTrmD compared with
on
AZ51 which leads to ~50-fold higher binding affinity (K ). This observation could be due to
D
the role of the hydrophobic alkyl chain in forming the hydrophobic core at the active site. To
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test this hypothesis, we replaced the long alkyl group with diethylene glycol to reduce the
hydrophobicity of the substituent but still retaining a certain level of structural flexibility
(compounds 25 and 26 in Table 1). Clearly, the ethylene glycol substituents reduce the enzyme
inhibitory activity by 4- to 8-fold, compared with the alkyl chain, further suggesting that
hydrophobicity or lipophilicity of the substituent facilitate TrmD inhibitory activity.
0
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Taken together, we show that 15 occupies the SAM binding site and induces a Tyr120 side-
chain flip. From the crystal structures, we hypothesize that the flipped side chain and the
flexible alkyl chain could interfere with tRNA binding. To further investigate this hypothesis,
we have performed a tRNA competition assay, in which the IC value was observed to increase
5
0
by 13-fold from 0.02 µM to 0.26 µM when the tRNA concentration was increased (Fig. 3F).
6
O -Substituted thienopyrimidinones. To extend the biochemical SAR study, we assessed the
6
effect of O -subtituents on the TrmD inhibitory activity of thienopyrimidinones (Fig. 1B). We
6
synthesized a series of O -substituted analogs with alkyl substituents of varying lengths by
6
treating 37, 38 and 10 with alkyl/arylalkyl halides in presence of K CO in DMF to afford O -
2
3
alkylated analogues 39-47 (Scheme 2). PaTrmD IC values were then determined for the
5
0
compounds using the in vitro assay described earlier (Table 4). The result shows that these
compounds do not inhibit PaTrmD, with the exception of 47, which is likely to undergo facile
degradation in which the aryl substituent is released to form 15. The basis for the lack of
6
activity of the O -substituted thienopyrimidinone derivatives likely results from the limited
6
space available to accommodate the O -group in the adenine-base binding site (Fig. 2B). In
addition to lower enzyme inhibitory activity compared to the other thienopyrimidinone, the O6-
substituted analogs also only show a minor impact on protein thermal stabilisation (ΔT = 1-2
m
°
C), suggesting weak interactions with PaTrmD (Supporting Information Fig. S4).
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Scheme 2. Synthesis of O -substituted thienopyrimidinones
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Conditions: (a) Et N, EtOH, reflux; (b) alkyl bromide, K CO , DMF; (c) 4 N HCl in dioxane.
3
2
3
6
Table 4. Structures and IC TrmD inhibition values for O -subsituted
5
0
thienopyrimidinone derivatives
R2
N
R1
O
R3
HN
O
N
N
S
Compound
39
R (R = H)
R3
IC (μM)
1
2
50
methyl
n-propyl
n-hexyl
NAa
NA
NA
4
0
4
4
43
1
2
n-octyl
n-octyl
n-octyl
methyl
n-propyl
n-hexyl
NA
NA
NA
4
4
O
N
4
5
methyl
NA
4
6
O
N
n-propyl
NA
47
n-octyl
1.8 ± 0.1
N3
aNA = not active up to 50 μM
Thienopyrimidinone analogues with long alkyl substituents possess antibacterial activity.
Based on the biochemical activity of the thienopyrimidinone analogues as TrmD inhibitors, we
next quantified the antibacterial activity of the compounds. The first analysis involved a single-
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dose (100 μM) growth inhibition assay against a collection of Gram-positive and Gram-
negative bacteria. Of the compounds listed in Table 1, only 15, 23 and 24 showed growth
inhibitory activity at 100 μM against Gram-positive bacteria and mycobacteria. We then used
these compounds to determine minimal inhibitory (MIC) (Table 5) and minimal bactericidal
0
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(MBC) concentrations (Table 5). The discovery of the activity of the 15 series analogues
against Gram-positive bacteria and mycobacteria is in agreement with the broad-spectrum
binding affinity of 15 to three TrmDs (Table 2; Supporting Information Fig. S5). It is worth
noting that 15 and 24 also exhibit antibacterial activity against the Gram-negative pathogens
Acinetobacter baumannii and Salmonella enteritidis with high MIC /MIC values. Thus, 15,
50
90
23 and 24 show signs of broad-spectrum antibacterial activity, possibly due to their multiple
TrmD targets. In an attempt to extend and improve the antibacterial activity to Gram-negative
2
4
bacteria, we either added primary amines to 15 and its series analogues (Scheme 1), or
conjugated with siderophores25,26 (Supporting Information Scheme S1), where we
synthesized compounds 31-34, 53 and 57, respectively (Table 1). These compounds retained
submicromolar TrmD inhibitory activity, though they did not show activity against Gram-
negative bacteria and even lost the activity to Gram-positive bacteria (data not shown).
1
5
As with the TrmD inhibitors developed by Hill et al. , there is little relationship between the
potency of TrmD inhibition by the new thienopyrimidinone compounds developed here and
their antibacterial activity. The most likely explanation is poor cell permeability, though it is
possible that inhibition of the otherwise essential TrmD by the compounds is incomplete in the
cellular environment. That bacterial uptake is responsible for the observed antibacterial activity
is supported by the observation of stronger activity against two Gram-positive pathogens (S.
aureus and S. pneumoniae) compared to the single Gram-negative pathogen, A. baumannii,
with its additional outer cell membrane (Table 5). However, mycobacteria have a complex
mycolic acid-rich cell wall that presents a drug permeability problem similar to Gram-negative
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bacteria yet M. smegmatis and M. tuberculosis show sensitivity to TrmD inhibitors similar to
Gram-positive S. aureus (Table 5). This idiosyncratic activity could result from mechanisms
of antibacterial activity other than TrmD inhibition, drug efflux pumps, or compound
degradation. The strong SAR for TrmD inhibition by thienopyrimidinone compounds
established here provides a foundation for pursuing antibacterial SAR.
1
1
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1
1
2
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2
2
2
2
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4
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4
4
4
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4
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
2
3
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9
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1
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0
Table 5. Antibacterial activities (M) for selected thienopyrimidinone analogues
15
MIC90
NA
95
NA
NA
NA
36
23
MIC90
NA
>100
NA
NA
NA
8.5
24
MIC90
NA
99
NA
>100
NA
8.5
Pathogen
a
a
MBC99.9b
NA
>100
NA
MIC50
NA
MIC50
NA
>100
NA
NA
NA
6
MBC99.9
NA
>100
NA
NA
NA
MIC50
NA
75
NA
>100
NA
5
MBC99.9
NA
>100
NA
>100
NA
34
c
P. aeruginosa
A. baumannii
Gram-negative K. pneumoniae
S. enteritidis
62
NA
NA
NA
31
NA
NA
54
E. coli
S. aureus
34
Gram-positive E. faecalis
S. pneumoniae
NA
60
58
NA
73
72
NA
NA
34
13
NA
56
18
NA
>100
36
NA
62
17
NA
74
23
NA
>100
>100
>100
>100
69
M. smegmatis
Mycobacteria
d
e
M. tuberculosis
78
>100
22
43
75
44
88
nd
aMIC50 and MIC90 represent hit concentrations inhibiting growth by 50% and 90%, respectively. MBC99.9
b
c
d
represents hit concentrations kill bacteria by 99.9%. NA = no bacterial activity. Denotes detectable growth
inhibition or bactericidal activity but less than 50% for MIC50, less than 90% for MIC90, and less than 99.9% for
e
MBC99.9. nd = not determined.
Haemolytic activity of the thienopyrimidinone compounds. To further explore the behavior
of the thienopyrimidinone analogs, we assessed the ability of the compounds to rupture red
blood cells as an index of membrane disrupting potential. The haemolytic activity of all
compounds is shown in Supporting Information Table S2. In general, most of the tested
compounds show no or weak haemolytic activity at the highest tested concentration (100 μM).
DISCUSSION AND CONCLUSIONS
Elaborating on a thienopyrimidinone scaffold, we prepared and analyzed a series of TrmD
inhibitors, which revealed a novel SAM-competitive, active site Tyr-flipping inhibition
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mechanism that distinguished Gram-negative TrmDs from Gram-positive and mycobacterial
counterparts. Several of these compounds showed nanomolar TrmD inhibition, tRNA-
competitive binding, and micromolar antimicrobial activity against Gram-positive bacteria
and, in some instances, Gram-negatives, and mycobacteria.
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EXPERIMENTAL SECTION
Protein expression and purification. Production of PaTrmD protein was described
1
4
previously . Briefly, a truncated fragment (spanning residues Leu5-Asp250) of PaTrmD was
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cloned into the vector pNIC28-Bsa4 and transformed into chemically competent E. coli BL21
(DE3) Rosetta T1R cells. For M. tuberculosis TrmD, S. aureus TrmD, E. faecalis TrmD and
S. pneumoniae TrmD, the full-length genes were inserted into vector pYUB28b-cHIS and
6
transformed into chemically competent BL21 (DE3). The following protein expression and
purification procedures were applied to all TrmD proteins. E. coli cells harboring a plasmid
were cultivated in Luria-Bertani (LB) medium at 37 C to OD600 0.6 - 0.8. The proteins were
then overexpressed by the addition of 0.5 mM IPTG at 16 C for 24 h. Cells were pelleted and
stored at -80 C.
All enzyme purification was performed on ÄKTA (GE Healthcare) chromatography system at
4 °C. The thawed pellets from 1-L cell cultures were resuspended in 30 ml of lysis buffer (20
mM Na-HEPES, pH 7.5, 0.3 M NaCl, 5% glycerol, 0.5 mM TCEP), sonicated in the presence
of one tablet of EDTA-free protease inhibitors (Roche), and the lysates cleared by
centrifugation at 22,000 rpm for 45 min in a JA-25.50 rotor. The proteins were purified by
immobilized metal affinity chromatography (IMAC) using HisTrap HP IMAC column (GE
Healthcare) and by size-exclusion on a HiLoad Superdex 200 16/60 column (GE Healthcare).
Purified proteins were concentrated to 20 mg/ml by ultrafiltration in 20 mM Na-HEPES, pH
7
.5, 0.3 M NaCl, 10% (v/v) glycerol, 0.5 mM TCEP, and were stored at -80 °C.
tRNA in vitro synthesis and purification. The preparation of tRNA substrates has been
reported previously . A PaTrmD substrate, tRNALeu(CAG), was synthesized using in vitro
transcription by MEGAshortscrip T7 Transcription Kit (Cat no. AM1354, Thermo Scientific)
according to the manufacturer’s instructions. DNA template and primers (Supporting
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Information Table S3) were obtained from Integrated DNA Technologies, Inc. (IDT). The
transcribed tRNA was desalted in RNase-free water and purified on an Agilent HPLC 1200
equipped with an Agilent Bio SEC-3 size-exclusion column (300 Å pore size, 3 µm particle
size, 7.8 mm i.d.) operated at 60 C eluted with 100 mM ammonium acetate buffer. The purity
of the tRNA product was further assessed on an Agilent 2100 Bioanalyzer with a small RNA
chip and the tRNA was quantified using a Nanodrop spectrophotometer. The final product was
desalted in RNase-free water, concentrated to 20 mg/ml and frozen at -20 °C.
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TrmD inhibitor assay. The tRNA methyltransferase activity of TrmD was measured by a
bioluminescence-based assay using the MTase-Glo Assay Kit (Promega, Cat no. V7601) in a
384-well format. Generation of SAH was measured by its conversion to ADP by the MTase-
Glo Reagent, with MTase-Glo Detection Solution applied to convert ADP to ATP, which is
then used by luciferase to generate a luminescent signal proportional to the coupled TrmD
methyltransferase activity. Luminescence was measured using a plate-reading luminometer
(BioTek Synergy 4 microplate reader) and was correlated to SAH concentration using an SAH
standard curve generated according to manufacturer’s instructions. The TrmD concentration
used in this assay is 8 nM. Thus, the estimated limit of IC detection for a substrate competitive
50
inhibitor is about 4 nM which is half of the molar concentration of the enzyme. The detailed
TrmD activity assay and kinetic studies have been described in our recent work14,17
.
The TrmD methyltransferase reaction was performed at 37 °C in 5 µl reaction mixtures
containing 8 nM TrmD enzyme, 50 mM Tris-HCl buffer, pH 8.0, 80 mM NaCl, 1 mM MgCl2,
0
.5 mM DTT, 0.01% (v/v) Triton X-100, 2 μM SAM and 0.5 μM tRNALeu(CAG), and 1x MTase-
Glo Reagent. TrmD enzyme in 1x assay buffer (50 mM Tris-HCl buffer, pH 8.0, 80 mM NaCl,
mM MgCl , 0.5 mM DTT, 0.01% v/v Triton X-100) was pre-incubated with an inhibitor in
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a serial dilution (0-500 μM) at ambient temperature for 15 min. The negative-control mix was
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prepared in an identical manner except that 1 × assay buffer was used in place of the inhibitor
solution. The reaction was initiated by adding the TrmD enzyme mixture to a final
concentration of 0.25 µg/ml. The plate was sealed and incubate at 37 °C for 45 min. After
reaction the mixture was mixed with 5 µl of MTase-Glo Detection Solution and further
incubated for 30 min at ambient temperature in the dark. The luminescent signal was read in
the microplate luminometer and background subtracted from all sample and control reads
before analyzing the net signal intensity in Prism GraphPad. The rate for each activity assay
with a series of compound titrations was expressed as a percentage of the control assay and
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analysed using nonlinear regression fit in Prism GraphPad to estimate IC values for each
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compound. The assay was done in two independent experiments performed in technical
duplicates.
Surface plasmon resonance (SPR). SPR analysis was performed using a BIACORE T200
instrument (GE Healthcare) equipped with a CM5-S sensor chip. The surfaces of all flow cells
were activated for 7 min with a 1:1 mixture of 0.1 M NHS and 0.1 M EDC at a flow rate of 10
μl/min. Anti-His antibody was captured on all surfaces to ~12,000 RU. Surfaces were then
blocked with a 7 min injection of 1 M ethanolamine, pH 8.0. The protein TrmD, at a
concentration of 50 μg/ml in 50 mM Tris-HCl pH8, 150 mM NaCl, 1 mM MgCl , 1 mM DTT
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and 0.05% Tween-20, was captured on the anti-His antibody surface to a density of ~2200 RU.
Flow cell #1 was left blank to serve as a reference surface. Different concentrations (2-fold
dilutions) of the test analyte were injected from lowest to highest concentration in the same
buffer over the flow cell at 30 μl/min for a contact time of 30 s and dissociation time of 240 s.
Analysis of inhibitor binding affinity to TrmD was quantified using 2-fold dilutions. Data were
collected at a rate of 10 Hz, and at a temperature of 25 °C. The K value was calculated using
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steady-state affinity fit.
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Crystallization and data collection: PaTrmD. The crystal screening and optimization for
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PaTrmD has been described in our recent work . Briefly, Crystals of PaTrmD were grew by
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mixing equal volumes of 20 mg ml protein and precipitant solution containing 0.1 M Tris-
HCl, pH 8.6-8.8, 20% (v/v) MPD, 20% (w/v) PEG 1000, and 5% (w/v) PEG200 and incubating
at 20 C using vapour diffusion method (hanging-drop). The crystals were soaked with 1-5 mM
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inhibitor in the precipitating solution supplemented with 20% (v/v) glycerol at 20 C for at
least 4 h and rapidly frozen in liquid nitrogen. X-ray diffraction data were collected at the PXIII
beamline in SLS (Villigen, Switzerland) for the PaTrmD-AZ51 crystal. For both PaTrmD-11
and PaTrmD-15 crystals, X-ray diffraction data were collected at the MX2 beamline in
Australian Synchrotron.
Crystallization and data collection: MtbTrmD. Crystallization conditions were screened at 20
°C with protein concentration at 10 mg/ml using the vapor diffusion method and commercial
crystallization screens in Intelli 96-3 wells sitting-drop plates. Three precipitant:protein ratios
(1:1, 1:2 and 2:1) were tested using the mosquito crystallization robot (Art Robbins
Instruments) and drop volumes of 0.2 µl. Optimized crystals of the MtbTrmD were obtained
using the vapor diffusion method (sitting drops) in 24-well trays, by mixing a volume of 2 µl
of protein with 1 µl of a precipitant solution containing 100 mM Bis-Tris propane at pH 6.5,
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0% (w/v) PEG3350, 0.1 M ammonium acetate. Prior to data collection, crystals were soaked
with 1-5 mM inhibitor (for obtaining inhibitor-bound structure) or 5 mM SAH in their
respective precipitating solution supplemented with 20% (v/v) glycerol and rapidly frozen in
liquid nitrogen. X-ray diffraction data were collected at the MX1 beamline in Australian
Synchrotron for apo MtbTrmD crystal, at the PXIII beamline in SLS (Villigen, Switzerland)
for the MtbTrmD-SAH and MtbTrmD-12 crystals, and at the MX1 and MX2 beamlines in
Australian Synchrotron for MtbTrmD-AZ51 and MtbTrmD-15 crystals, respectively.
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Structure determination and refinement: PaTrmD. Diffraction intensities were reduced with
XDS , scaled, merged and truncated with SCALA/TRUNCATE28,29. The structures were
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determined by molecular replacement using Phaser with the structure from PaTrmD-SAM
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(PDB ID 5WYQ) as search probe. Model for the 3D structure of PaTrmD was built iteratively
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at the computer graphics using COOT and refined using Autobuster . Waters and ligands
were added to the model after a few runs of refinement. The geometrical parameters for
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inhibitors were generated using program PRODRG .
Structure determination and refinement: MtbTrmD. Data reduction is the same as described
above for PaTrmD. The structures were determined by molecular replacement using Phaser30
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with the MtbTrmD structure modelled from I-TASSER server as search probe. A model for
the 3D structure of MtbTrmD was built using COOT31 and refined using Autobuster except
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that apo MtbTrmD structure was refined by Refmac . The geometrical parameters for
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inhibitors were generated using program PRODRG .
The quality of the structures was assessed using the MOLPROBITY server36
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(
molprobity.biochem.duke.edu) and figures were generated using the Pymol software . Data
collection and structure refinement parameters are summarized in Table 3.
Thermal stability assay. The thermal stability analysis of PaTrmD was performed in a 96-well
PCR plate (Bio-Rad) with 50 μl per reaction containing 5x SYPRO Orange dye (Invitrogen),
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μM test protein and the test ligand(s) at various concentrations. The assay buffer (50 mM
Tris-HCl pH8, 150 mM NaCl, 1 mM MgCl2, 1 mM DTT and 0.05% Tween-20) was added
instead of the test ligand as a negative control. The temperature was increased from 25 to 95
°
C in an i-Cycler iQ5 real-time PCR (Bio-Rad). The thermal stability curve and the temperature
midpoint T for the protein-unfolding transition was analyzed using the Bio-Rad iQ5 software.
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