Discovery of PqsE Thioesterase Inhibitors for Pseudomonas aeruginosa Using DNA-Encoded Small Molecule Library Screening

Article abstract of DOI:10.1021/acschembio.9b00905

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent inhibitors were resynthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that noncompetitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC₅₀ = 5 μM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well-defined.

Full text of DOI:10.1021/acschembio.9b00905

Subscriber access provided by Vanderbilt Libraries
Article
Discovery of PqsE Thioesterase Inhibitors for Pseudomonas
aeruginosa using DNA-Encoded Small Molecule Library Screening
Julie S Valastyan, Michael R Tota, Isabelle R. Taylor, Vasiliki Stergioula, Graham
A.B. Hone, Chari D Smith, Brad R. Henke, Kenneth G Carson, and Bonnie L Bassler
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00905 • Publication Date (Web): 27 Dec 2019
Downloaded from pubs.acs.org on December 28, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 30
ACS Chemical Biology
1
2
3
4
Discovery of PqsE Thioesterase Inhibitors for Pseudomonas aeruginosa using DNA-Encoded
5
6
7
Small Molecule Library Screening
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Julie S. Valastyan1,2, Michael R. Tota3, Isabelle R. Taylor1, Vasiliki Stergioula1, Graham A.B.
Hone3, Chari D. Smith1, Brad R. Henke4, Kenneth G. Carson3, and Bonnie L. Bassler1,2*
1Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
2Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
3Macroceutics Incorporated, Monmouth Junction, NJ 08852, USA (now HotSpot Therapeutics).
4Opti-Mol Consulting, LLC, Cary, North Carolina 27513, USA.
*To whom correspondence should be addressed. Email: bbassler@princeton.edu
Short Title: Small Molecule Screen for PqsE Inhibitors
ACS Paragon Plus Environment

ACS Chemical Biology
Page 2 of 30
1
2
3
4
ABSTRACT
5
6
7
8
9
Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States.
PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive
target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A
library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to
the purified PqsE protein. The structures of the bound molecules were identified by high
throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest
affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent
inhibitors were re-synthesized off DNA and examined for the ability to alter PqsE thermal melting
and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism
of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic
acids that non-competitively inhibit PqsE. A small set of analogs designed to probe initial
structure-activity relationships showed increases in potency relative to the original hits, the best
of which has an IC50 = 5 M. Compound refinement is required to assess their in vivo activities as
the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates
DNA-encoded compound library screening as a rapid and effective method to identify catalytic
inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins
revealed by genetic screening to have crucial in vivo activities but whose biological functions have
not been well defined.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that
colonizes a variety of niches, including the skin, lungs, plants, soil, and abiotic surfaces1. P.
aeruginosa is notorious for causing nosocomial infections, most notably of immune-compromised
individuals, cystic fibrosis (CF) sufferers, burn victims, and patients with indwelling medical
devices, creating a significant health burden2. P. aeruginosa is resistant to many commonly used
ACS Paragon Plus Environment

Page 3 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
antibiotics, hindering eradication of infections3,4. Antibiotic resistance is linked to the ability of P.
aeruginosa to form sessile communities of cells adhered to surfaces, called biofilms, a trait that
has propelled P. aeruginosa to become a leading cause of hospital-acquired infections in the
United States5–8. New approaches are urgently needed to combat P. aeruginosa pathogenesis.
P. aeruginosa virulence and biofilm formation are controlled by the cell-cell communication
process called quorum sensing that relies on the production, release, and group-wide detection
of extracellular signal molecules called autoinducers9–11. Quorum sensing enables bacteria to
orchestrate collective behaviors, and in the case of P. aeruginosa, these behaviors include biofilm
formation and the production of the virulence factor pyocyanin, among other pathogenicity traits
(recently reviewed in 12). Germane to the present work is the recent finding that the P. aeruginosa
RhlR quorum-sensing receptor responds to two autoinducers: a canonical homoserine lactone
(C4-homoserine lactone) that is synthesized by RhlI, and a second, alternative autoinducer of
unknown structure that is produced by the PqsE thioesterase enzyme13–17. Thus, RhlR-RhlI and
RhlR-PqsE form quorum-sensing receptor-autoinducer synthase pairs. What is crucial is that the
RhlR-PqsE pair, and not the RhlR-RhlI pair, is essential for virulence in both a nematode and a
mouse model of infection16. Specifically, both rhlR and pqsE mutants are highly attenuated for
virulence16. Indeed, the pqsE mutant is even more defective for virulence than the rhlR mutant,
making PqsE an interesting target for in vivo inhibition in efforts to develop new therapeutics16.
The conundrum is that the in vivo role of PqsE is unknown. In brief, the pqsE gene is a
member of the pqsABCDE operon responsible for synthesis of the signaling molecule 2-heptyl-3-
hydroxy-4-quinolone (PQS), the autoinducer for a different quorum-sensing pathway18. However,
PqsE, the final gene in the operon, is not required for PQS synthesis15. PqsE is known to be a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
thioesterase enzyme based on structural studies and in vitro enzyme activity assays19,20
.
However, neither the endogenous substrate(s) nor the product(s) of PqsE catalysis are known. It
is also known that PqsE, via an undefined mechanism, is required for production of the virulence
factor pyocyanin21,22. Specifically, a P. aeruginosa pqsE mutant and P. aeruginosa harboring the
ACS Paragon Plus Environment

ACS Chemical Biology
Page 4 of 30
1
2
3
4
5
6
7
8
catalysis-inactive PqsE S273A protein make no pyocyanin16. Curiously however, a previously
discovered small molecule, C1, that inhibits PqsE thioesterase activity in vitro does not affect in
vivo pyocyanin production levels23.
9
Given the critical role of PqsE in P. aeruginosa virulence coupled with the poor
understanding of PqsE function, we reasoned that an unbiased method of screening coupled with
a strategy for rapidly and efficiently testing very large numbers of compounds could deliver the
best opportunity to identify structurally diverse small molecule PqsE inhibitors. High-diversity
DNA-encoded libraries (DELs) can be used for unbiased, pooled screening to identify molecules
that bind to a purified, functional protein target of interest24–26. Several hundred million molecules
are screened rapidly and simultaneously (reviewed in 27,28). The approach yields molecules that
bind the protein target irrespective of the protein’s biochemical activity, in our case, enabling us
to subsequently interrogate their PqsE-related functions and correlate those functions with in vivo
effects relevant to antimicrobial activity. Sorting through hits to identify those compounds with
desired properties does demand robust, high-capacity, secondary screens that report on activities
of interest29. A final attractive feature of DELs is that the size of these libraries generally provides
sufficient numbers of structurally related hit compounds to support convincing structure-activity
relationships (SAR) and minimizes the risk of prosecuting ‘‘singleton” screening hits in which
structural analogs of the original hit molecule have no specific activity for the desired target30.
Here, we describe the affinity-based selection method we used to screen a DEL of drug-
like compounds for binders to purified PqsE. Multiple clusters of hits were identified with shared
chemical features, representing potential small molecule tools to study a range of catalytic and
regulatory roles of PqsE. Following a two-step validation strategy, we identified the subset of
binders that inhibit PqsE thioesterase activity. We report here the initial results from hit
compounds of one chemotype, a series of 2-(phenylcarbamoyl)benzoic acids. We prepared a
small set of analogs based on the structures of two hit molecules and performed an initial probe
of SAR. This strategy enabled identification of compounds with increased potency. Mechanism of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 5 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
action studies indicate these inhibitors exhibit non-competitive inhibition of PqsE-mediated
hydrolysis of a synthetic substrate. The compounds failed to inhibit in vivo production of pyocyanin
because they do not enter, are rapidly metabolized by, or are rapidly exported from P. aeruginosa.
Additional structural classes revealed by this screen may provide future opportunities for further
exploration of PqsE inhibition.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS
Development of a DNA-encoded small molecule screen for PqsE binders
To develop an affinity-based screen for DNA-encoded small molecules that bind to PqsE,
we first examined the requirements for the protein target. A plasmid carrying the P. aeruginosa
pqsE gene fused to the DNA encoding an N-terminal 6xHis tag was introduced into E. coli
BL2116,19. Protein production was induced and 6xHis-PqsE was purified to >90% on nickel resin
using FPLC. Purified 6xHis-PqsE protein was assessed for the ability to bind to cobalt resin and
to withstand the mandatory washing steps that would later be used in the screen (Figure S1A)31.
We selected cobalt resin on which to perform the screen to exploit resin binding by the 6xHis tag,
with the notion that contaminating proteins that co-purified with 6xHis-PqsE on nickel would be
eliminated (Figure S1A, designated FT)31. The majority of the 6xHis-PqsE protein could be eluted
from the cobalt resin with 500 mM imidazole (Figure S1A, Eluate 1), and a portion of the remaining
protein could be eluted in buffer containing 1 M imidazole (Figure S1A, Eluate 2). Nonetheless, a
fraction of the 6xHis-PqsE protein remained bound to the resin after these treatments. That
fraction could be recovered by heating the resin to 60°C for 5 min (Figure S1A Eluate 3). The
rationale was that heat treatment would enable recovery of the remainder of the protein and
simultaneously destabilize PqsE, resulting in release of the bound small molecules.
We next made two constructs as controls to verify that the covalently attached DNA
barcodes do not interfere with compound binding to PqsE. First, as a positive control, we needed
ACS Paragon Plus Environment

ACS Chemical Biology
Page 6 of 30
1
2
3
4
5
6
7
8
9
a small molecule PqsE binder that could be linked to DNA at a site of attachment on the molecule
that would not interfere with compound binding. Hartmann23 has published a set of small molecule
PqsE inhibitors, three of which have been co-crystallized with PqsE, providing key structural
information on potential points of attachment for a linker to DNA. We selected the highest affinity
inhibitor, 2-(pyridin-3’-yl) benzoic acid (called C1), for modification. Inspection of the X-ray co-
crystal structure of C1 complexed with PqsE suggested that a linker attached at the 4’ position
(Figure S1B) would likely project out of PqsE into a solvent-exposed region. We therefore
synthesized probe molecule P1 (Figure S1B, synthesis details in Supplementary Information) and
coupled the molecule to an azide-based 8-base pair DNA headpiece using click chemistry32. A
full-length DNA barcode (~120bp) was subsequently added by ligation (Figure S1B, called
Positive Control). As a negative control, we tested a ~120bp DNA barcode without an attached
small molecule.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Prior to examining binding of the two controls to PqsE, we validated our ability to quantify
each control by RT-PCR of the respective barcode, including in mixtures (Table S1 and
Supplemental Methods). The PCR primers used to amplify the positive control DNA barcode did
not amplify the un-modified negative control DNA barcode (Figure S1C, left). However, the PCR
primers used to amplify the negative control barcode DNA exhibited modest amplification of the
positive P1-attached barcode DNA (Figure S1C, right). Thus, we could expect a false elevation in
the PCR value from the negative control in mixtures quantified by PCR. Our linear range for PCR
amplification spans seven-logs, so we reasoned that so long as we could discern a significant
increase in amplification of the P1-attached positive control DNA barcode relative to that of the
un-modified negative control DNA barcode, we could accept some background noise in the
readout. Therefore, we went forward with the binding analysis.
A schematic of the overall screening process is shown in Figure 1A. In brief, to test if the
PqsE protein could selectively bind and retain a DNA-encoded molecule, we combined 100 pmol
of the un-modified negative control DNA barcode with 1 pmol of the positive control P1-DNA-
ACS Paragon Plus Environment

Page 7 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
encoded compound and assessed binding to 6xHis-PqsE-bound cobalt resin. This compound
mixture is referred to as the Input. First, the Input mixture was pre-incubated with the 6xHis-PqsE
purified protein. Following incubation, the 6xHis-PqsE-Input mixture was loaded onto cobalt resin.
The resin was washed five times to remove all unbound molecules, then 6xHis-PqsE and bound
molecules were eluted by heating in 500 mM imidazole. We call this the Round 1 selection. The
molecules isolated from the 6xHis-PqsE in Round 1 were incubated with DNA-binding magnetic
beads to remove imidazole. This process was repeated two more times, each time with fresh
6xHis-PqsE protein (designated Round 2 and Round 3 selection). After each round of selection,
the amounts of bound P1-encoded and un-modified barcode DNAs were quantified by RT-PCR
(Figure 1B).Starting with a 100-fold excess of negative control DNA, three rounds of selection
enabled us to recover a ~50-fold excess of positive control DNA, representing a 5,000-fold
enrichment, suggesting the potential for identification of specific PqsE binders using this protocol.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A
B
D
C
3700-fold
1600-fold
Input
103
102
104
103
102
101
100
10-1
10-2
10-3
Input
Round 1
Round 2
Round 3
101
Round 1
Round 2
Round 3
100
10-1
10-2
10-3
10-4
10-5
10-6
10-7
80-fold
25-fold
1-fold
9-fold
Negative
Control
Positive
Control
6xHis-PqsE
Resin Control
Head Piece Codon Codon Codon
(Constant)
Library
ID
Degenerate
Sequence
Experiment
ID
Closing Piece
(Constant)
1
2
3
ACS Paragon Plus Environment

ACS Chemical Biology
Page 8 of 30
1
2
3
4
5
6
7
8
9
Figure 1 a) Flow chart summarizing the screening method. b) Quantitation of positive and
negative control DNA barcodes following three rounds of selection with purified 6xHis-PqsE. Error
bars represent standard deviation (SD) of technical replicates, N=4. c) Quantitation of the amount
of library barcode DNA remaining following three rounds of selection with resin coated with 6xHis-
PqsE or naked resin. The fold changes in remaining molecules are denoted. Error bars represent
SD of technical replicates, N=5. d) Diagram showing the major motifs in the DNA-tag.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Production of the compound library
The Macroceutics DEL that was screened against PqsE contained approximately 550
million small molecules. This compound set was generated from five different library builds
(DEL01-DEL05), and all of the libraries were synthesized using traditional split-pool-mix
techniques outlined previously26,33. The various chemistries used to produce these libraries
included acylation, reductive amination, sulfonylation, and SN2-mediated alkylation34. Libraries
were made in both branched and linear geometries. This Macroceutics DEL was designed to
maximize diversity, while maintaining compound physical properties suitable for lead optimization
in follow-up medicinal chemistry efforts. An example schematic of DEL02, a library produced as
a branched dimer, is shown in Figure S1D. Cycle 1 (position c1) consisted of approximately 65
trifunctional cores, each of which had orthogonal protection moieties on two nitrogen atoms as
depicted in Figure S1D. These 65 cores were loaded on DNA, and using subsequent split-pool-
mix methodology26,33, reacted with ~1,000 building blocks each in Cycle 2 (position c2) and Cycle
3 (position c3), to provide a branched design with a final library size of 65 million compounds in
DEL02.
Screening the DNA-encoded compound library for PqsE binders
We used the above protocol to screen the DNA-encoded small molecule library for
compounds that bind to PqsE (Figure 1A). We pre-incubated 5,000 pmol of the library,
ACS Paragon Plus Environment

Page 9 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
representing a pool of 550 million unique molecules, with 6xHis-PqsE protein and performed the
three rounds of selection. Following each round of selection, an aliquot was removed and total
DNA bound to PqsE was quantified by RT-PCR to assess how many encoded molecules
remained (Figure 1C). We performed the identical procedure with cobalt resin to which no 6xHis-
PqsE had been added to assess the background level of encoded molecules binding to the resin.
Following three rounds of selection, 10-fold more DNA-encoded molecules remained bound to
the resin coated with 6xHis-PqsE than to the resin itself, suggesting that, while there may be
overlap, the resin containing 6xHis-PqsE protein shows high selectivity to bind a subset of our
DNA barcoded compounds. The library molecules remaining on both the 6xHis-PqsE-coated and
the naked resins were collected following heat treatment of the resins and the DNA barcodes
were sequenced (Figure 1A). Additionally, an aliquot of the naïve library that had not been
subjected to any selection was sequenced to eliminate any sequencing bias arising from the DNA
tags (Figure 1A).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Analysis of small molecule hits
The DNA from our selection experiments was assessed by Next-Generation sequencing25.
Using Macroceutics’ in-house proprietary software, the sequence data were analyzed and
compared to the naïve library and resin-binding control data. Important for this analysis is that
each DNA tag contained multiple motifs for decoding the screening results (Figure 1D). First, the
terminal DNA sequences, called the head and closing pieces, were constant. Primers were
designed to hybridize with these two regions to enable PCR amplification of the tags. Second,
each DNA sequence contained a barcode identifying the DEL from which the compound came
(Library ID), allowing multiple libraries to be screened simultaneously. In our screen, five DELs
were pooled and screened, so there were five different library ID sequences. Third, the tags
contained an experiment ID DNA sequence, unique to each screen. Fourth, individual chemical
building blocks incorporated into the compounds were encoded by unique DNA sequences called
ACS Paragon Plus Environment

ACS Chemical Biology
Page 10 of 30
1
2
3
4
5
6
7
8
9
codons. Since there were three chemical cycles used to produce each compound in each library,
the DNA tag for each compound contained all three of its corresponding codons. This feature
enables compound structure identification. Finally, a degenerate DNA sequence was included as
a unique ID for each individual compound. Since the libraries were amplified by PCR prior to
sequencing, the degenerate sequence allowed differentiation of unique hits from duplications
arising from PCR amplification. Our strategy for hit analysis is shown in Figure 2A.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 11 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
A
B
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C
60
40
20
0
c3
iii
ii
i
i
6xHis-PqsE
Empty Resin
ii
c1
c2
iii
01
02
03
04
5
05
DEL Library
D 1.5
-2
4
1
-1
14
17
5
5
7
11
11
14
13
21
1.0
21
22
24
28
38
41
45
43
47 47
47
50
55
62
A
62
E
66
D
0.5
0.0
83
1
I
y
J
L
F
T
Z
E
s
A
B
E
K
P
S
V
X
Y
B
l
C
D
H
N
R
U
C
G
O
Q
M
W
n
C
A
B
E
C
D
q
o
P
L
+
T
On-DNA Compounds
L
B
T
m
B
m
E
Figure 2 a) Flow chart summarizing the pipeline for assessment of screening hits. b) Fraction of
total reads from each DEL based on sequencing of DNA tags for compounds bound by 6xHis-
PqsE versus those bound to the empty resin. c) Data cube showing the screening hits from DEL02
that possessed codon combinations with hit counts of 75 or greater. The X, Y, and Z axes
represent, respectively, compound building blocks in the three codons representing synthetic
positions c1, c2 and c3 as shown in Figure S1D. Codon combinations that yielded hits in the resin
ACS Paragon Plus Environment

ACS Chemical Biology
Page 12 of 30
1
2
3
4
5
6
7
8
9
binding or in the naïve library controls have been removed from the view. Three related molecules
(i, ii, and iii) corresponding to strong features in the data cube are shown. The moieties designated
R1, R2, and R3 represent the various Cycle 3 capping moieties from the library. d) Thioesterase
inhibition by 10 M of the on-DNA candidate compounds in the Ellman’s assay. We note that the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
positive control inhibitor C1 was tested at 100 M explaining its higher potency in the graph. The
% inhibition caused by each compound is shown above its corresponding bar. Error bars
represent SD of technical replicates, n=2. e) General synthesis of compounds 1–16. Reagents
and conditions: (a) Diisopropyl azodicarboxylate (DIAD), Triphenylphosphine (Ph3P),
Tetrahydrofuran (THF), 0°C to RT, 12 h; (b) Iron (Fe), Ammonium chloride (NH4Cl), Ethanol
(EtOH), 80°C, 12 h; (c) Dichloromethane (CH2Cl2), RT, 6 h.
Regarding the success of our screen, the library ID sequences showed different fractions
of total reads came from each of the five DELs and each DEL had a distinct background profile
(Figure 2B) demonstrating that the libraries behave differently with respect to binding to PqsE and
to the control resin. Each codon combination encodes a unique compound and the number of
times that combination appears (hit count) can be determined using the unique sequences of the
codons and the corresponding degenerate sequence. A high hit count is assumed to represent
an enhancement of that compound during selection. Since each library has a three building-block
design, with three synthetic codons, it is convenient to represent the screening data as a cube,
with each axis composed of building blocks for one of the cycles (Figure 2C shows the cube for
DEL02). Patterns, or “features” in the cube can be interpreted as SAR. DEL02 contains a total of
65 million compounds. Assessment of the sequencing data from DEL02 is summarized in Table
S2 and Figure S2. Even with a comparable number of DNA sequences from the resin control and
a larger number of naïve DNA sequences, the PqsE-selected DNA sequences yielded a much
larger number of high hit count DNA sequences. After removing sequences that appeared in the
ACS Paragon Plus Environment

Page 13 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
resin-binding or naïve sets, there were 6,732 PqsE-selected sequences that exhibited a hit count
of 75 or greater, which was about 0.1% of the unique codon combinations recovered or about
0.01% of the compounds applied.
9
A representative compound cube from DEL02 is shown in Figure 2C. Three extremely
strong features can be observed representing three closely-related core structures (out of 65
cores) in this library (Figure 2C). In the Figure, the three features are the vertical lines representing
the three chemical structures: Cycle 1 is denoted by “c1”, Cycle 2 is “c2”, and Cycle 3 is “c3”. All
three core structures featured a central p-amino-benzoic acid with a pendant 5-, 6-, or 7-
membered nitrogen heterocycle. Note that the benzoic acid depicted in Figure 2C is the Cycle 2
moiety coded for in those features. The heavy vertical patterns in Figure 2C show that strong
binding to PqsE is independent of the identity of the Cycle 3 building block. Cycle 3 included
acylations, reductive aminations, and sulfonylations, and all three reactions led to hits. This result
corresponds to an SAR “dead-spot” that does not influence binding to PqsE. Based on these
results, we resynthesized hit compounds from these core structures using on-DNA re-synthesis.
For our initial retest, we focused on the compounds with the strongest and/or most
frequent hit features from the initial screening campaign. Thirty-one exemplar molecules attached
to their DNA barcodes were selected for assessment of inhibition of PqsE catalytic activity.
Although the in vivo substrate and product for PqsE are not known, PqsE can catalyze thioester
bond cleavage. We monitor this catalytic activity using a surrogate substrate called mBTL (meta-
bromo-thiolactone) that we previously identified as an agonist of the P. aeruginosa RhlR quorum-
sensing receptor35. The MW of mBTL is 357 g/mol and cleavage of the thiol bond yields a product
with a mass of 375.01 g/mol (Figure S3A) that can be followed by mass spectrometry. Indeed,
Figure S3B shows that mBTL, when incubated with purified PqsE protein, but not in its absence,
results in the appearance of the 375.01 m/z peak. This result establishes that we can use mBTL
as a convenient and inexpensive artificial substrate for PqsE, enabling efficient assessment of
screening hits for the ability to inhibit PqsE thioesterase activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

ACS Chemical Biology
Page 14 of 30
1
2
3
4
5
6
7
8
9
After confirming PqsE thioesterase activity with mass spectrometry, we developed an
efficient, inexpensive, high throughput plate-based method using Ellman’s reagent (5,5'-dithio-
bis-[2-nitrobenzoic acid]) for routine assessment of enzyme inhibition19. First, we monitored
inhibition of PqsE-driven cleavage of mBTL by compounds from the screen, still containing their
DNA tags (Figure 2D). Seventeen of the 31 test compounds, when assayed at 10 M with 500
nM 6xHis-PqsE, showed at least 20% inhibition of PqsE thioesterase activity, with five compounds
(on-DNA compounds J, AA, BB, DD, and EE, Figure 2D) showing at least 50% inhibition. We
chose to focus on the structural class represented by the on-DNA hit BB (Figure 2D). BB is in the
family of compounds depicted in Figure 2C. It contains an azepane ring (Figure 2C, compound
iii) with a tert-butoxycarbonyl (Boc) group in the R3 position. We selected this chemotype based
on consistency of hit features within the primary screen, synthetic tractability, and calculated
physicochemical properties such as molecular weight, polar surface area, and cLogP values of
the corresponding off-DNA analog of compound BB. Interestingly, on-DNA hit BB possesses
structural features similar to the known C1 inhibitor.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To confirm off-DNA activity of this chemotype, we synthesized an initial set of 4 analogs
of the hit designated BB in Figure 2D off of their DNA barcodes, which correspond to compounds
1–4 in Table 1. The point of attachment to DNA is at the 4’-position as shown for the three
compounds in Figure 2C. We synthesized the corresponding analog in which the attachment point
is removed altogether by substitution with a hydrogen atom (compound 1, Table 1) and the analog
with that attachment point capped by a simple N,N-dimethyl amide (compound 3, Table 1).
Compound 3 is the closest structural analog to on-DNA hit BB in our set. Because the Boc group
is easily removed under acidic conditions36, we sought to understand whether the presence of
that group was important to PqsE activity. Thus, we also prepared the corresponding compounds
2 and 4 (Table 1) in which the Boc group was removed.
Table 1. PqsE Enzyme Inhibition Activity of Compounds 1–16.
ACS Paragon Plus Environment

Page 15 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
5'
HO
O
6'
4'
O
B
6
5
N
O
2'
A
H
R
3
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound
Structure
PqsE Thioesterase IC50
(M)
HO
O
O
1
18.1 ± 1.7
N
O
H
N
Boc
HO
O
O
2
3
>100
N
H
O
NH
O
HO
O
O
N
19.1 ± 3.2
N
O
H
N
Boc
O
O
HO
O
4
>100
O
N
N
H
NH
HO
O
O
5
6
7
8
>100
>100
>100
>100
N
O
H
Cl
N
Boc
HO
HO
HO
O
O
O
O
N
O
H
N
Cl
Boc
O
Cl
O
N
H
O
O
N
Boc
Boc
N
H
N
ACS Paragon Plus Environment

ACS Chemical Biology
Page 16 of 30
1
2
3
4
5
HO
O
O
9
18.9 ± 4.8
11.5 ± 3.8
N
H
O
6
7
8
9
N
O
HO
O
O
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
O
H
HO
HO
O
O
11
12
13
10.3 ± 1.5
24.1 ± 8.9
32.1 ± 15.6
N
O
H
O
O
N
O
H
O
HO
O
O
N
O
H
N
Boc
Cl
HO
HO
HO
O
O
O
14
5.6 ± 1.0
O
O
N
O
O
O
H
N
N
N
Boc
Boc
Boc
15
16
9.0 ± 2.0
>100
N
H
Cl
Cl
O
N
H
The general synthetic scheme by which Compounds 1–4 and subsequent analogs 5–16
were prepared is shown in Figure 2E. In brief, 3-nitrophenol and the appropriate alcohol were
subjected to typical Mitsunobu reaction conditions37 using diisopropylazodicarboxylate to afford
the desired phenyl ether. The nitro group was reduced using elemental iron and ammonium
ACS Paragon Plus Environment

Page 17 of 30
ACS Chemical Biology
1
2
3
4
5
6
chloride38, and the corresponding aniline product was treated with the appropriate phthalic
anhydride to afford the desired targets.
7
8
9
Assessment of activity of compounds 1–16
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compounds 1–4 (Table 1) were tested both for the ability to bind to PqsE via a thermal
shift assay (Figure 3A) and for the ability to inhibit PqsE thioesterase activity (Table 1). All four
compounds were soluble under both assay conditions (Figure S4A, B). Compounds 1 and 3
significantly shifted the melting temperature of PqsE in a dose-dependent manner (Figure 3A)
indicating these compounds bind to and stabilize PqsE. Specifically, at 500 M, compounds 1
and 3 shift the PqsE melting temperature by >3°C. Compounds 2 and 4, in which the Boc group
has been removed, do not cause a melting temperature shift at concentrations below 500 M
indicating a considerable loss in affinity for PqsE. This result raised the question of whether the
decrease in binding is a consequence of the strongly basic amine moiety that is unmasked by
Boc removal or the loss of important binding interactions between PqsE and the Boc group. Since
the DEL02 screening results indicated that PqsE binding was largely independent of the
composition of the Cycle 3 building block, we surmised that loss of activity was due to the
presence of a strongly basic amine in compounds 2 and 4.
ACS Paragon Plus Environment

ACS Chemical Biology
Page 18 of 30
1
2
3
4
5
6
7
8
9
A
B
71
70
69
68
67
66
65
Compound
1
2
3
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
200
400
600
[Compound] (µM)
150
100
50
0
-6
-4
-2
0
2
4
log [Compound 1] (µM)
C
150
100
50
0
-6
-4
-2
0
2
4
log [Compound 3] (µM)
Figure 3 a) Melting temperatures for 6xHis-PqsE in the presence of the designated compounds.
Error bars are shown as SD of biological replicates, n=3. b) PqsE thioesterase inhibition curve for
compound 1. Percent maximum inhibition is defined as the fluorescence value in the presence of
the inhibitor divided by the fluorescence value in the absence of inhibitor. A curve was fit to these
data points to determine IC50 value, as reported in Table 1. Error bars represent SD of biological
replicates, n=3. c) Inhibition curve as in (b) for compound 3.
To more rigorously examine compounds for inhibition of PqsE, we developed a
thioesterase assay with an >5-fold larger dynamic range than that provided by the Ellman’s
reagent used in our initial enzyme inhibition test. We employed 7-diethylamino-3-(4’-maleimidyl-
phenyl)-4-methylcoumarin (CPM), a reagent that fluoresces following reaction with free sulfhydryl
ACS Paragon Plus Environment

Page 19 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
groups39. We incubated 100 M mBTL with 250 nM PqsE at room temperature for 20 min with a
range of concentrations of test compounds to determine their IC50 values. Compounds 1 and 3
profiled as modestly potent thioesterase inhibitors in this assay with IC50 values of 18 and 19 M,
respectively (Table 1 and Figure 3B, C). Compounds 2 and 4 did not yield IC50 values even when
tested at 100 M (Table 1). These data parallel that from the thermal shift assay. Verifying the
results of the CPM-based assay, analysis by mass spectrometry showed that PqsE-mediated
cleavage of mBTL was reduced in the presence of Compounds 1 and 3 relative to controls lacking
inhibitor compounds (Figure S3B).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We next prepared a small set of additional compounds in this chemotype to probe initial
SAR (compounds 5–16, Table 1) and evaluated them for inhibition of PqsE thioesterase activity
(Table 1 provides all of the IC50 values and Figure 4 shows the dose response curves for all
compounds exhibiting activity (compounds 9–15)). All compounds were soluble under the
thioesterase assay conditions (Figure S4B). Based on the results from compounds 1–4, the Boc
groups were retained in compounds 5–8 and 13–16. A chlorine atom was used as a ‘probe’ moiety
to evaluate the tolerance to substitution at each open position of the two aromatic rings contained
in the structure (compounds 5–7 and 14–16). Compounds 9–13 were designed to provide a
cursory examination of the importance of the t-butyl-3-hydroxyazepanecarboxylate group.
ACS Paragon Plus Environment

ACS Chemical Biology
Page 20 of 30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4 Thioesterase dose-response inhibition analyses for compounds 9–15 from Table 1,
which reports the IC50 values. Curve fits to these data were used to determine the IC50 values for
Table 1. Error bars represent SD of biological replicates, n=3.
ACS Paragon Plus Environment

Page 21 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
The screening results from this set of analogs reveal three key pieces of information. First,
it appears that phenyl ring A is quite intolerant of substitution, as compounds 5–7, in which a
chloride is placed in positions 3–5, (for compound position numbering see the structure at the top
of Table 1), are all inactive at the highest concentration tested. Additionally, compound 8, in which
the phenyl group has been replaced by the sterically larger naphthyl group, is also inactive. These
data suggest that positions 4 and 5 of the aromatic ring pack tightly against the PqsE protein. This
same case can be made for position 3; however, loss of activity in analog 7 could also be due to
a shift in the preferred relative conformation of the carboxylic acid and amide moieties.
Substitution of hydrogen with the sterically larger chlorine atom will affect the conformation of
those functional groups, and it is likely that the relative spatial positions of those groups are
important aspects of compound binding to PqsE. We were unable to prepare the analog with a
chlorine in the 6 position, thus effects of substitution at that position remain unknown.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Second, the data suggest that the 2’ and 5’ positions of phenyl ring B are tolerant to
substitution (see compounds 14 and 15). In fact, compounds 14 and 15 both show a modest
increase in potency relative to the hit compounds 1 and 3, suggesting substitution at either of
these two positions may be beneficial. By contrast, substitution at the 6’ position is detrimental to
activity (compound 16). Compound 14 is the most potent inhibitor we have discovered to date in
this chemical series. We did not prepare the analog with a chlorine atom in the 4’ position because
compound 3 had already shown us that substitution in this position was tolerated.
Finally, it appears that the substituent on the phenol is also tolerant to substitution.
Compound 9, in which the t-butylcarbamate moiety in compound 1 has been altered to an
acetamide, is equipotent to compound 1. Replacement of the azepane ring with a cycloheptyl ring
(compound 10) or a phenyl ring (compound 11) provided a slight increase in inhibitory potency
relative to hit compound 1, while replacement with tetrahydropyranyl (compound 12) or piperidinyl
(compound 13) moieties led to no change in potency relative to compound 1. These results
ACS Paragon Plus Environment

ACS Chemical Biology
Page 22 of 30
1
2
3
4
5
6
suggest that nonpolar groups are slightly more preferred than polar groups in this region, although
the differences in potency between these compounds are minor.
7
8
9
Overall, the data from this small set of analogs provide proof of SAR with respect to PqsE
thioesterase inhibition with this chemotype and suggest that the 2’, 4’, and 5’ positions in ring B,
along with the substituent on the ring B phenol are the most productive positions on the molecule
for additional structure-activity work. The importance of the carboxylic acid and the amide
functional groups to biological activity still need to be determined. While these compounds are
somewhat similar in structure to the small heterocyclic ligands reported by Hartmann23 to be active
site inhibitors, we do not know where on the PqsE protein these compounds bind, and it would be
premature to assume they bind analogously to the Hartmann inhibitors.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Assessment of activities in vivo in P. aeruginosa
To discover whether the active thioesterase inhibitors (Compounds 1, 3, 9–15) have
effects in P. aeruginosa, we examined them for their influence on PqsE-dependent pyocyanin
production by P. aeruginosa. On the assumption that catalytic activity is required for pyocyanin
production16, reduction of pyocyanin would be an indication that our PqsE thioesterase inhibitors
are soluble, stable overnight at 37oC, permeable to the bacterial membrane, and actively inhibiting
cytoplasmic PqsE enzyme activity in the critical period when pyocyanin is produced as a
consequence of quorum sensing. P. aeruginosa cells were back-diluted 1:1,000 from a saturated,
overnight culture and compounds were added at 100 M. All compounds are soluble in these
conditions (Figure S4C). The cultures containing the test compounds were incubated overnight
at 37°C with aeration to allow the cells to regrow. Pyocyanin production was monitored by OD695.
Unlike mBTL, a known inhibitor of pyocyanin production, none of these new compounds exhibit
in vivo activity (Figure 5A).
ACS Paragon Plus Environment

Page 23 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
A
B
0.8
0.6
0.4
0.2
0.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
L
1
3
9
0
1
2
3
4
5
1
O
1
1
1
1
1
T
S
B
M
m
D
Compound
C
150
100
50
0.2
0.1
0.0
BQL
BQL
BQL
BQL
0
0 0.5 3 7 O/N 0 0.5 3 7 O/N 0 0.5 3 7 O/N
0 0.5 3 7 O/N 0 0.5 3 7 O/N 0 0.5 3 7 O/N
mBTL
Compound 1 Compound 3
mBTL
Compound 1 Compound 3
D
F
E
5×10-6
4×10-6
3×10-6
2×10-6
1×10-6
8×10-6
6×10-6
4×10-6
2×10-6
DMSO
DMSO
25 µM compound
50 µM compound
25 µM compound
50 µM compound
-5
-2×10-6
5
10
15
20
-5
-1×10-6
5
10
15
20
1/[S] (nM)-1
1/[S] (nM)-1
Compound 1 Compound 1 Compound 3 Compound 3
DMSO
25 µM
50 µM
25 µM
50 µM
Vmax
(RFU/min)
5.1x106
2.0x106
1.7x106
1.9x106
1.2x106
Km (µM)
261
331
328
390
310
Figure 5 a) Pyocyanin levels as judged by OD695 per OD600 (growth) following treatment of P.
aeruginosa with the designated compounds at 100 M. Error bars represent SD of biological
replicates, n=3. b) Concentrations of mBTL, compound 1, and compound 3 in cell-free culture
fluids following incubation with P. aeruginosa for the designated times. O/N designates 16 h of
treatment. BQL = below the quantitation limit. Error bars represent SD of biological replicates,
n=4. c) As in panel (b) for the concentrations of mBTL, compound 1, and compound 3 in cell
lysates. d) Lineweaver-Burk plots of thioesterase inhibition by compound 1 at a range of
concentrations of the mBTL substrate. The inhibitor was tested at 25 M and 50 M. Error bars
ACS Paragon Plus Environment

ACS Chemical Biology
Page 24 of 30
1
2
3
4
represent SD of biological replicates, n=3. e) Lineweaver-Burk plots as in (d) for compound 3. (F)
5
6
Calculated Vmax and Km for each condition from the data in panels D and E.
7
8
9
Analysis of compound stability and metabolism
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compounds 1 and 3 are micromolar inhibitors of PqsE thioesterase activity. However,
given that they do not reduce pyocyanin production in P. aeruginosa, which requires in vivo PqsE
activity, we suspected that the compounds may not reach the target at concentrations sufficient
to elicit a response. Moreover, the overnight incubations necessary for laboratory measurements
of pyocyanin production provide a high bar for metabolic stability of PqsE inhibitors in vivo. To
investigate the fates of compounds 1 and 3 in our assays, we used mass spectrometry to quantify
their levels over time following administration to P. aeruginosa. mBTL, which is known to be
internalized and maintained in P. aeruginosa, was used as the positive control. Indeed, as
expected, over time, the concentration of mBTL progressively decreased in the cell-free culture
fluid (Figure 5B) and increased in the cell lysate (Figure 5C). By contrast, for the first 7 h of
incubation, the amounts of compounds 1 and 3 in the cell-free culture fluids remained steady
(Figure 5B) and neither compound could be detected in the cell lysate (Figure 5C). Following
overnight incubation, we could not detect compound 1 or 3 in the cell-free culture fluid or the cell
lysate (Figure 5B, C, respectively). We conclude that compounds 1 and 3 are unable to enter P.
aeruginosa, or if they are internalized, they are subsequently metabolized or effluxed. Neither
compound has sufficient stability to withstand overnight incubation under our assay conditions.
These results apparently explain why compounds 1 and 3 do not inhibit in vivo pyocyanin
production.
Mechanism of action (MoA) of compounds 1 and 3
We assessed the MoA of compounds 1 and 3 by quantifying initial rates of mBTL cleavage
over a range of concentrations (Figure 5D, E). In the absence of inhibitor, the Vmax of the reaction
ACS Paragon Plus Environment

Page 25 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
is 5.1 x 106 RFU/min and the Km is 261 μM. Modest changes in Km occurred in the presence of
the inhibitors, but the changes did not depend on inhibitor concentration (Figure 5F). In contrast,
the Vmax for PqsE catalysis decreased in the presence of each inhibitor and in a dose-dependent
manner (Figure 5D-F). We therefore conclude that compounds 1 and 3 act non-competitively to
inhibit the ability of PqsE to cleave mBTL.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hartmann et. al. have previously reported on a set of PqsE thioesterase inhibitors which
coincidentally have some structural similarity to our series23. X-ray co-crystal structures of those
compounds revealed they bound in the active site of PqsE. The authors posited a competitive
inhibition mechanism for their inhibitors; however, no formal MoA studies were reported. Our MoA
studies with compounds 1 and 3 indicate that these inhibitors are non-competitive. However, we
do not know whether our compounds are orthosteric or allosteric inhibitors. Either our inhibitory
compounds bind at a site that is distinct from the previously reported compounds, or both
chemotypes bind in the active site but they do not block substrate binding. We have begun
structural analyses to further characterize the compounds we identified here, and we are also
investigating additional hit chemotypes from the DEL screening campaign to identify compounds
that exhibit other mechanisms of interference with PqsE function.
Conclusions
P. aeruginosa infection is a significant health burden, especially to the immune
compromised population40. PqsE is now established as a therapeutic target as it is essential for
P. aeruginosa pathogenicity and it has a binding pocket that should be susceptible to small
molecule inhibition19. In this initial work, we, like others in the field, have encountered long-
standing challenges regarding cell penetration, metabolism, and efflux of interesting anti-virulence
compounds41–43. Future SAR expansion of the hits from this screen as well as characterization of
hits from the other DELs screened as part of the current study could reveal potent PqsE inhibitors
ACS Paragon Plus Environment

ACS Chemical Biology
Page 26 of 30
1
2
3
4
that are also internalized by and exhibit in vivo PqsE inhibition in P. aeruginosa, either through
5
6
inhibition of thioesterase activity or other potential activities of the PqsE protein.
7
8
9
Supplementary Information Available: This material is available free of charge via the Internet and
contains four supplemental figures, two supplemental tables, methods, compound syntheses, and
analytical chemistry data.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACKNOWLEDGEMENTS
We thank members of the Bassler laboratory for helpful discussions. Sequencing analysis was
performed at the Lewis-Sigler Genomics Core Facility. Analyses of compound stability and
metabolism were performed by WuXi AppTec. This work was supported by the Howard Hughes
Medical Institute and NIH grant 5R37GM065859. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the National Institutes of Health.
The authors declare that they have no competing financial interests.
REFERENCES
(1) Moradali, M. F., Ghods, S., and Rehm, B. H. A. (2017) Pseudomonas aeruginosa Lifestyle: A
Paradigm for Adaptation, Survival, and Persistence. Front. Cell. Infect. Microbiol. 7.
(2) Sadikot, R. T., Blackwell, T. S., Christman, J. W., and Prince, A. S. (2005) Pathogen-host
interactions in Pseudomonas aeruginosa pneumonia. Am. J. Respir. Crit. Care Med. 171, 1209–
1223.
(3) Tacconelli, E., Carrara, E., Savoldi, A., Harbarth, S., Mendelson, M., Monnet, D. L., Pulcini,
C., Kahlmeter, G., Kluytmans, J., Carmeli, Y., Ouellette, M., Outterson, K., Patel, J., Cavaleri, M.,
Cox, E. M., Houchens, C. R., Grayson, M. L., Hansen, P., Singh, N., Theuretzbacher, U., Magrini,
N., Aboderin, A. O., Al-Abri, S. S., Awang Jalil, N., Benzonana, N., Bhattacharya, S., Brink, A. J.,
Burkert, F. R., Cars, O., Cornaglia, G., Dyar, O. J., Friedrich, A. W., Gales, A. C., Gandra, S.,
Giske, C. G., Goff, D. A., Goossens, H., Gottlieb, T., Guzman Blanco, M., Hryniewicz, W., Kattula,
D., Jinks, T., Kanj, S. S., Kerr, L., Kieny, M.-P., Kim, Y. S., Kozlov, R. S., Labarca, J.,
Laxminarayan, R., Leder, K., Leibovici, L., Levy-Hara, G., Littman, J., Malhotra-Kumar, S.,
Manchanda, V., Moja, L., Ndoye, B., Pan, A., Paterson, D. L., Paul, M., Qiu, H., Ramon-Pardo,
P., Rodríguez-Baño, J., Sanguinetti, M., Sengupta, S., Sharland, M., Si-Mehand, M., Silver, L. L.,
Song, W., Steinbakk, M., Thomsen, J., Thwaites, G. E., van der Meer, J. W., Van Kinh, N., Vega,
ACS Paragon Plus Environment

Page 27 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
S., Villegas, M. V., Wechsler-Fördös, A., Wertheim, H. F. L., Wesangula, E., Woodford, N.,
Yilmaz, F. O., and Zorzet, A. (2018) Discovery, research, and development of new antibiotics: the
WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 18, 318–327.
(4) Botelho, J., Grosso, F., and Peixe, L. (2019) Antibiotic resistance in Pseudomonas aeruginosa
– Mechanisms, epidemiology and evolution. Drug Resist. Updat. 44, 100640.
(5) Costerton, J. W., Stewart, P. S., and Greenberg, E. P. (1999) Bacterial biofilms: a common
cause of persistent infections. Science 284, 1318–1322.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(6) Singh, P. K., Schaefer, A. L., Parsek, M. R., Moninger, T. O., Welsh, M. J., and Greenberg, E.
P. (2000) Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial
biofilms. Nature 407, 762–764.
(7) Klevens, R. M., Edwards, J. R., Richards, C. L., Horan, T. C., Gaynes, R. P., Pollock, D. A.,
and Cardo, D. M. (2007) Estimating Health Care-Associated Infections and Deaths in U.S.
Hospitals, 2002. Public Health Rep. 122, 160–166.
(8) Ciofu, O., and Tolker-Nielsen, T. (2019) Tolerance and Resistance of Pseudomonas
aeruginosa Biofilms to Antimicrobial Agents—How P. aeruginosa Can Escape Antibiotics. Front.
Microbiol. 10.
(9) Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W., and Greenberg,
E. P. (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm.
Science 280, 295–8.
(10) Pearson, J. P., Feldman, M., Iglewski, B. H., and Prince, A. (2000) Pseudomonas aeruginosa
cell-to-cell signaling is required for virulence in a model of acute pulmonary infection. Infect.
Immun. 68, 4331–4334.
(11) Mukherjee, S., Moustafa, D., Smith, C. D., Goldberg, J. B., and Bassler, B. L. (2017) The
RhlR quorum-sensing receptor controls Pseudomonas aeruginosa pathogenesis and biofilm
development independently of its canonical homoserine lactone autoinducer. PLoS Pathog. 13,
e1006504.
(12) Papenfort, K., and Bassler, B. L. (2016) Quorum sensing signal–response systems in Gram-
negative bacteria. Nat. Rev. Microbiol. 14, 576.
(13) Winson, M. K., Camara, M., Latifi, A., Foglino, M., Chhabra, S. R., Daykin, M., Bally, M.,
Chapon, V., Salmond, G. P., and Bycroft, B. W. (1995) Multiple N-acyl-L-homoserine lactone
signal molecules regulate production of virulence determinants and secondary metabolites in
Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 92, 9427–9431.
(14) Brint, J. M., and Ohman, D. E. (1995) Synthesis of multiple exoproducts in Pseudomonas
aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with
homology to the autoinducer-responsive LuxR-LuxI family. J. Bacteriol. 177, 7155–7163.
(15) Farrow, J. M., Sund, Z. M., Ellison, M. L., Wade, D. S., Coleman, J. P., and Pesci, E. C.
(2008) PqsE Functions Independently of PqsR-Pseudomonas Quinolone Signal and Enhances
the rhl Quorum-Sensing System. J. Bacteriol. 190, 7043–7051.
(16) Mukherjee, S., Moustafa, D. A., Stergioula, V., Smith, C. D., Goldberg, J. B., and Bassler, B.
L. (2018) The PqsE and RhlR proteins are an autoinducer synthase–receptor pair that control
virulence and biofilm development in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. 115,
E9411–E9418.
(17) McCready, A. R., Paczkowski, J. E., Cong, J.-P., and Bassler, B. L. (2019) An autoinducer-
independent RhlR quorum-sensing receptor enables analysis of RhlR regulation. PLoS Pathog.
15, e1007820.
(18) Pesci, E. C., Milbank, J. B., Pearson, J. P., McKnight, S., Kende, A. S., Greenberg, E. P.,
and Iglewski, B. H. (1999) Quinolone signaling in the cell-to-cell communication system of
Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 96, 11229–11234.
(19) Yu, S., Jensen, V., Seeliger, J., Feldmann, I., Weber, S., Schleicher, E., Häussler, S., and
Blankenfeldt, W. (2009) Structure elucidation and preliminary assessment of hydrolase activity of
ACS Paragon Plus Environment

ACS Chemical Biology
Page 28 of 30
1
2
3
4
5
6
7
8
9
PqsE, the Pseudomonas quinolone signal (PQS) response protein. Biochemistry 48, 10298–
10307.
(20) Drees, S. L., and Fetzner, S. (2015) PqsE of Pseudomonas aeruginosa Acts as Pathway-
Specific Thioesterase in the Biosynthesis of Alkylquinolone Signaling Molecules. Chem. Biol. 22,
611–618.
(21) Gallagher, L. A., McKnight, S. L., Kuznetsova, M. S., Pesci, E. C., and Manoil, C. (2002)
Functions Required for Extracellular Quinolone Signaling by Pseudomonas aeruginosa. J.
Bacteriol. 184, 6472–6480.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(22) Diggle, S. P., Winzer, K., Chhabra, S. R., Worrall, K. E., Cámara, M., and Williams, P. (2003)
The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-
dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of
stationary phase and can be produced in the absence of LasR. Mol. Microbiol. 50, 29–43.
(23) Zender, M., Witzgall, F., Drees, S. L., Weidel, E., Maurer, C. K., Fetzner, S., Blankenfeldt,
W., Empting, M., and Hartmann, R. W. (2016) Dissecting the Multiple Roles of PqsE in
Pseudomonas aeruginosa Virulence by Discovery of Small Tool Compounds. ACS Chem. Biol.
11, 1755–1763.
(24) Brenner, S., and Lerner, R. A. (1992) Encoded combinatorial chemistry. Proc. Natl. Acad.
Sci. 89, 5381–5383.
(25) Mannocci, L., Zhang, Y., Scheuermann, J., Leimbacher, M., De Bellis, G., Rizzi, E., Dumelin,
C., Melkko, S., and Neri, D. (2008) High-throughput sequencing allows the identification of binding
molecules isolated from DNA-encoded chemical libraries. Proc. Natl. Acad. Sci. U. S. A. 105,
17670–17675.
(26) Clark, M. A., Acharya, R. A., Arico-Muendel, C. C., Belyanskaya, S. L., Benjamin, D. R.,
Carlson, N. R., Centrella, P. A., Chiu, C. H., Creaser, S. P., Cuozzo, J. W., Davie, C. P., Ding, Y.,
Franklin, G. J., Franzen, K. D., Gefter, M. L., Hale, S. P., Hansen, N. J. V., Israel, D. I., Jiang, J.,
Kavarana, M. J., Kelley, M. S., Kollmann, C. S., Li, F., Lind, K., Mataruse, S., Medeiros, P. F.,
Messer, J. A., Myers, P., O’Keefe, H., Oliff, M. C., Rise, C. E., Satz, A. L., Skinner, S. R.,
Svendsen, J. L., Tang, L., van Vloten, K., Wagner, R. W., Yao, G., Zhao, B., and Morgan, B. A.
(2009) Design, synthesis and selection of DNA-encoded small-molecule libraries. Nat. Chem.
Biol. 5, 647–654.
(27) Neri, D., and Lerner, R. A. (2018) DNA-Encoded Chemical Libraries: A Selection System
Based on Endowing Organic Compounds with Amplifiable Information. Annu. Rev. Biochem. 87,
479–502.
(28) Favalli, N., Bassi, G., Scheuermann, J., and Neri, D. (2018) DNA-encoded chemical libraries
- achievements and remaining challenges. FEBS Lett. 592, 2168–2180.
(29) Cochrane, W. G., Malone, M. L., Dang, V. Q., Cavett, V., Satz, A. L., and Paegel, B. M.
(2019) Activity-Based DNA-Encoded Library Screening. ACS Comb. Sci. 21, 425–435.
(30) Faver, J. C., Riehle, K., Lancia, D. R., Milbank, J. B. J., Kollmann, C. S., Simmons, N., Yu,
Z., and Matzuk, M. M. (2019) Quantitative Comparison of Enrichment from DNA-Encoded
Chemical Library Selections. ACS Comb. Sci. 21, 75–82.
(31) Chaga, G., Hopp, J., and Nelson, P. (1999) Immobilized metal ion affinity chromatography
on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification
of lactate dehydrogenase from chicken breast muscle. Biotechnol. Appl. Biochem. 29 ( Pt 1), 19–
24.
(32) El-Sagheer, A. H., and Brown, T. (2010) Click chemistry with DNA. Chem. Soc. Rev. 39,
1388–1405.
(33) Halpin, D. R., Lee, J. A., Wrenn, S. J., and Harbury, P. B. (2004) DNA Display III. Solid-
Phase Organic Synthesis on Unprotected DNA. PLOS Biol. 2, e175.
(34) Cui, J., Hao, J., Ulanovskaya, O. A., Dundas, J., Liang, J., and Kozmin, S. A. (2011) Creation
and manipulation of common functional groups en route to a skeletally diverse chemical library.
Proc. Natl. Acad. Sci. U. S. A. 108, 6763–6768.
ACS Paragon Plus Environment

Page 29 of 30
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
(35) O’Loughlin, C. T., Miller, L. C., Siryaporn, A., Drescher, K., Semmelhack, M. F., and Bassler,
B. L. (2013) A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm
formation. Proc. Natl. Acad. Sci. 110, 17981–17986.
(36) Greene, T. W., and Wuts, P. G. M. Protective Groups in Organic Synthesis. John Wiley &
Sons, New York, 1999.
(37) Reynolds A. J, and Kassiou, M. (2009) Recent Advances in the Mitsunobu Reaction:
Modifications and Applications to Biologically Active Molecules. Curr. Org. Chem. 13, 1610–1632.
(38) Chen, X., Chen, T., Xiang, Y., Zhou, Y., Han, D., Han, L.-B., and Yin, S.-F. (2014) Metal-free
regioselective hydrobromination of alkynes through CH/CBr activation. Tetrahedron Lett. 55,
4572–4575.
(39) Chung, C. C., Ohwaki, K., Schneeweis, J. E., Stec, E., Varnerin, J. P., Goudreau, P. N.,
Chang, A., Cassaday, J., Yang, L., Yamakawa, T., Kornienko, O., Hodder, P., Inglese, J., Ferrer,
M., Strulovici, B., Kusunoki, J., Tota, M. R., and Takagi, T. (2008) A Fluorescence-Based Thiol
Quantification Assay for Ultra-High-Throughput Screening for Inhibitors of Coenzyme A
Production. ASSAY Drug Dev. Technol. 6, 361–374.
(40) Giske, C. G., Monnet, D. L., Cars, O., and Carmeli, Y. (2008) Clinical and Economic Impact
of Common Multidrug-Resistant Gram-Negative Bacilli. Antimicrob. Agents Chemother. 52, 813–
821.
(41) Nicas, T. I., and Hancock, R. E. W. (1983) Pseudomonas aeruginosa outer membrane
permeability: Isolation of a porin protein F-deficient mutant. J. Bacteriol. 153, 281–285.
(42) Hancock, R. E. (1998) Resistance mechanisms in Pseudomonas aeruginosa and other
nonfermentative gram-negative bacteria. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 27 Suppl
1, S93-99.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(43) Breidenstein, E. B. M., de la Fuente-Núñez, C., and Hancock, R. E. W. (2011) Pseudomonas
aeruginosa: all roads lead to resistance. Trends Microbiol. 19, 419–426.
ACS Paragon Plus Environment