Polypharmacology Approaches against the Pseudomonas aeruginosa

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Polypharmacology approaches against the Pseudomonas aeruginosa MvfR regulon and their application in blocking virulence and antibiotic tolerance Damien Maura, Steffen L. Drees, Arunava Bandyopadhaya, Tomoe Kitao, Michele Negri, Melissa Starkey, Biliana Lesic, Sylvain Milot, Eric Deziel, Robert Zahler, Mike Pucci, Antonio Felici, Susanne Fetzner, Francois Lepine, and Laurence G. Rahme ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01139 • Publication Date (Web): 05 Apr 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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Polypharmacology approaches against the Pseudomonas aeruginosa MvfR

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regulon and their application in blocking virulence and antibiotic tolerance

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Damien Maura a,b,c, Steffen L. Drees d, Arunava Bandyopadhaya a,b,c, Tomoe Kitao a,b,c, Michele Negri e,

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Melissa Starkey a,b,c, Biliana Lesic a,b,c,, Sylvain Milot f, Eric Déziel f, Robert Zahler g, Mike Pucci g,

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Antonio Felici e, Susanne Fetzner d, François Lépine f, and Laurence G. Rahme a,b,c

*

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a

Department of Surgery, b Department of Microbiology and Immunobiology, Harvard Medical School

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and Massachusetts General Hospital,

Shriners Hospitals for Children Boston, Boston, MA, USA,

d

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Institute for Molecular Microbiology and Biotechnology, University of Münster, Münster, Germany,

e

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Aptuit, Verona, Italy,

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Cambridge, MA, USA.

f

c

INRS Institut Armand Frappier, Laval, QC, Canada,

g

Spero Therapeutics,

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*

Corresponding author: Laurence G. Rahme, Ph.D.

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E-Mail: [email protected]

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Tel. +1 (617) 724-5003; Fax +1 (617) 724-8558

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Short title: Dual inhibitors target MvfR and PqsBC

1 ACS Paragon Plus Environment

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Abstract

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Pseudomonas aeruginosa is an important nosocomial pathogen that is frequently recalcitrant to

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available antibiotics, underlining the urgent need for alternative therapeutic options against this

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pathogen. Targeting virulence functions is a promising alternative strategy as it is expected to generate

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less selective resistance to treatment compared to antibiotics. Capitalizing on our non-ligand based

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benzamide – benzimidazole (BB) core structure compounds reported to efficiently block the activity of

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the P. aeruginosa multiple virulence factor regulator MvfR, here we report the first class of inhibitors

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shown to interfere with PqsBC enzyme activity, responsible for the synthesis of the MvfR activating

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ligands HHQ and PQS, and the first to target simultaneously MvfR and PqsBC activity. The use of these

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compounds reveals that inhibiting PqsBC is sufficient to block P. aeruginosa’s acute virulence functions,

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as the synthesis of MvfR ligands is inhibited. Our results show that MvfR remains the best target of this

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QS pathway, as we show that antagonists of this target block both acute and persistence related

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functions. The structural properties of the compounds reported in this study provide several insights

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that are instrumental for the design of improved MvfR regulon inhibitors against both acute and

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persistent P. aeruginosa infections. Moreover, the data presented offer the possibility of a

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polypharmacology approach of simultaneous silencing two targets in the same pathway. Such a

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combined anti-virulence strategy holds promise in increasing therapeutic efficacy, and providing

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alternatives in the event of a single target’s resistance development.

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Keywords: Anti-virulence, Pseudomonas aeruginosa, Quorum Sensing Inhibitors, MvfR, PqsBC, Dual

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Inhibitors, Polypharmacology, Antibiotic Tolerance, Acute Infections



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Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen responsible for more

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than 50,000 infections in the US each year, primarily causing nosocomial infections in

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immunocompromised and cystic fibrosis patients (1). P. aeruginosa rapid expansion of resistance to

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almost all available antibiotics urges the development of alternative therapeutic strategies (2). One

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promising strategy, especially in the case of multi-drug resistant or pan antibiotic resistant P. aeruginosa

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clinical strains, is anti-virulence drugs that target bacterial virulence systems or master virulence

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regulators (3). One such master virulence regulator is the quorum sensing (QS) cell-to-cell bacterial

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communication system.

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P. aeruginosa possesses three major QS systems: LasR (4), RhlR (5, 6) and MvfR (7-10). The MvfR QS

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system is a promising anti-virulence target due to its critical role in inducing the expression of multiple

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P. aeruginosa virulence systems that promote both acute and chronic infections (7, 11-14). Moreover, as

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opposed to LasR no clinical isolates with frequent mutations in MvfR were reported to date (15). The

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transcriptional regulator MvfR (also known as PqsR) binds to 37 loci and regulates the expression of the

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associated genes (16) including the pqsABCDE operon, whose encoded proteins catalyze the

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biosynthesis of MvfR inducers and of ~60 distinct low-molecular-weight compounds (7-9, 17, 18), part of

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which are the 4-hydroxyl-2-alkyl-quinolines (HAQs)

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aminoacetophenone (2-AA) (12, 14, 20). This multi-step biosynthetic pathway is summarized in Figure

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1a. The first step is the conversion of the HAQs precursor anthranilic acid by PqsA and PqsD into 2-

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aminobenzoylacetyl-CoA (2-ABA-CoA) (21, 22), which then either spontaneously cyclizes into 2,4-

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dihydroxyquinoline (DHQ) (22-24) or is hydrolyzed by the thioesterase PqsE (or TesB) into 2-

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aminobenzoylacetate (2-ABA) (23). 2-ABA and octanoyl-CoA are then condensed by the PqsBC enzyme

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into the MvfR activating ligand 4-hydroxy-2-heptyl-quinoline (HHQ) (24, 25), which is later hydroxylated

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into the second MvfR activating ligand 3,4-dihydroxy-2-heptyl-quinoline (PQS) by PqsH (26). 2-ABA can

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also decompose into DHQ (24) or undergo decarboxylation to 2-AA (24) (Fig 1a).

(19)

and the

non-HAQ molecule 2-


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Importantly, HHQ and PQS are critical for MvfR activity in vitro, however, only HHQ appears to be

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essential for acute infection in vivo, as the absence of PQS assessed by using a pqsH mutant causes WT

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mortality in mice (9). On the contrary, 2-AA silences the acute infection branch of the MvfR QS system

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by binding and inhibiting the activity of PqsBC (13, 25) and promotes antibiotic tolerance as well as

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chronic/persistent infections by interfering with the bacterial translation apparatus and modulating

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epigenetically the host immune system to promote host tolerance to infection respectively (12-14, 27).

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The role of the MvfR QS pathway in both acute and chronic infections has motivated several drug

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discovery studies that generated inhibitors of PqsA (28, 29), PqsD (30, 31) and MvfR (32-35). However,

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no synthetic PqsBC inhibitor has been identified to date. We previously reported the identification and

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development of a new family of molecules with a Benzamide-Benzimidazole (BB) core structure as

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highly cell-permeable inhibitors of the MvfR QS system (32). One of our most potent inhibitors, M64,

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was shown to target MvfR and inhibit HAQs and 2-AA synthesis with an IC50 in the nanomolar range (32).

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The present work describes our effort to obtain further mechanistic knowledge on the BB family

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inhibitors’ capability in inhibiting MvfR circuitry. This work provides novel insights that are critical in the

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design of improved MvfR regulon inhibitors and reports the identification of the unprecedented first

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class of dual inhibitors of MvfR and PqsBC activities.

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Results and Discussion

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HAQs and 2-AA inhibition profiling reveals MvfR regulon inhibitors with dual targets.

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We previously reported an initial target assessment of few of our most potent BB compounds and

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demonstrated the physical interaction of the most potent inhibitor, M64, with MvfR (32). However, the

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profile of a BB compound, M51, was ambiguous which could suggest that this chemical family might

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target other proteins in the MvfR QS system besides the transcriptional regulator MvfR itself. In order to



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address this point, we performed a systematic target assessment for representative BB compounds in

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our collection.

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First, we assessed the ability of each compound to interfere with the activity of MvfR, PqsA, PqsBC or

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PqsD by quantifying the production levels of the MvfR-regulated molecules HHQ, PQS, DHQ and 2-AA.

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We used an mvfR isogenic mutant strain that constitutively expresses the pqsABCDE genes (mvfR-

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pPqsABCD) (32) and thus has MvfR-independent HHQ, PQS, 2-AA and DHQ production. In this strain,

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inhibitors targeting PqsA or PqsD enzymes result in decreased production of all four MvfR-regulated

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molecules, while PqsBC inhibition causes decreased production of HHQ and PQS level and an

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accumulation of 2-AA or DHQ; in the mvfR- pPqsABCD strain MvfR inhibition has no impact on the

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production of any of these molecules. Interestingly, our inhibitors’ collection contained compounds

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exerting at least two types of inhibitory patterns in the mvfR- pPqsABCD strain (Fig. 1b). The first group

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of molecules (green) exhibits the same inhibition pattern previously described with the MvfR inhibitor

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M64 (32) that is, no inhibition of HHQ, PQS, DHQ, and 2-AA. These results suggest that inhibitors M50,

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M62, M34, M61, M53 and M57 do not target the PqsA, PqsBC or PqsD enzymes pointing to MvfR as the

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potential target. In contrast, however, the second group of compounds (blue), although inhibiting HHQ

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and PQS, led to a considerable accumulation of 2-AA and DHQ (Fig. 1b), clearly suggesting that they are

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targeting the PqsBC enzyme.

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The ability of each compound to interfere with the activity of either MvfR or PqsBC was further

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interrogated in the parental strain PA14 where the expression of the pqs operon is MvfR dependent (Fig.

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1c). In this strain, inhibition of MvfR results in a reduced production of HHQ, PQS, 2-AA and DHQ

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whereas PqsBC inhibition only decreases HHQ and PQS production but not 2-AA or DHQ. Figure 1c

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shows the 2-AA, DHQ, PQS and HHQ levels produced in the WT strain PA14 in presence of each

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compound. We observed two different inhibition profiles: Compounds of the first profile (red) – M50,


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M62, M34, M61, M53, M57, M59, M58, M51, B1 and M52 – exhibit the inhibition pattern we previously

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observed with the MvfR inhibitor M64 (32) that is, inhibition of HHQ, PQS, 2-AA and DHQ in this wild

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type PA14 strain, suggesting they are targeting MvfR. Interestingly, however, five of these compounds –

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M59, M58, M51, B1 and M52 – were identified as PqsBC inhibitors in Figure 1b, indicating that they may

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act as dual inhibitory compounds that also target MvfR in addition the PqsBC enzymatic activity.

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Compounds from the second profile (orange), identified as PqsBC inhibitors in Figure 1b – M27, M26,

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M23 M4, M8 and M55 – partially inhibit the synthesis of 2-AA and DHQ in addition to fully blocking the

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synthesis of HHQ and PQS (Fig. 1c). M22 also blocks 2-AA and DHQ production but requires higher

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concentrations to do so (Fig. S5). These data suggest that compounds M27, M26, M23 M4, M8 and M55

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and M22 may also interfere with MvfR activity in addition to that of PqsBC. However, their lower

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efficacy at reducing 2-AA and DHQ production compared to M59, M58, M51, B1 and M52 suggests that,

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overall, they are less potent MvfR inhibitors (Fig. 1c). While compounds M23, M4 and M8 reduce 2-AA

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production, they induce the production of DHQ in the wild type PA14 strain (Fig. 1c). Although the

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reason for this effect is not clear, changes in the bacterial culture pH and inactivation of PqsE have been

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shown to affect the ratio between 2-AA and DHQ (23, 24).

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Taken together, these data suggest that the BB compounds can be classified in three categories: 1) MvfR

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inhibitors (M64, M50, M62, M34, M61, M53 and M57); 2) MvfR – PqsBC dual inhibitors with high anti-

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MvfR and high anti-PqsBC activity (M59, M58, M51, B1 and M52); and 3) MvfR – PqsBC dual inhibitors

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with low anti-MvfR and high anti-PqsBC activity (M27, M26, M23, M4, M8, M55).

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We report here a combination of two HAQs quantification assays in live cells that provide valuable

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insights on compound target(s). The mvfR isogenic mutant strain constitutively expressing the pqs

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operon (mvfR- pPqsABCD) is particularly useful in discriminating between MvfR and PqsA/D inhibitors,



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and can also help identify PqsBC inhibitors that were overshadowed by the MvfR/PqsA/PqsD inhibition

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phenotype dominant in the PA14 wild type strain. Accordingly, it is possible that some MvfR, PqsA or

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PqsD inhibitors reported in previous studies such as (28, 30, 33) might also have other targets within the

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MvfR QS pathway. We believe that such assays would benefit the community in discriminating the

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target(s) of MvfR QS system inhibitors and allow a better understanding of the determinants driving the

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interaction of compounds with various targets in this pathway.

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Target validation

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To validate the inhibitors’ targets, we selected representative compounds from each category and

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assessed their ability to bind to MvfR using surface plasmon resonance (SPR). As expected, all tested

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inhibitors bind to MvfR (Fig. 2a, c and S2). Compounds with a low anti-MvfR activity (M27, M26 and

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M23) bind 9 to 111 times less efficiently to MvfR than those with high anti-MvfR activity (M64, M50,

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M62, M59, M51) (Fig. 2a). This lower binding is nonetheless significant since the KD of those inhibitors is

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in the same order of magnitude as that of HHQ and PQS, two well established MvfR native ligands (10).

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We then assessed the ability of one compound in each category to interfere directly with PqsBC

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enzymatic activity by quantifying the in vitro conversion of 2-ABA into HHQ using purified PqsBC protein.

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As expected, the MvfR – PqsBC dual inhibitors M59 and M27 block the ability of PqsBC to convert 2-ABA

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into HHQ, with an EC50 of 13.4 µM and 12.5 µM respectively (Fig. 2c, d). Moreover, Figure 2e,f show that

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M59 binds to PqsBC and displaces 2-AA, a natural PqsBC inhibitor structurally unrelated to BB

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compounds acting competitively with the physiological substrate 2-ABA, confirming further M59 ability

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to interfere with PqsBC. These data indicate that the compounds reported here are significantly more

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potent PqsBC inhibitors than 2-AA whose EC50 for PqsBC inhibition was previously found to be 46 µM

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(25). Moreover, the anti-PqsBC activity of 2-AA in live cells is weak as its IC50 for HHQ inhibition is around

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400 µM (13). Interestingly, Figure 2c shows that the MvfR inhibitor M64 also interferes with PqsBC


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activity although the inhibition is weaker (EC50 ~185µM) compared to that of M59 and M27. This

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suggests that other inhibitors from the first category (M50, M62, M34, M61, M53 and M57) may also be

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MvfR – PqsBC dual inhibitors with a weak anti-PqsBC activity. To exclude any direct reactions of

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inhibitors with the PqsBC substrates, we analyzed inhibitor-substrate mixtures by UV spectroscopy and

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assessed 2-ABA and octanoyl-CoA stability by HPLC. Data verified the absence of reactivity and

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confirmed the stability of both, 2-ABA and octanoyl-CoA in the presence of any of the inhibitors (Fig. S1

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and Table S1). Overall, these physical interaction and enzymatic activity data confirm the existence of

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MvfR – PqsBC dual inhibitors.

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One puzzling question is how compounds with the same core structure can bind to such different

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targets, one being a transcriptional regulator and the other a biosynthetic enzyme. It is worth noting

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that the common point between both proteins is HHQ. Indeed, PqsBC catalyzes HHQ production while

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MvfR binds to HHQ. Therefore, it is possible that compounds with the BB core structure share some

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physical properties with HHQ allowing them to bind HHQ related proteins/targets.

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Polypharmacology, involving a single or multiple drugs acting on the same pathway has been shown to

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have a significant impact on the treatment efficacy for various diseases (36, 37), including bacterial

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infections (38, 39). Although there seems to be no synergistic benefit with this series of inhibitory

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compounds, the possibility of blocking two different targets in the same pathway offers an advantage in

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the event of a single target’s resistance development (39, 40). For example, if a resistance mutation

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occurs in PqsBC, a dual inhibitor would retain both anti-virulence and anti-persistence potency via its

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anti-MvfR activity. In the case of a resistance mutation in MvfR, dual inhibitors would still retain an anti-

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virulence potency thanks to their anti-PqsBC activity although they might not be able to retain the anti-

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persistence activity. The current view in the anti-virulence field is that resistance is still expected to

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occur, but at much lower frequencies than that promoted by traditional antibiotics. By definition

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virulence contributes to pathogen fitness in vivo implying that an anti-virulence resistant mutant would



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theoretically outcompete sensitive cells during treatment. However, some important microbial

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population genetics and ecological concepts such as public/private use of virulence factors, fitness

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localization and population structure suggest that selective pressure to anti-virulence may only be

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applied in specific settings, as opposed to that of antibiotics which apply selective pressure in all settings

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(3, 41, 42). It is worth pointing that thus far no MvfR mutations have been reported in P. aeruginosa

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clinical isolates sequenced suggesting that a functional MvfR QS system is critical for P. aeruginosa

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infections. However, one cannot exclude this from occurring once MvfR QS system inhibitors are used in

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the context of long term treatment regiments (i.e. CF patients). Therefore a polypharmacology anti-

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virulence approach could present a significant advantage for the treatment of P. aeruginosa infections.

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Inhibition of P. aeruginosa virulence and antibiotic tolerance

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To assess the potential of our compounds to reduce P. aeruginosa virulence, we evaluated the ability of

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selected inhibitors from each category to block P. aeruginosa acute virulence against A549 human lung

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epithelial cells and RAW264.7 macrophages in vitro using cell viability as a readout. Cell survival was

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quantified 3 hours post-infection with PA14 in the presence or absence of each inhibitor. Infection with

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P. aeruginosa resulted in 79.2% lung epithelial cell death (Fig. 3a) and 74.4% macrophage cell death (Fig.

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3b). In contrast, treatment with inhibitors from all three categories increased 3.1 to 3.8 times lung

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epithelial cell survival and 1.9 to 2.6 times macrophage survival to PA14 infection (Fig. 3a-b). These

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compounds show no cytotoxic effect (Fig. S4) and importantly reduce P. aeruginosa virulence in an MvfR

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QS system dependent manner, as they do not significantly change the lung cell survival rate when added

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to the mvfR mutant cells (Fig. S3), indicating no off-target effects (Fig. S4). Moreover, no significant

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difference in the survival of lung epithelial cells or murine macrophages was observed between

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compounds that exhibit low (M27, M26, M23) and high anti-MvfR activity (M64, M50, M59, M58, M51)

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(Fig. 1c). 9 ACS Paragon Plus Environment

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Next, we evaluated the potential of our compounds to inhibit antibiotic tolerance by assessing bacterial

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survival to the β-lactam antibiotic Meropenem. Data presented in Figure 4 indicate that the dual

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inhibitors with low anti-MvfR activity (M27, M26, and M23) do not significantly reduce tolerance to

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Meropenem. However, the dual inhibitors that exhibit high anti-MvfR activity and consequently block

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2AA production (M64, M50, M59, M58 and M51) reduce antibiotic tolerance by more than 70%

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compared to vehicle control (Fig. 4).

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Overall, these data indicate that all three categories of inhibitors have a similar therapeutic potential in

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the context of acute P. aeruginosa infections likely because they all efficiently block HHQ and PQS

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production. However, dual inhibitors with a high anti-MvfR activity are more potent at blocking

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antibiotic tolerance than those with a low anti-MvfR activity since they restrict 2-AA production more

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efficiently. Therefore, MvfR – PqsBC dual inhibitors with a high anti-MvfR activity have an increased

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therapeutic potential against pro-acute infection related molecules HHQ and PQS, as well as the pro-

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persistent and immunomodulatory molecule 2-AA.

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Role of inhibitors structure in target recognition

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Finally, we assessed whether some compound structural determinants could be associated with

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selective target recognition. Figure 5 shows the chemical structure for every inhibitor of the three

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categories. Notably, almost all the compounds with a high anti-MvfR activity (columns 1 and 2) contain a

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nitro group at the position 5 of the benzimidazole ring. In contrast, this nitro group is lacking from all

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compounds with a low anti-MvfR activity (column 3). This suggests that the nitro group may play a

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critical role for the interaction with MvfR. This is most obvious when comparing the binding and

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inhibitory activity of M26 versus M51, two identical compounds but for the nitro group which is lacking

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in M26 (Fig. 5). Indeed, the addition of the nitro group on M26 increases 25 times the compound

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binding to MvfR (Fig. 2a) and 14.8 times the inhibition of 2-AA production in PA14 (Fig. 1c). Moreover,



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replacing the methyl group at the position 5 of the benzimidazole ring of M27 by a nitro group as in M50

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(Fig. 5) increases 9.5 times the binding to MvfR (Fig. 2a) and 14.3 times the inhibition of 2-AA production

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in PA14 (Fig. 1c). Similarly, the addition of the nitro group on M55, as in M50 (Fig. 5), increases 15.4

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times the inhibition of 2-AA production in PA14 (Fig. 1c). Notably, HHQ ligand based analogues

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harboring strong electron withdrawing groups such as NO2, CN or CF3 on the benzene moiety of the

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quinolone structure were also found to be strong MvfR inhibitors (35, 43), supporting our data on the

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importance of the nitro group for BB compounds to interact with MvfR. Future in depth protein –

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inhibitor interaction studies will provide more insights on this aspect. Interestingly, the presence of the

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nitro group also appears to decrease the compounds anti-PqsBC activity. Indeed, adding the nitro group

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to B1 as in M62, or to M55 as in M50, or replacing the methyl group of M27 by a nitro group as in M50

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reduce 4.6, 8.9 or 2.3 times respectively HHQ production in the mvfR mutant strain constitutively

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expressing the pqs operon (Fig. 1b). Overall, these data demonstrate that the nitro group at the position

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5 of the benzimidazole ring is critical for the compounds to selectively recognize MvfR over PqsBC, and

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suggest modifications on the benzamide moiety to further modulate target selectivity.

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Conclusions

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This study provides exciting new insights into the mode of action and therapeutic potential of

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compounds with a BB core structure in the context of quorum sensing inhibition in P. aeruginosa. We

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previously reported a series of non-ligand based BB compounds interfering with the P. aeruginosa MvfR

250

QS system (32). In this study, we assessed further the mode of action of this BB compound series in this

251

pathogen. While they all inhibit the MvfR QS system, we discovered that the transcriptional regulator

252

MvfR is not their only target in the MvfR pathway. Our analysis shows that several of our BB compounds

253

also interfere with the PqsBC enzyme, inhibiting its ability to convert 2-ABA into the MvfR activating

254

ligand HHQ. These compounds represent the first class of inhibitors ever reported to interfere with


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255

PqsBC, and are the first synthetic inhibitors ever shown to interfere with both MvfR and PqsBC. These

256

dual inhibitors harbor an exciting therapeutic potential due to their ability to block both acute and

257

chronic virulence related functions in P. aeruginosa, and to simultaneously inhibit more than one target.

258

As such they offer new avenues to overcome the probability of resistance that might be presented with

259

single target inhibitor in this pathogen.



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260

Methods

261

Bacterial strains, plasmids and growing conditions

262

PA14 (UCBPP-PA14) is a P. aeruginosa human clinical isolate (44). The strain mvfR-pPqsABCD which has

263

constitutive and MvfR-independent pqs operon expression was previously described in (32). Unless

264

noted otherwise, all bacterial strains were grown in 5mL LB Lenox medium (Fisher Scientific) at 37˚C

265

under 200rpm orbital shaking using glass tubes (VWR). 75µg/mL Tetracycline was added when growing

266

the mvfR- pPqsABCD strain to maintain the pDN18 plasmid.

267 268

HAQs and 2-AA quantification

269

HAQs and 2-AA levels were quantified in bacterial culture supernatants by LC/MS as described in (9, 45).

270 271

Binding to MvfR via surface plasmon resonance

272

Purification of the MvfR ligand binding domain (MvfRc87) was performed as described in (32). MvfRc87

273

protein (50µg/mL) was diluted in 10mM Sodium Acetate buffer (pH5.5) and immobilized on a CM7

274

Series S Sensor Chip using an Amine Coupling reagent kit (GE Healthcare) at the level of 3,000-5,000

275

Response Units (RU). PBS (pH 7.4) containing 0.05% P20 surfactant was used as the running buffer

276

during protein immobilization.

277

The interactions between test compounds and MvfRc87 ligand binding domain were analyzed by

278

Biacore™ T200 evaluation software 2.0 (GE Healthcare). 10mM HEPES (pH 7.4) containing 150 mM NaCl,

279

3mM EDTA, 0.05% P20 surfactant and 4% DMSO was the running buffer. Injections were performed at a

280

flow rate of 30µL/min with 60s of contact time and 420s (Single-cycle kinetics) or 180s (Multi-cycle

281

kinetics) of dissociation time. Each injection was followed by an extra wash with 50% DMSO. Solvent

282

correction was performed according to sensorgram analysis. The zero-concentration curve was


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283

subtracted from the other sensorgrams. The affinities of compounds were determined with the “Steady

284

State Affinity” yielding the Binding Affinity Constant (KD) and the maximum binding capacity (Rmax)

285

expressed as Response Units (RU).

286 287

Inhibition of PqsBC enzymatic activity

288

PqsBC was purified and catalytic activity was determined as described previously (25). 2-ABA was

289

synthesized according to (24, 25). Octanoyl-CoA and 2-AA were purchased from Sigma-Aldrich (St. Louis,

290

MO, USA). For evaluating enzyme inhibition, 20-100 nM PqsBC was incubated with various

291

concentrations of the respective inhibitor for 5 minutes before measuring the residual activity. 2-ABA

292

and octanoyl-CoA concentrations were 150-200 µM and 20-40 µM, respectively, and the DMSO

293

concentration was 1 %. All assays were conducted in triplicate.

294 295

Binding to PqsBC via fluorescence spectroscopy

296

Dissociation constants of PqsBC-inhibitor complexes were determined by fluorescence spectroscopy or

297

fluorescence polarization spectrometry, using the fluorescence properties of the respective inhibitor

298

molecule. 2-AA displacement from PqsBC by inhibitors was monitored by the 2-AA fluorescence

299

intensity change resulting from binding of the molecule to PqsBC (25). All assays were conducted in a

300

buffer containing 50 mM HEPES, pH 8.0, 50 mM NaCl and 1 % DMSO, using a Jasco FP-6500 fluorescence

301

spectrometer with polarization accessory.

302 303

Cell viability assays

304

Cells survival to PA14 infection was assessed as previously described in (32). Briefly, bacterial cells were

305

grown until mid-exponential phase (OD600nm = 2) in the presence or absence of each inhibitor at 50 µM,

306

then washed and added to host cells at a MOI of 100. Three hours post-infection, bacterial cells were 14 ACS Paragon Plus Environment


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307

killed with 500 µg/mL Gentamycin and washed away twice with PBS. Cells were incubated in 100 µg/mL

308

MTT for 16 hours at 37 °C in 5% CO2, then MTT was dissolved in DMSO and OD570nm was measured. All

309

cells were maintained in 5% CO2 at 37 °C. A549 (human lung epithelial cell line, ATCC, USA) and

310

RAW264.7 cells (mouse macrophage cell line, IMGENEX, USA) were maintained in F12K and DMEM

311

medium (Life Technologies, USA) respectively. The media were supplemented with 10% heat-inactivated

312

FBS, penicillin/streptomycin, 2 mM L-glutamine, and 10 mM HEPES (all from Gibco). The cells were

313

seeded in T-75 tissue culture flasks (Falcon, USA) and used between passages 2 and 3.

314 315

Antibiotic tolerance

316

P. aeruginosa cells were grown at 37˚C 200rpm in 10g/L TSB media until mid-exponential phase (OD600nm

317

2) then exposed to 10ug/mL Meropenem (Sandoz, USA) for 24 hour under the same incubating

318

conditions. Before (t=0) and after (t=24h) Meropenem addition, a 200µL sample of each culture was

319

collected, diluted and plated on LB agar plates to quantify the total number of bacteria (t=0) and the

320

surviving bacteria (t=24h). Colony forming units (CFUs) were counted after 24h incubation at 37˚C. The

321

fraction of antibiotic tolerant cells was then calculated as the ratio of the amount of total bacteria (t=0)

322

divided by the amount of surviving bacteria (t=24h). Data are expressed as the percentage of antibiotic

323

tolerant cells relative to the DMSO vehicle control, which represents a survival fraction of 2.3x10-6 cells.

324 325

Statistical analyses

326

Statistical significance was assessed using unpaired One Way ANOVA + Dunnett’s post-test or One Way

327

ANOVA + Tukey post-test as indicated using GraphPad Prism software.

328 329

Author contributions 15 ACS Paragon Plus Environment

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330

DM, SLD, AB, TK, MN, MS, BL, RZ, MP, AF, SF, FL and LGR designed experiments. DM, SLD, AB, TK, MN,

331

SM and ED performed experiments. DM and LGR wrote the manuscript and prepared figures. All authors

332

reviewed the manuscript.

333 334

Acknowledgements

335

This work was supported by Shriners Hospital Postdoctoral Fellowship #84206 to DM and by the

336

research grants, Shriners #8770, Cystic Fibrosis Foundation #11P0, NIAID R33AI105902 to L.G.R and

337

grant FE 383/23-2 from the Deutsche Forschungsgemeinschaft to S.F. Funding sources had no role in

338

study design, data analysis and interpretation or decision to publish.

339 340

Competing financial interest

341

LGR is the scientific founder and scientific advisory board member of Spero Therapeutics LLC. MP is

342

Executive Director, Early Drug Discovery at Spero Therapeutics. RZ is an independent consultant. AF is

343

Director and Head of Microbiology Unit at Aptuit (Verona). MN is Research Scientist, Microbiology

344

Department at Aptuit (Verona). LGR, SF, FL and corresponding lab members received no funding from

345

Spero Therapeutics.

346 347 348



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References

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491 492 493


45.

Lepine, F., Deziel, E., Milot, S., and Rahme, L. G. (2003) A stable isotope dilution assay for the quantification of the Pseudomonas quinolone signal in Pseudomonas aeruginosa cultures, Biochim Biophys Acta 1622, 36-41.

494 495

Figure legends

496 497

Figure 1: HAQs and 2-AA biosynthesis pathway and inhibition in live P. aeruginosa cells

498

a) HAQs and 2-AA biosynthesis pathway. PqsABCDE are encoded by the pqs operon. PqsH and TesB

499

encoding genes are located elsewhere in P. aeruginosa chromosome. TesB is another thioesterase also

500

able to convert 2-ABA-CoA into 2-ABA. b, c) HHQ, PQS, 2-AA and DHQ levels measured by LC/MS in an

501

mvfR mutant strain constitutively expressing the pqs operon (b) or in PA14 wild type strain (c) in the

502

presence or absence of various BB inhibitors at 100µM. Levels are normalized to that of the DMSO

503

vehicle control. Results show the average ± SD of at least two independent replicates.

504 505

Figure 2: Target validation

506

Compounds binding intensity to MvfR was measured via SPR (a, b). Binding to MvfR was assessed with a

507

wide range of inhibitors concentrations. Compounds interference with PqsBC was measured via enzyme

508

kinetics by assessing the inhibitory activity on the condensation of 2-ABA and octanoyl-CoA to HHQ by

509

PqsBC (c, d). 2-ABA conversion into HHQ was assessed with a wide range of inhibitors concentrations.

510

Shown is the average ± SEM of three independent replicates. e. Binding of M59 assayed with

511

fluorescence polarization spectrometry. The autofluorescence of M59 was used to probe binding of the

512

molecule to PqsBC (gray fit) and octanoyl-PqsBC (black fit), each of which were titrated stepwise to the

513

inhibitor solution. The calculated dissociation constants were 2.5µM and 2.9µM, respectively, indicating

514

that M59 binds to both forms of the enzyme. f. The displacement of 2-AA, a PqsBC inhibitor competitive

515

with 2-ABA, by M59 was analyzed by measuring the 2-AA fluorescence intensity change in response to 20 ACS Paragon Plus Environment


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Page 26 of 27

516

PqsBC binding. In an experiment where 2-AA was titrated into a solution containing 1 µM PqsBC, the KD

517

was 6.8µM (upper fit). When 10µM M59 was present in the protein solution, the apparent KD of the

518

PqsBC-2-AA complex increased (lower fit), indicating that M59 interferes with 2-AA binding.

519 520

Figure 3: Anti-virulence efficacy in lung epithelial cells and macrophage infection assays

521

Survival of A549 human lung epithelial cells (a) or RAW264.7 macrophage cells (b) to PA14 infection in

522

presence of 50 µM of dual inhibitors with high anti-MvfR activity and low anti-PqsBC activity (green),

523

dual inhibitors with high anti-MvfR activity and high anti-PqsBC activity (red), dual inhibitors with low

524

anti-MvfR activity and high anti-PqsBC activity (orange) or the DMSO vehicle control (black). Results

525

show the average ± SEM of at least 3 independent replicates. Statistical significance to the DMSO control

526

was assessed using one way ANOVA + Dunnett’s post-test. No statistical difference was observed when

527

comparing the inhibitors with each other (p>0.05, One Way ANOVA + Tukey post-test).

528 529

Figure 4: Inhibition of antibiotic tolerance

530

Tolerance to 10µg/mL of the β-lactam antibiotic Meropenem in presence of 10µM of dual inhibitors

531

with high anti-MvfR activity and low anti-PqsBC activity (green), dual inhibitors with high anti-MvfR

532

activity and high anti-PqsBC activity (red), dual inhibitors with low anti-MvfR activity and high anti-PqsBC

533

activity (orange) or the DMSO vehicle control (black). Results show the average ± SEM of at least 3

534

independent replicates. Statistical significance to the DMSO control was assessed using one way ANOVA

535

+ Dunnett’s post-test.

536 537

Figure 5: Dual inhibitors structures sorted based on their potency on each target

538 21 ACS Paragon Plus Environment

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539

Supporting information

540

Further supporting data on compounds targets, cytotoxicity and off-target assessments.


Polypharmacology Approaches against the Pseudomonas aeruginosa

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