Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca

Article abstract of DOI:10.1021/acsinfecdis.0c00326

Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 29 ± 4 μM) through the inhibition of NpsA (KD = 29 ± 4 nM).

Full text of DOI:10.1021/acsinfecdis.0c00326

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Article
Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin
Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca
Evan M. Alexander, Dale F. Kreitler, Valeria Guidolin, Alexander K Hurben, Eric J
Drake, Peter W. Villalta, Silvia Balbo, Andrew M. Gulick, and Courtney C. Aldrich
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.0c00326 • Publication Date (Web): 02 Jun 2020
Downloaded from pubs.acs.org on June 3, 2020
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Biosynthesis, Mechanism of Action, and Inhibition of the
Enterotoxin Tilimycin Produced by the Opportunistic
Pathogen Klebsiella oxytoca
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Evan M. Alexander, Dale F. Kreitler, Valeria Guidolin, Alexander K. Hurben, Eric
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1,3
3,4*
2*
1*
Drake, Peter W. Villalta, Silvia Balbo, Andrew M. Gulick, Courtney C. Aldrich
1Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA
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Department of Structural Biology, Jacobs School of Medicine and Biomedical Sciences
University at Buffalo, Buffalo, New York 14203, USA
3Masonic Cancer Center, University of Minnesota, Minneapolis, MN, 55455, USA
4Division of Environmental Health Sciences, School of Public Health, University of Minnesota,
Minneapolis, MN, 55455, USA
KEYWORDS: Nonribosomal peptide synthetase, adenylation, microbiome, tilimycin,
pyrrolobenzodiazepine, Klebsiella oxytoca
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ABSTRACT
Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that
causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD)
natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway
comprised of three proteins: NpsA, ThdA and NpsB. We describe the functional and structural
characterization of the fully reconstituted NRPS system and report the steady-state kinetic
analysis of all natural substrates and cofactors as well as the structural characterization of both
NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics
techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to
eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in
cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin
biosynthesis in whole cell K. oxytoca (IC = 29 ± 4 µM) through the inhibition of NpsA (K =
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D
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9 ± 4 nM).
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INTRODUCTION
The gastrointestinal (GI) tract of every human houses an extraordinarily complex ecosystem
of microorganisms, collectively known as the GI microbiota, with representation of over one
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thousand different species from all domains of life. Advances in metagenomic screening over
the past decade have begun to reveal the profound influence of the GI microbiota on human
health and disease.2–7 Normally, these commensal microorganisms work harmoniously with the
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,9
human host to maintain a symbiotic relationship, but disturbance of the gut microbiota leads to
dysbiosis, which can have far reaching effects.10,11 While many chronic diseases including
inflammatory bowel disease, obesity, diabetes, and neurodegenerative disorders have been
correlated to dysbiosis of gut microbial communities,12–22 the causative links between microbiota
and disease are often shrouded by the complexity of the host-microbiota interactions.
Klebsiella oxytoca and its role in antibiotic-associated hemorrhagic colitis (AAHC) serves as
a model for understanding the molecular basis of a disease caused by an imbalance of the gut
microbiota.23–25 K. oxytoca is an opportunistic gram-negative bacterial species that resides at low
abundance in the colon of 2–10 percent of healthy individuals.23,26 Following β-lactam antibiotic
treatment, K. oxytoca utilizes its constitutively expressed β-lactamase to survive while
commensal bacteria are eliminated. In the absence of an otherwise competitive microbial
community, K. oxytoca thrives, triggering severe dysbiosis. Subsequent expansion of K. oxytoca
in the colon leads to overproduction of tilivalline and related small molecule enterotoxins that
cause apoptosis of colonic epithelium cells.27–30 Originally, the cytotoxicity and corresponding
AAHC was attributed solely to tilivalline,23,31,32 but further analysis led to the identification of
tilimycin,27–29 which is a more potent cytotoxin and likely plays a greater role in the
pathogenicity of K. oxytoca.30,33
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Tilimycin is a tricyclic pyrrolobenzodiazepine synthesized by K. oxytoca via a nonribosomal
peptide synthetase (NRPS) pathway (Figure 1).24,28,29 NRPSs are large multi-modular proteins
responsible for the synthesis of short peptides in an assembly line like fashion, independent of
mRNA ribosomal machinery. The formation of a single amide bond in a NRPS module requires
three unique domains. First, an adenylation domain (A) is responsible for amino acid selection,
activation and transfer onto the downstream thiolation domain. The thiolation domain (T)
contains a flexible and mobile phosphopantetheinyl cofactor arm which carries the amino acid
that is connected via a thioester linkage. Finally, a condensation domain (C) catalyzes amide
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bond formation between two loaded amino acids. Tilimycin biosynthesis is performed by a
bimodular NRPS comprised of NpsA, ThdA and NpsB. NpsA and ThdA are stand-alone
proteins for adenylation and thiolation respectively while NpsB contains a complete C-A-T
module along with a C-terminal reductase (Re) domain for release of tilimycin. Bioinformatic
analyses of signature sequences within the adenylation domains suggest 3-hydroxyanthranilic
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acid is the substrate for NpsA while NpsB confers specificity for L-proline. Thus, NpsA is
predicted to activate and load the stand alone thiolation protein ThdA with 3-hydroxyanthranilic
acid, while NpsB activates and loads L-proline on to its corresponding thiolation domain. The
condensation domain is predicted to catalyze amide bond formation between the 3-
hydroxyanthranilate thioester and the free amine of L-proline to afford the L-N-(3-
hydroxyanthraniloyl)prolyl-STNpsB acyl-peptide intermediate followed by reductive release of
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the thioester to L-N-(3-hydroxyanthraniloyl)prolinal mediated by the reductase domain. Ring-
chain tautomerization of the amino aldehyde to the close-chain hemiaminals yields tilimycin.
which can undergo non-enzymatic reaction with indole, produced in large quantities
endogenously in K. oxytoca, to afford tilivalline.33
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Figure 1. The NRPS pathway for the biosynthesis of tilimycin. Reductive release of the
dipeptide affords L-N-(3-hydroxyanthraniloyl)prolinal that spontaneously cyclizes to a mixture
of diastereomeric aminals known as tilimycin that are in equilibrium with the imine species.
Non-enzymatic condensation with indole yields tilivalline. A = adenylation domain, T =
thiolation domain, C = condensation domain, Re = thioester-reductase domain.
Herein we report the detailed functional and structural characterization of the tilimycin
biosynthetic pathway that extends prior metabolic profiling and mutasynthesis studies to define
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the overall pathway and substrate tolerance. We then describe the synthesis of two classes of
rationally designed bisubstrate inhibitors which enabled structural characterization of NpsA and
a NpsA-ThdA fusion protein in the adenylation and thioester conformations, respectively. One of
the bisubstrate inhibitors was shown to selectively inhibit tilimycin synthesis in cell culture
without impairing bacterial replication. Finally, to investigate the molecular mechanism of action
of the pyrrolobenzodiazepine class of natural products, we utilized DNA adductomic techniques
to successfully detect and structurally characterize alkylated DNA from eukaryotic cells exposed
to tilimycin, helping to further clarify the causative link between a microbiota derived natural
product and human disease.
RESULTS
Cloning, Expression and Purification of NpsA, holo-ThdA and holo-NpsB. The thdA and
npsB genes were PCR amplified from K. oxytoca strain MIT-5243 genomic DNA and cloned
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into a pET15b vector containing a TEV cleavage site and a 5-His affinity tag at the N-terminus.
The npsA gene was codon optimized for expression in E. coli, synthesized and cloned into a
pET15b vector containing an identical 5-His TEV cleavage site. The plasmids were transformed
and overexpressed in E. coli BL21(DE3) cells. Two step purification was performed starting
with immobilized metal affinity chromatography (IMAC). The partially purified apo-ThdA and
apo-NpsB were incubated with the promiscuous phosphopantetheinyl transferase Sfp from
Bacillus subtilis to ensure conversion to the holo protein form. Subsequent size exclusion
chromatography afforded 20 mg/L of NpsA, 27 mg/L of holo-ThdA and 3 mg/L of holo-NpsB.
The molecular mass of the proteins determined by SDS-PAGE were consistent with their
calculated molecular weight: ~56 kDa (55,772 kDa calculated) for NpsA, ~10 kDa (9,486 kDa
calculated) for holo-ThdA, and ~150 kDa (164,015 kDa calculated) for holo-NpsB (Figure S1).
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Biochemical Characterization of NpsA and ThdA. NpsA catalyzes a two-step ATP-
dependent acylation of the phosphopantetheine prosthetic group of holo-ThdA to yield 3-
hydroxyanthraniloyl-ThdA (acyl-ThdA) that proceeds through a 3-hydroxyanthraniloyl
adenosine monophosphate (3HA-AMP) intermediate (Figure 2A). Catalysis of these two steps
requires dynamic movement of the C-terminal hinge region of NpsA as observed in other
adenylating enzymes.35–37 Initially, the “closed” adenylation conformation allows for binding and
ligation of aryl-acid and ATP substrates. The “open” thiolation conformation enables holo-ThdA
recruitment and subsequent acylation. While prior in vitro mutasynthesis studies have revealed
that NpsA possesses relaxed substrate specificity for anthranilic acid derivatives27–29, steady-state
kinetic characterization of NpsA has not been reported. To assess the steady-state kinetics of the
complete adenylation-acylation reaction, we used a continuous coupled assay wherein the
pyrophosphate (PPi) byproduct generated in the initial adenylation reaction is converted to
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phosphate by inorganic pyrophosphatase and then purine nucleoside phosphorylase catalyzes the
phosphorolysis of 7-methylthioguanosine to 7-methylthioguanine (MesG), whose formation can
be monitored at 360 nm.38,39 We confirmed the reaction velocity was linearly dependent on
NpsA concentration for this coupled assay. ThdA was also required for full activity and the
reaction rate decreased 140-fold in the absence of ThdA. Initial velocity experiments were
performed by varying one substrate at fixed saturating concentrations of the other two substrates
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and provided apparent K values of 63, 15 and 3.4 µM for 3-hydroxyanthranilic acid, ATP and
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holo-ThdA, respectively, with an average apparent kcat equal to 0.89 s (Figure 2A, Table 1).
Because of reduced rates at high concentrations of ThdA, the kinetic data were fit to a substrate-
inhibited model providing a substrate inhibition constant, K of 15 µM. Interestingly, substrate
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inhibition by thiolation domains has not been previously observed. We next explored the
substrate specificity of 3-hydroxyanthranilic acid with 3-hydroxybenzoic acid and anthranilic
acid to probe the importance of the 2-amino or 3-hydroxyl substituents. Removal of the 3-
hydroxy group in anthranilic acid led to a 52-fold decrease in the specificity constant (k /K ),
cat
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while deletion of the 2-amino group in 3-hydroxybenzoic acid provided a more pronounced 200-
fold decrease in specificity constant indicating the 2-amino group is more critical for catalytic
efficiency than the 3-hydroxy group.
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Figure 2: A) Adenylation-ligation enzymatic reaction catalyzed by NpsA. B) Initial velocity vs
substrate concentration [3HA], [ATP] and [ThdA]. Data were fit to the Michaelis-Menten
equation or substrate inhibition model. Each reaction contained 5 mM ATP and 20 µM ThdA
(for varying 3HA), 500 µM 3-HA and 20 µM ThdA (for varying 3-ATP), 500 µM 3HA and 5
mM ATP (for varying ThdA) in 50 mM Tris, 150 mM NaCl, 10 mM MgCl pH 8 buffer with 20
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nM NpsA. Data are average (±SD) of triplicate experiments.
Table 1. Apparent Kinetic parameters for NpsA.
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Substrate
K
M
(µM)
k
cat (s )
M
kcat/K (M ·s )
(6.3 ± 0.2  101
(1.8 ± 0.1)  104
3
3
-Hydroxyanthranilic acid
-Hydroxybenzoic acid
0.78 ± 0.01
0.35 ± 0.07
0.29 ± 0.01
0.82 ± 0.01
(4.0 ± 0.9  103
(8.8 ± 2.6  101
(8.5 ± 0.7  102
(3.4 ± 0.3)  102
Anthranilic acid
ATP
(1.5 ± 0.1  101
(5.6 ± 0.4)  104
(3.2 ± 0.2)  105
3
.4 ± 0.2,
holo-ThdA
1.08 ± 0.02
(K
i
= 15 ± 2)
Characterization of NpsB and Fully Reconstituted Assay. The next downstream enzyme
in the NRPS pathway is NpsB with a C-A-T-Re multidomain architecture. Unlike the stand-
alone proteins NpsA and ThdA, the enzymatic activities of NpsB cannot be easily decoupled. We
initially attempted to interrogate the activity of the embedded adenylation domain of NpsB
employing hydroxylamine to facilitate enzyme turnover by reaction with the acyl-adenylate
intermediate using the coupled assay employed for NpsA.40,41 Addition of hydroxylamine to the
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NpsB assay containing saturating L-proline and ATP did not stimulate enzyme turnover as
measured by PPi release suggesting acyl transfer to the prolyl-loaded thiolation domain (L-Pro-
STNpsB) was rapid or the initial L-Pro-adenylate was inaccessible for reaction with
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hydroxylamine (Figure S2). The observed steady-state rate for PPi formation of 0.01 sec (that
is dependent on both L-proline and ATP) is consistent with the leak of the acyl-adenylate from
the adenylation domain, which enables slow enzyme turnover. We next explored whether
addition of NADPH to the NpsB assay containing saturating L-proline and ATP could induce
turnover of L-prolyl-STNpsB through reduction of the proline thioester by the reductase (Re)
domain to a prolinal shunt product (Figure 3A, red arrow). However, addition of NADPH did
not stimulate enzyme turnover as measured by PPi release or NADPH consumption which
suggests the Re domain is only operative on the native L-N-(3-hydroxyanthraniloyl)prolyl-
STNpsB substrate. To test this hypothesis, we developed a fully reconstituted assay with
stoichiometric 3-hydroxyanthraniloyl-SThdA as substrate, which could be generated by pre-
incubating stoichiometric ThdA with catalytic NpsA and 3-hydroxyanthranilic acid. Incubation
of the substrates 3-hydroxyanthraniloyl-SThdA, L-proline, ATP and NADPH with catalytic
NpsB afforded tilimycin as confirmed with an authentic synthetic standard (Figure 3A, blue
arrow). To investigate the apparent steady-state enzyme kinetic parameters of NpsB, we
measured NADPH consumption at 340 nm and varied the concentration of one substrate (i.e. L-
proline) at fixed saturating concentration of the other two substrates (i.e. ATP and NADPH)
along with 20 µM 3-hydroxyanthraniloyl-SThdA. Initial velocity experiments provided
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apparent K values of 1.8  10 , 21 and 57 µM for of L-proline, ATP and NADPH, respectively
M
-1
with an average apparent k equal to 0.050 s (Figure 3B, Table 2)
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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
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4
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5
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3
4
5
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7
8
9
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1
2
3
4
5
6
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8
9
0
Figure 3. A) Overall enzymatic reaction catalyzed by NpsB. The A-domain of NpsB activates
L-proline to the prolyl-adenylate species and transfers it to the downstream T-domain of NpsB.
The intermediate L-Pro-ST can undergo two competitive reactions: direct reduction by the
reductase domain (red arrow) or condensation with acyl-ThdA (blue arrow), which can
subsequently be reduced by the Re domain to afford tilimycin. B) Initial velocity vs substrate
concentration [L-Proline], [ATP] and [NADPH]. Data were fit to the Michaelis-Menten equation.
Each reaction contained 5 mM ATP and 0.5 mM NADPH (for varying L-Proline), 5 mM L-
Proline and 0.5 mM NADPH (for varying ATP), 5 mM L-Proline and 5 mM ATP (for varying
NADPH) in 50 mM Tris, 150 mM NaCl, 10 mM MgCl pH 8 buffer with 250 nM NpsB and 20
2
µM 3-hydroxyanthraniloyl-SThdA. Data are average (±SD) of triplicate experiments.
Table 2. Apparent Kinetic parameters for NpsB.
cat (s-1)
-1 -1
Substrate
K
M
(µM)
k
M
kcat/K (M ·s )
(1.8 ± 0.3  103
(2.1 ± 0.3  101
(5.7 ± 1.7  101
(2.1 ± 0.4)  101
(2.7 ± 0.4  103
(1.0 ± 0.3)  103
L-Proline
ATP
0.037 ± 0.002
0.056 ± 0.002
0.058 ± 0.007
NADPH
Rational Design and Synthesis of NpsA Inhibitors. Adenylation enzyme inhibitors that
mimic the acyl-AMP intermediate (Figure 4A and 4B) can be developed by replacing the labile
phosphate moiety of the acyl-AMP with a stabile non-cleavable bioisostere such as sulfamide to
4
2
afford an acyl adenosine monosulfamide (acyl-AMS) inhibitor. The sulfamide moiety closely
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mimics the tetrahedral geometry and charge distribution of the ionized phosphate group, but is
chemically and enzymatically stabile. Acyl-AMS inhibitors derive their high affinity by
simultaneously interacting with the carboxylic acid and ATP binding pockets of the adenylation
domain while their specificity arises from the acyl group. The NRPS assembly line responsible
for tilimycin biosynthesis contains two potential adenylation domain targets, NpsA and NpsB.
The stand-alone adenylating enzyme NpsA was selected as a target because it uses the esoteric
substrate, 3-hydroxyanthranilic acid, ensuring higher selectivity than NpsB, whose adenylation
domain is functionally related to prolyl tRNA synthetases found in all domains of life.43
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
4
4
4
5
5
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
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
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0
1
2
3
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6
7
8
9
0
A
NH2
SH
N
O
O
O
N
O
P
N
N
O
O
S
O
NH2
NH2
O H
OH
OH
OH
3HA-AMP
B
C
NH2
N
SH
OH
O
N
N
O
O O
S
S
N
N
N
O
H
NH2
NH2
OH
O H
OH
3HA-AMS
NH2
N
NH2
N
SH
OH
N
N
N
N
O
S
O
S
O O
S
N
N
N
N
O
O
H
H
NH2
NH2
O H
O H
OH
OH
OH
3HA-AVS
Figure 4. A) The mechanism in which 3-hydroxyanthraniloyl-adenylate is loaded to the holo-
ThdA phosphopantetheinyl arm yielding acyl-ThdA. B) Non-cleavable bioisostere 3-
hydroxyanthraniloyl-AMS mimicking the adenylate intermediate, inhibiting acylation of the
downstream ThdA. C.) Vinyl-sulfonamide 3-hydroxyanthraniloyl-AVS participates in a hetero-
Michael addition with the terminal phosphopantetheinyl thiol of holo-ThdA resulting in covalent
modification and irreversible inhibition.
Based on the above considerations, we designed the inhibitor 3-hydroxyanthraniloyl-AMS 5.
The synthesis commenced from 2,3-isopropylideneadenosine 1 that was converted to the 5-
1
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44
azido nucleoside 2 followed by catalytic hydrogenation to the intermediate amino derivative,
which was directly condensed with sulfamide using our recently developed method by refluxing
4
5
in 1,4-dioxane to afford 3 (Scheme 1A). Acylation of 3 with N-hydroxysuccinimidyl (NHS)
0
1
2
3
4
5
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7
8
9
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ester 7 promoted by Cs CO in DMF furnished acyl-sulfamide 4. Catalytic hydrogenolysis of the
2
3
3
-benzyloxy ether and concomitant reduction of the 2-nitro group using Pd/C in methanol
followed by TFA deprotection of the acetonide afforded the final product 3-hydroxyanthraniloyl-
AMS 5 in a 45% overall yield after purification by preparative reverse-phase high-performance
liquid chromatography (HPLC). The N-hydroxysuccinimidyl ester 7 was synthesized from the 3-
benzyloxy-2-nitrobenzoic acid 646 by DCC-mediated coupling with N-hydroxysuccinimide
(Scheme 1B). To explore the SAR of 3-hydroxyanthraniloyl-AMS 5, we also prepared two
closely related analogues, 3-hydroxybenzoyl-AMS 8 and anthraniloyl-AMS 9 through deletion
of the 2-amino and 3-hydroxy functional groups present in the aryl acid moiety (Scheme 1C).
These were prepared analogously to 5 as described in the Supporting Information starting from
2
-anthranilic acid and 3-hydroxybenzoic acid.
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Scheme 1. Synthesis of 3-hydroxyanthraniloyl-AMS (5).
A
NH2
N
NH2
N
N
N
N
N
O
O
S
R
N
a) Pd/C, H2
MeOH, 6 h
H2N
N
N
7, Cs2CO3
O
O
H
DMF
O
O
O
O
23 °C, 16 h
b) sulfamide
1,4-dioxane
76%
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
4
4
4
5
5
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
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
7
8
9
0
1
00 °C, 16 h
1
, R = OH
3
ref 44
8%
64%
9
2, R = N3
NH2
N
NH2
N
N
N
N
O
O
O
O
O O
S
S
N
N
N
N
N
a) Pd/C, H2
EtOH, 3 h
N
N
O
O
O H
H
H
H
H
NO2
NH2
O
O
b) 80% aq. TFA
45% (2 steps)
OH
OBn
OH
4
5
B
C
O
O
O
N
O
OH NHS, DCC
THF
O
NO2
NO2
23 °C, 5 h
OBn
OBn
90%
6
7
NH2
N
NH2
N
N
N
N
N
O
O
O
O
O O
S
S
N
N
N
N
N
N
O
O
H
H
H
H
NH2
O H
O H
OH
OH
OH
8
9
We next synthesized a second class of adenylation inhibitor that incorporates a
vinylsulfonamide isostere of the acyl-phosphate linkage.47,48 Adenosine vinylsulfonamide (AVS)
inhibitors mimic the acyl-adenylate intermediate, but contain a reactive Michael acceptor at the
precise position of the incoming phosphopantetheine thiol that irreversibly binds the downstream
thiolation domain. AVS inhibitors thus cross-link the adenylation and thiolation domains,
stabilizing their transient interaction and are useful for capturing the adenylation domain in the
thioester conformation (Figure 4C).49,50
We designed 3-hydroxybenzoyl-AVS 13 for structural characterization of NpsA in complex
with ThdA. Notably, 13 lacks a 2-amino group on the aryl ring due to the oxidative instability of
the ortho-aminophenol moiety observed under the extended crystallization trials. The 3-hydroxy
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2
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4
4
4
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5
5
5
5
5
5
5
5
5
5
6
group was retained since it is more important for NpsA binding (vide infra). Synthesis of ligand
1
3 was achieved in three steps beginning with Horner-Wittig condensation of 3-
hydroxybenzaldehyde 10 and N-Boc-diphenylphosphoryl sulfonamide51 to afford E-
vinylsulfonamide 11 (Scheme 2). Subsequent Mitsunobu coupling of 11 with 2,3-
isopropylideneadenosine 1 gave 12. Global deprotection of the MOM, Boc and acetonide groups
using TFA yielded the final 3-hydroxybenzoyl-AVS 13.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Synthesis of 3-hydroxybenzoyl-AVS (13).
O
O
O
O
O O
Ph2P
S
S
Boc
H
NHBoc
N
1, PPh3, DIAD
H
NaH, DMF
0° to 24 °C, 16 h
92%
DCM
23 °C, 15 h
OMOM
OMOM
75% brsm
10
11
NH2
N
NH2
N
N
N
N
N
O
S
O
O O
S
8
0% aq TFA
N
N
N
N
O
O
H
Boc
23 °C, 2 h
95%
O H
O
O
OH
OMOM
OH
12
13
Biophysical and Biochemical Characterization of AMS inhibitors. Acyl-adenylate
inhibitors 5, 8, and 9 were evaluated for enzyme inhibition of NpsA using our coupled assay with
20 nM NpsA and 20 µM ThdA under initial velocity conditions in the presence of saturating
concentrations of 3-hydroxyanthranilic acid (500 µM) and ATP (5 mM). The concentration-
response plots were fit to the Morrison equation for tight binding inhibition to determine the
inhibition constants (Figure 5A). The lead inhibitor 3-hydroxylanthraniloyl-AMS 5 exhibited an
appK = 54 nM (Table 3). Analog 8 lacking the 2-amino group had a modest 4-fold loss of
i
potency while analog 9 devoid of the 3-hydroxyl group was 22-fold less potent than 5.
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1
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2
2
2
2
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. A) Concentration-response plot for NpsA inhibition by 5, 8 and 9: 3-
hydroxyanthraniloyl-AMS 5 (circle), 3-hydroxybenzoyl-AMS 8 (square), anthraniloyl-AMS 9
(triangle). The curve represents the best non-linear fit to the Morrison equation. Data are average
(
(
±SD) of triplicate experiments. B) Representative ITC binding isotherm of binding 5 to NpsA.
Top) Data obtained by titration of 25 nM NpsA with 0.25 µM 5. (Bottom) The integrated curve
showing experimental points and the best fit (–).
Table 3: Summary of characterization of all three inhibitors against purified NpsA.
TΔS
Cmpd
appK
i
(nM)
K
D
(nM)
ΔH (kcal/mol)
ΔG (kcal/mol)
(1.04 ± 0.16)  101
9.45 ± 0.23
n
(kcal/mol)
(5.4 ± 0.8  101
(2.3 ± 0.4  102
(1.2 ± 0.1)  103
(2.93 ± 0.28  101
(1.12 ± 0.68  102
(4.85 ± 2.50  102
(-1.61 ± 0.16  101
(-1.64 ± 0.29  101
(-1.09 ± 0.35)  101
5
8
9
-5.67 ± 0.21
-6.92 ± 2.91
-2.31 ± 0.47
1.06 ± 0.03
0.94 ± 0.01
1.17 ±0 .02
8.59 ± 0.31
We also determined the binding affinities of 5, 8 and 9 to NpsA by isothermal titration
calorimetry to provide the dissociation constant (K ), Gibbs free energy (ΔG), enthalpy (ΔH),
D
and entropy (ΔS) of the protein-ligand interactions (Table 3, Figure 5B). Compound 5 binds
tightly with a K of 29 nM, a value in close agreement to the apparent K value of 54 nM
D
i
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2
2
2
2
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4
4
4
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5
5
5
5
5
5
5
5
6
measured using the functional kinetic assay. Binding is enthalpically driven with a ΔH of -16.1
kcal/mol with a small unfavorable entropic component. Deletion of the 2-amino group in
compound 8 resulted in a 4-fold decrease in K relative to 5 due to an increased entropic penalty,
D
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8
9
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8
9
0
while deletion of the 3-hydroxyl in 9 provided a 17-fold decrease in K compared to 5 caused by
D
a 5.5 kcal/mol loss in enthalpy that was partially compensated by an entropic gain. The relative
binding affinities of 8 and 9 compared to 5 determined by ITC closely mirror the relative
inhibition constants from the steady-state kinetic assay.
Structures of NpsA and ThdA. To help gain further insight into molecular interactions
underpinning the observed trends in ligand affinities, we obtained an X-ray crystal structure (2.9
Å) of NpsA co-crystallized with 3-hydroxyanthraniloyl-AMS 5 (Figure 6A). The structure
features a single chain of NpsA with 3-hydroxyanthraniloyl-AMS 5 occupying the active site and
the C-terminal subdomain, A , positioned in the adenylate forming conformation identifiable
sub
by the contact between Lys492 within the Asub and the pro-R oxygen atom of the acyl-
sulfamide.42 The moderate resolution of this initial structure precluded unequivocal
determination of the orientation of the aryl ring. Therefore, we obtained X-ray crystal structures
of the NpsA N-terminal domain (in which the C-terminal subdomain of NpsA was truncated
from the Asn405 hinge residue onward) in complex with compounds 5, 8 and 9 (1.7-2.2 Å). The
resolution of the 3-hydroxyanthraniloyl-AMS 5 containing structure unequivocally displays the
orientation of the aryl ring suggested by the full-length structure (Figure 6A) in which the 3-
hydroxyl group form a tripartite H-bond with Asn207 and Ser271 (Figure 7B). This pose was
used to deduce the orientation of the ligand in the full-length structure (Figure 7A). Each
liganded structure features canonical nucleotide A domain interactions including: Asp388 with
the ribose 2,3-hydroxyls; Thr298 with the pro-S sulfamide oxygen; Asn293 with the adenine 6-
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amino group. In the aryl ring of the ligands, the 3-hydroxy group presumably participates as an
H-bond acceptor with an Asn207 donor and an H-bond donor with a Ser271 acceptor, while
anthraniloyl derivatives contain the additional 2-amino group that could potentially participate
as an H-bond donor with a Ser271 acceptor.
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
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
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8
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0
1
2
3
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We also generated an engineered NpsA-ThdA fusion construct and subsequently determined
the 2.9 Å crystal structure of NpsA-ThdA bound to 3-hydroxybenzoyl-AVS 13 (Figure 6B). The
final model contains four NpsA adenylation domains and four ThdA thiolation domains. The
linker joining the NpsA and ThdA domains are disordered in all four chains however the termini
of the respective molecules suggest that the proteins have adopted a domain swapped
configuration in which the carrier domain of one protein chain is donated to a neighboring
adenylation domain. This configuration was seen previously in the similar fusion construct from
4
8
enterobactin biosynthesis. Despite the disorder in the linker and ThdA, the interface between
the adenylation and thiolation domains is consistent and is similar to the interface seen in other
structures of trapped adenylation-thiolation structures.48–50 The Asub residues Arg411 and Arg414
have moved from a surface exposed loop in the adenylation conformation (Figure 6A) to form a
clamp over the Ser542 phosphodiester and 13, respectively in the thiolation conformation.
(Figure 6B). The majority of the NpsA-ThdA contact is mediated by a parallel helix-helix
interaction between ThdA helix 2 and a helix on the NpsA N-terminal domain that runs from
Gly248 to Val257. The residue interactions between the two appear as Arg411/Arg445/Asn541
that is analogous to the Arg437/Lys473/Asp574 motif, which is conserved among the
enterobactin, acinetobactin, bacillibactin and vibriobactin systems.48
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2
3
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0
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0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. A) 3-hydroxyanthraniloyl-AMS 5 bound to full length NpsA showing Asub domain in
the adenylation conformation. B) 3-hydroxybenzoyl-AVS 13 bound to NpsA-ThdA fusion
protein showing A sub domain in the acylation configuration binding auxiliary protein ThdA.
Figure 7. A) Full-length NpsA and 3-hydroxyanthraniloyl-AMS 5, B) NpsA-N and 3-
hydroxyanthraniloyl-AMS 5, C) NpsA-N and 3-hydroxybenzoyl-AMS 8, and D) NpsA-N and 2-
anthraniloyl-AMS 9. Each panel displays conserved A domain nucleotide binding residues
(Asn293, Thr298, Asp388). Simulated annealing electron density is calculated with coefficients
of the form mFo-DFc and contoured at 3
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Inhibition of Tilimycin Biosynthesis in Klebsiella oxytoca. 3-Hydroxyanthraniloyl-AMS 5
was next evaluated for its ability to inhibit tilimycin production in the clinical isolate K. oxytoca
strain MIT 10-5243. Due to the nonenzymatic addition of indole observed in endogenous
1
1
1
1
1
1
1
1
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2
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production of tilimycin, a high-performance liquid chromatography tandem mass spectrometry
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LC-MS/MS) method was developed to monitor the MS transition of tilivalline (334.4199.1
m/z) instead. We observed tilivalline production commenced as the K. oxytoca culture
transitioned to stationary phase at 6 hours, with peak concentrations being detected after 27
hours before declining due to degradation (Figure S4). We first showed 3-hydroxyanthraniloyl-
AMS 5 does not inhibit growth of K. oxytoca at 300 µM highlighting the selectivity and
nonmicrobicidal activity of this adenylation inhibitor (Figure S5). Tilivalline levels were
subsequently measured at 8-hours following incubation of 300 µM 5, 8, and 9 with K. oxytoca.
Unfortunately, inhibition of tilivalline was not detected for compound 5 and more careful
examination of the growth curves revealed 5 paradoxically stimulated growth of K. oxytoca in
earlier timepoints likely leading to the modest increase of tilivalline production observed (Figure
S5). We speculated metabolism or degradation of 5 under the culture conditions may have been
responsible for the lack of tilivalline inhibition. We quantified 5 in the culture of K. oxytoca
during the full time course and observed 75% degradation of 3-hydroxyanthraniloyl-AMS 5 by
the 8-hour timepoint (Figure S4). Deletion of either the 2-amino or 3-hydroxy groups of the
ortho-aminophenol in 5 with analogs 8 and 9 was expected to abolish the potential oxidative
instability and subsequent degradation. Gratifyingly, 3-hydroxybenzoyl-AMS 8 and 2-
anthraniloyl-AMS 9 reduced tilivalline levels with 8 completely ablating tilivalline production
under the experimental conditions (Figure 8A). The concentration-response plot for inhibition of
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tilivalline production of the most promising inhibitor 3-hydroxybenzoyl-AMS 8 yielded an IC50
value of 29 µM (Figure 8B).
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Figure 8. A) Cultures inoculated with K. oxytoca to a starting OD600 = 0.01 with 300 µM of 5, 8,
9 or 0.6% DMSO as a negative control were grown for 8 hours at 37 °C in CASO medium
shaking at 250 rpm. Tilivalline production was quantified using LC-MS/MS and the m/z
transitions 334.4199.1 [tilivalline (analyte)] and 424199.1 [O-benzyltilivalline (internal
standard)]. B) Concentration-response plot of the normalized % production of tilivalline at 8
hours under the incubation conditions described in panel A relative to a DMSO-only control
versus the concentration 3-hydroxybenzoyl-AMS 8. The curve was fit to a four-parameter Hill
equation to provide an IC value of 29 ± 4 µM and a Hillslope of -2.1 ± 0.5. The data points
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represent the mean (±SD) of two independent experiments.
DNA Adductomics. Tilimycin belongs to a class of NRPS secondary metabolites called
pyrrolobenzodiazepines.52,53 These compounds have been observed to react in vitro with DNA
via the electrophilic imine at the C-11 position and the nucleophilic N-2 of guanine forming a
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covalent aminal bond.54–56 Tilimycin has been predicted to react in an analogous fashion and
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ultimately results in single and double stranded DNA breaks. Downstream effects of DNA
adduct formation have been observed, however, direct detection of the DNA adducts in cells has
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33
n
not been observed. To further verify tilimycin’s mechanism of toxicity we used an LC-MS
DNA adductomics approach to simultaneously detect both anticipated DNA adducts from direct
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alkylation of DNA by tilimycin, and any unanticipated DNA adducts which may form. Data
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acquisition consists of MS and MS data dependent acquisition with MS fragmentation triggered
upon neutral loss of 2'-deoxyribose (116.0473 m/z) or one of the four nucleobases (151.0494,
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35.0545, 126.0429, 111.0433 m/z).
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Figure 9. Mass spectral data for ctDNA treated with tilimycin. A.) MS EIC of 484.1939 m/z.
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B.) MS data dependent event for 484.1939 m/z. C.) MS triggered event for
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84.1939368.1466 m/z (neutral loss of deoxyribose). D.) MS mass spectrum from
fragmentation of 484 m/z at retention time = 15.19 min. E.) MS mass spectrum from
fragmentation of 484.1939368.1466 m/z at retention time = 15.20 min.
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We began with exposure of either purified calf thymus DNA or N bacterial DNA with
tilimycin at 37 °C for 24 hours. The DNA was then enzymatically hydrolyzed, and the resulting
mixture was purified and enriched by solid phase extraction and fractionated into three portions
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10% MeOH/H O, 100% MeOH, 2% formic acid/MeOH). Each fraction was analyzed to detect
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and identify DNA adducts which formed due to the exposure. The proposed adduct dG-tilimycin
(484.1939 m/z) was observed in the 100% MeOH fraction with two distinct chromatographic
peaks (retention times of 14.5 and 15.1 min), likely corresponding to the two diastereomers
which could form upon nucleophilic addition of dG to the C-11 imine of tilimycin (Figure 9A,
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Figure S6). MS fragmentation of the parent ion of dG-tilimycin (484.1939 m/z) resulted in the
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neutral loss of deoxyribose (368.1466 m/z) which triggered MS fragmentation that was
dominated by a product ion of 269.0789 m/z (Figure 9E). Confirmation of the adduct identity
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was aided by the N bacterial DNA exposure which resulted in an isotopically labeled dG-
tilimycin (489.1791 m/z; +4.9852 m/z for 5 15N atom incorporation in the nucleobase) with
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fragmentation to 373.1323 m/z and 274.0637 m/z product ions which maintain all 5 N atoms of
guanine (Figure 10). Along with the proposed dG-tilimycin adduct, we observed a dA-tilimycin
adduct in very low abundance (Figure S7). In parallel, we analyzed calf thymus DNA exposed
tilivalline and did not detect any adducts, consistent with the hypothesis of other laboratories that
tilivalline neither degrades back into tilimycin nor interacts with DNA.
To determine if these metabolites are detectable from whole cell exposure, HEK cells were
treated with 1 µM tilimycin (IC ; Figure S13) and incubated at 37 °C for 24 hours. The cells
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were lysed, and the DNA was extracted, hydrolyzed, purified and enriched identically to the
DNA exposure studies as describe previously. Targeted mass spectrometry of the MS2
fragmentation of 484.1939 m/z yielded distinct extracted ion chromatogram (EIC) peaks of
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68.1446 m/z (neutral loss of 2'-deoxyribose) and 350.1360 m/z (neutral loss of 2'-deoxyribose
and H O) between 13.2-14.0 min (Figure 11) consistent with the proposed dG-tilimycin adduct
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observed in the tilimycin-treated ctDNA (Figure 9). The dA-tilimycin adduct was not observed,
likely indicating it is below the limit of detection of the assay.
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Figure 10. MS Fragmentation pattern of dG-tilimycin adduct (possible structure) from tilimycin
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exposure to ctDNA (black) overlaid with identical exposure to N bacterial DNA (blue).
Possible product ions F0-F4 shown.
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Figure 11. MS EIC of 484.1939368.1466 m/z (neutral loss of deoxyribose) for tilimycin-
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treated HEK cells (A), DMSO-only HEK cells (B), and tilimycin treated ctDNA (C). MS EIC of
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84.1939350.1360 m/z (neutral loss of deoxyribose, H O) for tilimycin-treated cells (D),
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DISCUSSION
Tilimycin belongs to a large class of NRPS secondary metabolites called
pyrrolobenzodiazepines (PBD) that contain a tricyclic ring system formed by condensation of
anthranilic acid and L-proline derivatives.59 PBDs are largely produced by the Streptomyces
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genus of bacteria and their exceptional cytotoxicity has been exploited to design synthetic PBD
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dimers as investigational chemotherapeutic agents. Targeted delivery of PBD dimers to tumor
cells through antibody-drug conjugate techniques has been investigated in multiple Phase I-III
clinical trials.60–62 Thus, the presence of an endogenously produced PBD within the human
microbiota is concerning.24
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63
The biosynthesis of prototypical PBDs including anthramycin , tomaymycin , and
sibiromycin64,65 have been elucidated by functional assignment of the corresponding biosynthetic
gene clusters through homology analysis, gene inactivation, and chemical complementation
studies. The PBD core is assembled by a bimodular NRPS comprised of a loading module with
an adenylation and thiolation domain, either as a single protein or separate stand-alone proteins,
and an extension module with a C-A-T-Re architecture. Müller and co-workers recently reported
the first attempt to biochemically reconstitute a complete PBD NRPS system using LC-MS to
directly detect phosphopantetheine tethered substrates including anthranilic acid and proline
derivatives. A dipeptide intermediate attached to the thiolation domain of the extension module
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was also observed. However, only trace quantities of PBD were produced, which the authors
speculated may be due to insufficient channeling between the recombinant NRPS proteins under
the in vitro assay conditions.63 We successfully reconstituted the complete tilivalline NRPS
employing recombinant NspA, ThdA, and NspB proteins and were able to dissect the activities
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of each module using continuous coupled assays that measured production of pyrophosphate or
NADPH consumption.
We first interrogated the activity of the loading module, which was greatly facilitated by the
unique organization of the tilimycin NRPS system wherein the adenylation and thiolation
domains, NpsA and ThdA, are stand-alone proteins, enabling use of stoichiometric quantities of
ThdA to promote turnover. Typically, adenylation domain activity is evaluated using a non-
physiologically relevant pyrophosphate exchange assay that only measures the first half-reaction
in the reverse direction through incorporation of pyrosphosphate into ATP. Instead, we evaluated
the loading of ThdA by NpsA by quantifying formation of the pyrophosphate byproduct using a
coupled enzyme system consisting of pyrophosphatase and purine nucleoside phosphorylase.38,40
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NpsA catalyzed the efficient loading of ThdA with 3-hydroxyanthranilic acid yielding a k of
cat
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0
.89 s and K values of 63, 15 and 3.4 µM for 3-hydroxyanthranilic acid, ATP and holo-ThdA,
M
respectively. The K for holo-ThdA is commensurate with other values reported for thiolation
M
domains with their cognate adenylation domains; however, the apparent K of ATP and 3-
M
hydroxyanthranilic acid are an order of magnitude lower and higher than related substrates with
aryl acid adenylation domains, respectively.36,41,66 Apparent K values determined for these
M
NRPS adenylation enzymes are strongly affected by the assay utilized (hydroxamate-MesG
versus the classical pyrophosphate exchange) as well as substrate concentrations, which could
explain the observed discrepancies.
We also observed ThdA possessed strong substrate inhibition with a K = 15 ± 2 µM. The
i
three dimensional structure of our NpsA:ThdA fusion construct shows the interface between the
ThdA loop 1 and the Asub domain of NpsA are composed of many electrostatic and non-specific
hydrophobic interactions. These interactions are also conserved in the homologous natural
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didomain of the Xenorhabdus species.67 These observations support the hypothesis that the
interface observed in the NpsA-ThdA crystal structure is physiologically relevant. This
interaction also explains the observed inhibition at high concentrations of ThdA; the substrate
binding pocket is occluded by ThdA and Asub when ThdA is bound to NpsA in the thiolation
conformation. There may be an equilibrium between adenylation and thiolation conformations
that shift in favor of thiolation in the presence of holo-ThdA.
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Interrogation of NpsB activity proved much more challenging and initial attempts using our
coupled assay systems for pyrophosphate or NADPH consumption failed to show any turnover.
However, catalytic turnover could be achieved employing stoichiometric concentrations of pre-
loaded ThdA as the substrate for NpsB in the presence of the other co-substrates L-proline and
NADPH. The time-dependent formation of tilivalline was confirmed by LC-MS analysis
employing an authentic synthetic standard. We were able to demonstrate for the first time the
-1
efficient production of a PBD in vitro with an overall kobs of 0.013 s using this product
formation assay (Figure S16). The inability of NpsB to turnover L-proline in the presence of
ATP and NADPH suggests the reductase domain possesses high specificity for the native L-N-
(3-hydroxyanthraniloyl)prolyl-STNpsB intermediate over L-prolyl-STNpsB, which stands in stark
contrast to other reported Re domains that more rapidly reduce the amino thioester monomers
over the corresponding dipeptide thioesters.68 The close correspondence between the kobs
-1
determined in the product formation and the kcat of 0.05 s independently determined using a
NADPH consumption assay confirm reduction is tightly coupled to dipeptide formation. While
the overall kcat is low, it represents a composite of the microscopic rate constants for L-proline
adenylation and thioesterification onto the thiolation domain of NpsB, condensation of the
resulting L-prolyl-STNpsB with the upstream 3-hydroxyanthraniloyl-SThdA, and thioester
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reduction of the L-N-(3-hydroxyanthraniloyl)prolyl-STNpsB intermediate to a dipeptide aldehyde
species that spontaneously cyclizes to tilimycin as a diastereomeric mixtures of cyclic aminals.
We hypothesize the rate-limiting step is likely reductive release since previous studies with Re
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domains68,69 show low turnover numbers (apparent k ’s ranging from 0.03 to 0.003 sec-1). By
cat
contrast, the reaction rates of isolated adenylation and condensation domains are typically two-
to-three orders of magnitudes greater than our observed kcat.39,50,68,70,71
The major metabolite isolated from K. oxytoca in cell culture as well as the gut microbiome
is not tilimycin, but rather tilivalline due to non-enzymatic and irreversible reaction of tilimycin
with indole by electrophilic aromatic substitution.24,29,33 The reaction proceeds through an
iminium species produced by dehydration of tilimycin. The initial report by Zechner and co-
workers identified tilivalline as the major metabolite produced by K. oxytoca; however,
subsequent studies have shown tilimycin is also formed in vitro and in vivo.29,33 Tilimycin is an
order of magnitude more cytotoxic than tilivalline and is also a genotoxic DNA-damaging agent,
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whereas tilivalline inhibits microtubule formation. Consequently, tilimycin likely plays a more
significant role in AAHC and may additionally lead to cellular transformation upon long-term
exposure.33
Interestingly, the relationship between the dipeptides tilimycin and tilivalline, which are
produced by enzymatic and non-enzymatic processes and mediate different cellular effects, bears
similarity to other NRPS systems. Fischbach et al. has shown that NRPS biosynthetic gene
clusters harboring terminal reductase domains that afford dipeptide aldehydes are ubiquitous in
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the gut microbiota. Dipeptide aldehydes produced in the gut can potentially modulate host-
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microbe interactions through inhibition of cysteine proteases. Cyclization of typical dipeptide
aldehydes lacking an anthranilate moiety occurs slowly over several hours in the gut yielding
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dihydropyrazinone and pyrazinone metabolites, which are predicted to have important
physiological roles in mediating microbe-microbe interactions.68,73
In parallel to our characterization of NpsA we synthesized the bisubstrate inhibitors 3-
hydroxyanthraniloyl-AMS 5, 3-hydroxybenzoyl-AMS 8 and anthraniloyl-AMS 9. Biophysical
characterization by isothermal titration calorimetry revealed the 3-hydroxyl of the aryl ring is
more important than the 2-amino substituent since removal of the 3-hydroxyl group in 8 led to a
greater than 20-fold loss of affinity and concordant 4 kcal/mol decrease in binding enthalpy. The
ITC result is consistent with the removal of the two hydrogen bonds (Asn207/Ser271) that could
contribute ~2 kcal/mol each to binding enthalpy as seen in the co-crystal structures. By contrast,
deletion of the 2-amino group in 9 led to no reduction in binding enthalpy and a modest 4-fold
reduction in affinity that can be attributed to an entropic penalty from removal of the possible
prearrangement via intramolecular hydrogen bonding between the 2-amino and the N-acyl
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sulfamide. The trend in binding affinities for 3-hydroxybenzoyl ligand 8 (K = 112 nM) and
D
anthraniloyl ligand 9 (K = 485 nM) is counterintuitively opposite the trend in K values for the
D
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respective substrates 3-hydroxybenzoic acid (K = 4,000 µM) and anthranilic acid (K = 854
M
M
nM) with NpsA. These results indicate the 3-hydroxy is more critical for binding, whereas the 2-
amino group is important for catalytic efficiency.
The design of inhibitors of specific enzymes in gut microbial pathways mechanistically
linked to human diseases represents a novel strategy to develop a new class of pharmacological
agents.74–76 The tilimycin pathway has been genetically validated for causing AAHC.24
33
Moreover, tilimycin and many PBDs are potent genotoxins, thus small-molecule inhibitors of
tilimycin biosynthesis could have therapeutic utility for not only treating AAHC, but also to
prevent transformation of colorectal cells in the 2–10% of people colonized with K. oxytoca.
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Attempts to ‘drug the microbiome’ are still in the early stages of conception; however, non-
antibiotic approaches may have utility, especially in light of the rise of antimicrobial resistance
and the revolution of precision medicine.77,78 As a prelude to future studies for treating AAHC,
we evaluated our bisubstrate inhibitors for inhibition of tilimycin in K. oxytoca cell cultures. We
initially examined our most potent enzyme inhibitor 3-hydroxyanthraniloyl-AMS 5 against K.
oxytoca and were dismayed to observe no inhibition of tilimycin production. We speculated the
lack of activity may be due to oxidative degradation of the ortho-aminophenol moiety of 3-
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hydroxyanthraniloyl-AMS 5 under the aerobic culture conditions. Indeed, we observed rapid
degradation of 5 under the culture conditions, so we examined 3-hydroxybenzoyl-AMS 8 and
anthraniloyl-AMS 9 that lack the sensitive ortho-aminophenol moiety. Consistent with its
improved biochemical potency, 3-hydroxybenzoyl-AMS 8 was found to possess superior activity
inhibiting 50% tilimycin production at 29 µM, but no observable growth inhibition of K. oxytoca
at 300 µM, the highest concentration evaluated. Previous studies of related adenylation inhibitors
have shown they possess poor bioavailability and thus are expected to accumulate to high levels
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in the gastrointestinal tract. Consequently, inhibitors such as 8 may have ideal characteristics
for selective inhibition of tilimycin inhibition while sparing both the host and the microbiome.
PBDs share a common mechanism of action through the covalent binding to guanosine in
double-stranded DNA, which was established by a series of seminal studies in the 1970s.57,80
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single X-ray co-crystal structure of anthramycin bound to the synthetic oligonucleotide 5-
CCAACGTTGG-3 confirmed anthramycin binds in the minor groove and alkylates N-2 of the
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guanine nucleobase. However, limited binding is available for most PBDs and DNA interaction
is inferred using molecular modeling and thermal stabilization assays measuring unfolding of
double-strand DNA with short synthetic oligonucleotides.82–86 A few recent studies have
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