Inhibition of the transcriptional repressor LexA: Withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials

Journal Pre-proof Inhibition of the transcriptional repressor LexA: Withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials

Pierangelo Bellio, Alisia Mancini, Letizia Di Pietro, Salvatore Cracchiolo, Nicola Franceschini, Samantha Reale, Francesco de Angelis, Mariagrazia Perilli, Gianfranco Amicosante, Francesca Spyrakis, Donatella Tondi, Laura Cendron, Giuseppe Celenza PII:

S0024-3205(19)31043-4

DOI:

https://doi.org/10.1016/j.lfs.2019.117116

Reference:

LFS 117116

To appear in:

Life Sciences

Received date:

9 October 2019

Accepted date:

27 November 2019

Please cite this article as: P. Bellio, A. Mancini, L. Di Pietro, et al., Inhibition of the transcriptional repressor LexA: Withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials, Life Sciences(2019), https://doi.org/10.1016/ j.lfs.2019.117116

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© 2019 Published by Elsevier.

Journal Pre-proof TITLE Inhibition of the transcriptional repressor LexA: withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials.

AUTHORS Pierangelo Bellioa, Alisia Mancinia, Letizia Di Pietroa, Salvatore Cracchioloa, Nicola Franceschinia, Samantha Realeb, Francesco de Angelisb, Mariagrazia Perillia, Gianfranco Amicosantea, Francesca Spyrakisc, Donatella Tondid, Laura e a* Cendron , Giuseppe Celenza . a

Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy

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Department of Physical and Chemical Sciences, University of L’Aquila, L’Aquila, Italy

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Department of Drug Science and Technology, University of Torino, Torino, Italy.

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Department of Biology, University of Padova, Padova, Italy

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Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy

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CORRESPONDENCE *

Giuseppe Celenza

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Email: [email protected]

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Telephone: +390862433444

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Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, Via Vetoio, 1, 67100, L’Aquila, Italy

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Journal Pre-proof ABSTRACT Aims LexA protein is a transcriptional repressor which regulates the expression of more than 60 genes belonging to the SOS global regulatory network activated by damages to bacterial DNA. Considering its role in bacteria, LexA represents a key target to counteract bacterial resistance: the possibility to modulate SOS response through the inhibition of LexA autoproteolysis may lead to reduced drug susceptibility and acquisition of resistance in bacteria. In our study we investigated boron-containing compounds as potential inhibitors of LexA self-cleavage. Main methods

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The inhibition of LexA self-cleavage was evaluated by following the variation of the first-order rate constant by LC-MS at several concentrations of inhibitors. In silico analysis were applied to predict the binding orientations assumed by the inhibitors in the protein active site, upon covalent binding to the catalytic Ser-119. Bacterial filamentation assay was used to confirm the ability of (3-aminophenyl)boronic acid to interfere with SOS induced activation. Key findings

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Boron-containing compounds act as inhibitors of LexA self-cleavage, as also confirmed by molecular modelling where the compounds interact with the catalytic Ser-119, via the formation of an acyl-enzyme intermediate. A new equation for the description of the inhibition potency in an autoproteolytic enzyme is also disclosed. Bacterial filamentation assays strongly support the interference of our compounds with the SOS response activation through inhibition of septum formation Significance

KEYWORDS

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The obtained results demonstrated that phenylboronic compounds could be exploited in a hit-to-lead optimization process towards effective LexA self-cleavage inhibitors. They would sustain the rehabilitation in therapy of several dismissed antibiotics.

Bacterial SOS response, LexA inhibition, phenylboronic acid derivatives, covalent inhibition, RecA protein.

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Journal Pre-proof INTRODUCTION Multi-Drug Resistant Bacteria represent nowadays a global emergency which limits the efficacy of the treatment of bacterial infections. The World Health Organization considers bacterial resistance among the three main risks for human health, and the development of new strategies for fighting bacterial infections are strongly desirable. In 2018 report, WHO evokes a post-antibiotic era in which "common infections and minor injuries can kill" [1] . The economic and social implications of bacterial resistance are enormous: only in Europe it costs 1.5 billion euro each year in antiinfective therapy and 600 million per day in productivity loss [2].

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The situation is even more dramatic if we consider the limited number of antimicrobial agents currently in clinical phase. The costs of research and development of new antimicrobial drugs are unsustainable in view of the rapid spread and evolution of antibiotic resistance, which has significantly shortened the useful life of old and new antimicrobial drugs. Bacteria play an active role in their own evolution and looking at bacterial resistance from this new prospective, would change our way of facing the problem as something that can finally be avoidable.

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The SOS pathway has been recently validated as a key target for combating the evolution of antibiotic resistance. Briefly, when DNA damage occurs, single-stranded DNA (ssDNA) accumulates and activates the DNA damage sensor of the system, RecA protein. Subsequently, RecA activates the self-cleavage of the transcriptional repressor LexA. As a result, the SOS genes (more than 60 identified) under LexA control are derepressed, leading to DNA repair and recombination [3,4] SOS genes include error-prone DNA polymerases, which catalyse translational replication over damaged DNA [5,6]. Moreover, the expression of integrases in integrons are under LexA control [3,7,8].

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LexA binds a specific palindromic sequence of 16-19 bp, named lexA-binding box. The affinity of LexA to its binding sequence allows a fine regulation of gene expression. It has been demonstrated that inactivation of LexA self-cleavage makes bacteria unable to initiate the SOS response, as well as to develop a resistant phenotype [9–11].

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LexA consists of two domains separated by a short flexible linker and in solution exists as a homodimer. The Nterminal domain (NTD) contains specific DNA binding activity, and the C-terminal domain (CTD) contains the protease activity [12,13]. In the self-cleavage mechanism, LexA undergoes a large conformational change in its C-terminal domain between inactive and active states that moves the cleavage loop within the active site, adjacent to the catalytic serine. The cleavage occurs between the residues Ala-84 and Gly-85. The possibility to inhibit the activation of the SOS response was previously investigated at three different levels: the RecA/LexA axis (Mo et al., 2018), RecA protein [16–21] and recently, LexA protein [14]. This paper, via a multidisciplinary approach, aims to contribute to the investigation in this field through the investigation of boron containing compounds as hit for the design of inhibitors of LexA self-cleavage. Importantly, considered the peculiar autoproteolytic activity of LexA, we elaborated an ad hoc equation for the description of the inhibition potency in an autoproteolytic enzyme.

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Journal Pre-proof MATERIALS AND METHODS Compounds Phenylboronic acid (1), (3-hydroxyphenyl)boronic acid (2), (3-aminophenyl)boronic acid (3), (3-carboxyphenyl)boronic acid (4), were from Sigma-Aldrich; [3-(2-carboxyvinyl)phenyl]boronic acid (5) was synthesized as already reported [22]. Cloning and protein purification

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Gene lexA, encoding for LexA, protein was amplified via polymerase chain reaction from genomic DNA of Escherichia coli BP6 (Re) with specific oligonucleotides: LexA_NdeI_for (5’-GGGGGGCATATGAAAGCGTTAACGGCC-3’) and XhoI_LexA_stop_rev (5’-GGGGGCTCGAGTTACAGCCAGTCGCCGTTGCG-3’) containing NdeI and XhoI restriction sites (in bold underlined). The amplification was carried out in 100 µL reaction: 2 ng of DNA template, 800 pmol of each oligonucleotide, 2 µl of 10 mM dNTPs mix, 2 units of Q5 Hot Start High-Fidelity DNA Polymerase from New England Biolabs, 1X of Q5 Reaction Buffer and 1X of Q5 High GC Enhancer.

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Amplicon was digested with NdeI and XhoI restriction enzymes (New England Biolabs) and was cloned into pET-28b(+) expression vector (Merck Millipore) and His-tagged at the N-terminal. E. coli JM109(DE3) competent cells (Promega) + having genotype endA1, recA1, gyrA96, thi, hsdR17 (rk , mk ), relA1, supE44, λ–, Δ(lac-proAB), [F´, traD36, proAB, q lacI ZΔM15], λDE3, were transformed with the pET28b-lexA plasmid for protein expression and purification.

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Terrific Broth medium (sigma-Aldrich) containing 50 mg/mL kanamycin (Sigma-Aldrich) was inoculated with 1 mL of overnight culture of transformed cells and incubated at 37°C in orbital shaker (240 rpm) until optical density at 600 nm reached 0.5. Protein expression was induced with 1 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside, from SigmaAldrich) and then incubated 16 h at 20°C in aerobic condition. Cells were harvested by centrifugation at 4,000 rpm for 40 min at 4°C and the pellet washed twice with phosphate buffer 25 mM, pH 7.0 and then lysed with lysozyme (2 mg/mL) at room temperature for 1 hours followed by centrifugation at 20,000 rpm for 1 hour. Supernatant was loaded in HiTrap FF column (GE Healthcare), pre-equilibrated with binding buffer (25 mM phosphate buffer pH 7.0, NaCl 150 mM, imidazole 45 mM, and 10% glycerol). Bound proteins were eluted in a single step with elution buffer containing high imidazole concentration (500 mM) and the protein quantified by Bradford assay. Protein pureness was estimated by visual examination of SDS-polyacrilamide gel stained with Coomassie Blue. Protein was dialysed in Storage Buffer (10 mM pipes-NaOH pH 7.0, EDTA 0.1 mM, NaCl 200 mM and 10% glycerol) and concentrated up to 1 mg/mL using Corning Spin-X UF Concentrators (Sigma-Aldrich). Cleavage assay and LC-MS analysis

LexA self-cleavage was induced by alkalinisation of the buffer as already reported [23]. Briefly, LexA in Storage Buffer was mixed with an equal volume of 2X Cleavage Buffer (100 mM tris-glycine, NaCl 300 mM, pH 10.6) to obtain a final LexA concentration of 25.0 µM. Mix was incubated at 37°C in the UPLC autosampler and 2 µL aliquots were withdrawn and injected into the chromatographic column at 15 min intervals from 0 to 120 min. Inhibitors were added at several inhibitor:LexA ([I]:[LexA]) molar ratios (100:1, 500:1, 1000:1 and 1500:1). The UPLC system consisted of a Waters ACQUITY I-Class with autosampler. Samples were analysed on a C18 reverse phase column Jupiter 5 µm 150 mm length × 20 mm i.d., pore size 300 Å (Phenomenex). The sample was eluted at a flow rate of 0.2 mL/min by a gradient of 0.01% TFA in water (solvent A) and 0.01% TFA in acetonitrile (solvent B) with B ramping from 30% to 80% within 8 minutes. The initial conditions 70% A/30% B were then restored in 2 minutes and equilibrated in 5 minutes. Effluent was monitored by ESI-MS detection. All mass spectrometric experiments were performed with a Waters XEVO G2 QTof (Quadrupole/Time-of-Fligth) mass spectrometer equipped with an ESI Z-spray ion source. The LC Z-spray interface was operated in the electrospray positive ionization mode (ES+) with the following parameters: capillary voltage = 3.0 kV, sampling cone = 35, extraction cone = 2; source temperature = 120°C, desolvation temperature = 250°C, desolvation gas (N2) flow rate = 800 L/h. 4

Journal Pre-proof Kinetic model The autoproteolysis of LexA is a unimolecular reaction that can be described as: →

Equation 1

where NTD and CTD are the N-terminal and C-terminal domains, respectively. The integrated rate law equation is: ]⁄[

[

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Equation 2 where k is the first order rate constant, [LexA] and [LexA]0 are, respectively, the concentration of LexA at any time and its initial concentration. The ratio [LexA]/[LexA]0 is obtained from LC-MS data. Once the autoproteolysis of LexA was induced by alkalinisation, as previously described, the residual fraction (f) of uncleaved LexA was calculated as the peak area of uncleaved LexA normalised to the total peak areas of NTD, CTD and LexA obtained from the extracted ion chromatogram (XIC) (Figure 2A): Equation 3

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As shown in Figure 3A, the reaction is described by an exponential decay (equation 2) and the first order rate constant is calculated by non-linear fitting of time-course autohydrolysis in absence (k0) and presence of inhibitors (ki). For instance, the first order rate constant k in exponential decay models decreases as the concentration of the inhibitor I increases.

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As stated before, autoproteolytic enzymes behave as non-Michaelian enzymes, through a unimolecular reaction where enzyme and substrate are coincident in a one-to-one ratio. In this case Michaelis-Menten equation cannot be applied. To better describe the potency of inhibitors in autoproteolytic enzymes, ki was calculated at several inhibitorLexA molar ratios ([I]:[LexA]) in the range from 0:1 to 1500:1. ki values obtained at each [I]:[LexA] ratio were plotted (Figure 4) and the curve fitted with the following equation empirically determined:

]

Equation 4

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[ ] [

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where ϕ is the [I]:[LexA] ratio that gives half of the first order rate constant k0. The ϕ value was determined for each phenylboronic acid derivatives to determine their ability to inhibit LexA autoproteolysis. (table 1). Data are reported as mean ± SE of at three independent experiments. Determination of the Minimum Inhibitory Concentrations (MICs) MICs of levofloxacin (Sigma-Aldrich) and phenylboronic acid derivatives were determined by microdilution method in a 96-well microplate. E. coli BL21(DE3) with genotype F- ompT gal dcm lon hsdSb(rb mb ) λ(DE3 [lacI lacUV5-t7p07 ind1 + s sam7 nin5]) [malB ]k-12(λ ), was grown in 10 mL Luria-Bertani (LB) broth (Sigma-Aldrich) and incubated at 37°C for 4 hours in orbital shaker. Mueller-Hinton (MH) broth (Sigma-Aldrich) was subsequently inoculated with an aliquot of the preculture and incubated overnight in orbital shaker at 37°C. Compounds were dissolved in water and tested in serial double dilution at concentrations ranging from 1 µg/mL to 2048 µg/mL, while levofloxacin was ranging from 4.88×10 -4 µg/mL to 1 µg/mL. Each well containing 50 µL of MH and boronic acids derivatives or levofloxacin was inoculated with 6 5 10 CFU/mL of the overnight culture to reach the 5×10 CFU/mL. Microplates were then incubated at 37°C for 16 hours before MIC determination. The growth in each well was quantified spectrophotometrically at  595 nm by a microplate reader iMark, BioRad (Milan, Italy). The percentage growth in each well was calculated as:

in which the background was obtained from the microorganism‐free plates (background), processed as the inoculated plates. The MIC for each compound and levofloxacin was defined as the concentration of drug that reduced growth by 5

Journal Pre-proof 80% with respect to organisms grown in the absence of drug (drug free well). MIC value was reported as the median of three independent experiments. Filamentation assay

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E. coli BL21(DE3) was used for whole cell filamentation assay. A combination of compound 3 and levofloxacin at 1/16 of the MIC values (128 µg/mL and 0.0039 µg/mL, respectively) was added to 1 mL of LB broth inoculated with 106 CFU/mL of overnight culture obtained as described above. Drug free control (Figure 6A), compound 3 at 128 µg/mL (Figure 6B) and levofloxacin at 0.0039 µg/mL (Figure 6C) were also included. Bacterial cultures were incubated in sterile glass tubes for 3 hours at 37°C in orbital shaker. Cultures were then transferred in 1.5 mL microtubes and centrifuged at 14,000 rpm for 5 min at 4°C and washed with Dulbecco’s Phosphate Buffered Saline (PBS) (SigmaAldrich). Pellets were resuspended in paraformaldehyde (4% v/v in PBS) and kept in ice for 30 min before centrifugation to remove paraformaldehyde. Aliquots of the resuspended pellets in 100 µL PBS were spotted on polylysine coated glass slides and then left to air-dry overnight at room temperature. Slides were washed with distilled water before adding 15 µL of fluoroshield with DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich) and then observed at fluorescent microscope. Molecular Modelling

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The molecular docking of selected phenylboronic acids within the protein binding site was performed with FLAPdock implemented in the software FLAP [24]. The high-resolution structure of LexA apo form (PDB code 1jhf) was used for all simulations [13]. The used docking approach is based on GRID Molecular Interaction Field similarities [25] combined with classical energetics. FLAP has been applied in different drug design campaigns, giving successful performances in terms of structure-, ligand- and pharmacophore-based virtual screening [26,27]. FLAPdock follows a molecular fragmentation approach, subsequent placement of each fragment in the site of the target, followed by incremental construction of the molecule [24]. For the phenylboronic derivatives, compounds 1-5, a modification of the approach was applied to simulate the covalent interaction between the catalytic serine Ser 119 and the boronic acid as already reported [28]. The set of boronic acids is imported into a virtual reaction software, and the reaction with a serine residue simulated to generate a library of boronic acyl-serine compounds. During the fragmentation step, the serine substructure was used to generate the initial fragment and then the subsequent fragments from the boronic acyl-serine structure added incrementally. The generated docking poses, and relative scoring, are reported in Table 1 and Figure 5. Software and data analysis

Chromatograms and mass spectra were analysed with MassLynx 4.1 (Waters). Data analysis and non-linear fitting were performed with OriginPro 2018 SR1 (OriginLab).

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Journal Pre-proof RESULTS LexA protein was cloned into pET-28b(+) overexpression vector and purified in a single step with HisTrap FF column, providing protein with a molecular weight approximatively of 24 kDa and a degree of purity higher than 95%, as determined by SDS-PAGE (Figure 1, lane 1). The identity of the protein was ascertained by SDS-PAGE of the cleavage products obtained by alkalinisation of the buffer (Figure 1, lane 3) which gives three spots corresponding to uncleaved LexA and the N-terminal and C-terminal domains as products of autohydrolysis.

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The average molecular weight of uncleaved LexA and its products of autohydrolysis were confirmed by LC-MS analysis. ESI MS chromatogram of LexA autohydrolysis after 30 min of incubation (Figure 2A) shows the presence of one peak at about 5 min which corresponds to the partially resolved N-terminal and C-terminal domains, and a second peak at about 7 min, consistent with uncleaved LexA. By deconvolution of the multicharged ion ESI mass spectra corresponding to these chromatographic peaks, the average mass of uncleaved LexA was determined as 24,375 Da, while for N-terminal and C-terminal domain the average molecular weight was 11,226 Da and 13,167 Da, respectively (Figure 2B).

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Self-cleavage of LexA induced by alkalinisation of the buffer was followed by LC-MS. Data were collected every 15 min up to 160 min and plotted as the fraction of the area of residual uncleaved LexA and the sum of the peak areas of uncleaved LexA and its products of hydrolysis (equation 3). Non-linear fitting of the data with equation 2 (Figure 3A) was used to estimate the first order rate constant k0 which is equal to 2.42×10-2 ± 8.87×10-4 min-1.

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Once the first order rate constant in absence of inhibitors was determined, LexA was incubated at 37°C with increased concentrations of compounds 1-5 (Table 1) at inhibitor-enzyme ([I]:[LexA]) molar ratios 1:100, 1:500, 1:1000 and 1:1500. Data were analysed as described above and plotted as fraction of residual uncleaved LexA (Figure 3B). The first order rate constants (ki) at each [I]:[LexA] ratio was calculated by non-linear fitting using equation 2. The obtained ki values were then plotted as function of [I]:[LexA] ratio, as shown in Figure 4. The function ki=f([I]:[LexA]) is well described by equation 4 from which the ϕ value, defined as the [I]:[LexA] ratio that gives half of the first order rate constant k0, was determined by non-linear fitting. As shown in Table 1, ϕ values are ranging from 181.2 ± 6.7 for compound 1, to about 40 for compound 3 and 5, 42.7 ± 7.6 and 43.5 ± 4.8, respectively.

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Molecular docking simulations were performed to get insight about the orientation assumed by the compounds in the protein cavity where the catalytic Ser-119 lies. Interactions of boron containing derivatives with serine hydrolases typically feature boron in an anionic tetrahedral form covalently bound to the protein [22,27]. According to their welldocumented chemical behaviour, the formation of a covalent bond between the boron and the Ser-119 hydroxyl group was assumed and the boronate moiety, upon the covalent reaction, was modelled in a tetrahedral sp3 hybridization (Figure 5). The predicted binding geometry showed the tetrahedral intermediate interacting with the εamino group of Lys-156 and with the backbone of the same Ser-119. This configuration can be found in general for all phenylboronic derivatives, while additional and more specific interactions were predicted for compound 2 and 3. The 3-hydroxy group of compound 2 forms a hydrogen bond with the β-hydroxyl of Thr-154, while compound 3 hydrogen bonds the backbone oxygen of Val-153 via its 3-amino phenyl group. The susceptibility of E. coli BL21(DE3) to levofloxacin and boronic acids derivatives was ascertained by microdilution method. MIC value for levofloxacin was 0.0624 µg/mL while for all compounds was determined as higher than 2048 µg/mL. To assess the ability of compound 3, the compound with the lowest ϕ value, to permeate bacterial cell and to modulate SOS response, an in vitro whole cell filamentation assay was carried out. Filamentation was induced by treating E. coli BL21 (DE3) with 0.0039 µg/mL of levofloxacin, a subinhibitory concentration equivalent to 1/16 of MIC value. As shown in Figure 6C levofloxacin is able to induce filamentation with respect to the untreated control (Figure 6A). No filamentation is observed with compound 3 at 128 µg/mL, equivalent to 1/16 of MIC value (Figure 6B). When levofloxacin and compound 3 are combined (levofloxacin 0.0039 µg/mL + compound 3 128 µg/mL), no filamentation is

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observed (Figure 6D). This result provides the evidence that the phenylboronic derivative 3 is able to permeate bacterial cell and to inhibit SOS response, presumably by inhibition of LexA self-cleavage.

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Journal Pre-proof DISCUSSION

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This study aims to explore the possibility to hinder SOS response in bacteria through inhibition of the transcriptional repressor, and autoproteolytic protein LexA. Our hypothesis is that the direct inhibition of LexA self-cleavage can be achieved via the identification of molecules able to interfere at the LexA active site level. LexA undergoes a catalysed self-cleavage induced and modulate by the presence of activated form of RecA protein or, in vitro, by alkalinisation of the reaction buffer [29,30]. The intramolecular reaction is mediated by the catalytic serine 119 through the formation of a transient tetrahedral intermediate between Ser-119 and the peptide bond between Ala-84 and Gly-85, followed by hydrolysis of the amide bond. This mechanism, involving a catalytic nucleophilic serine activated by a proton relay, can be observed with several variations in a large number of enzymes collectively known as serine hydrolases: examples are serine proteases [31], lipases [32], recombinases [33] and serine beta-lactamases [34]. Nowadays, a small group of inhibitors of serine hydrolases are known [35,36] and among them boron-containing compounds are actually the most promising inhibitors [37,38]. It is not by chance that three boron-containing compounds, bortezomib, tavaborole and crisaborole were approved for the treatment of multiple myeloma, onychomycosis and atopic dermatitis, respectively [37], while a combination of the antibiotic meropenem and the boron-derivative vaborbactam was recently introduced for use in patients with complicated urinary tract infections due to β-lactamase producing Enterobacteriaceae [39]. Moreover, boron-containing compounds like phenylboronic acid derivatives were successfully studied as scaffolds for the development of potential serine β-lactamase inhibitors [22,27,40].

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Therefore, we reasonably hypothesised that boron-containing compounds, specifically phenylboronic derivatives, might act as inhibitor of LexA self-cleavage via the formation of an acyl-enzyme intermediate with the catalytic Ser119. In this regard five phenylboronic acid derivatives (Table 1) were studied as potential inhibitor of the transcriptional repressor, and autoproteolytic protein, LexA.

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The first and most important issue to address, was how to define the “potency” of inhibitors in autoproteolytic enzymes that, by its own nature, behave as non-Michaelian enzymes. In this case, there is no compliance with Michaelis-Menten statement of steady state, which can be reached only when the concentration of substrate molecules is in great excess of the free enzyme concentration ([S]>>[E]). In autoproteolytic enzymes the equimolarity between enzyme and substrate makes unfeasible, indeed, to meet Michaelis-Menten assumptions. Moreover, although LexA is de facto an enzyme, turnover cannot be measured since the product of the enzymatic reaction is the consumption of the enzyme itself. For instance, LexA self-cleavage is defined by equation 1 which describes a pure first order reaction whose first order rate constant k can be obtained by non-linear fitting of an exponential decay (equation 2). In characterizing inhibitors of LexA autoproteolytic activity, the dissociation constant Kd (or inhibition constant Ki) can be easily calculated as it can be easily misconceived. We should first of all consider that while in normal enzyme kinetics characterization the concentration of the protein is well below the micromolar scale, usually around picomolar, the concentration of LexA in our experiments was 25 µM. In this case even an equimolar inhibitor would have a Ki value at least equal to 25 µM. In interpreting this data, one might paradoxically conclude that the inhibitor is not good as expected. However, the large excess of protein with respect to a normal enzyme kinetics, must be considered in defining the potency of inhibition. This can be easily understood observing Table 1 where Kd values for each compound is reported. For instance, the Kd value of compound 4, one of the best performing inhibitors, is equal to 1.09 mM. It means that to reduce by half the first order rate constant k of LexA self-cleavage, more than 1 mM of substance is needed. In a previous paper [22] the same compound was shown to behave as serine beta-lactamase KPC-2 inhibitor, with a Ki value equal to 31.17 µM. What happen if we normalise the Ki value with the concentration of the enzyme? In latter case the result of the normalisation gives about 10,000, while in LexA only 43.5. To overcome this issue, we decided to perform all experiments by using the molar ratio between the inhibitor and LexA ([I]:[LexA]). We also derived equation 4 where Kd (Ki) value is replaced by the ϕ value which represents the (*I]:[LexA]) ratio that gives half of the first order rate constant k0. As a matter of fact, the described issue deeply influenced the choice of the analytical method able to determine LexA self-cleavage in the best way. The UPLC-MS was chosen for its high sensitivity and specificity, which allow to detect 9

Journal Pre-proof unambiguously analytes even in trace amount. However it is well-known that mass spectrometry is not inherently quantitative since ionization efficiencies can be highly variable depending on the analyte [41]. Usually, in this respect, mass tags can be introduced, and stable isotope labelling applied. Nevertheless, mass labelling methods are expensive, awkward and time-consuming. On the other hand, in the study of LexA self-cleavage, all the analytes under investigation are peptides, thus we can assume that the efficiency of ionization is the same for both LexA and its selfcleavage peptides. Moreover, we monitored the LexA self-cleavage following the disappearance of the uncleaved LexA, thus no absolute quantification, where mass labelling is mandatory, is required. Based on these considerations we used a label free relative quantification of LexA and its self-cleavage peptides, calculating the extracted ion chromatograms (XIC) peak areas related to uncleaved LexA and its self-cleavage products and normalised to the total reconstructed peak area [42]. As shown in equation 3 the residual fraction of uncleaved LexA is calculated as the XIC of uncleaved LexA normalised to the total area of NTD, CTD and LexA (Figure 2A).

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Our experimental setting has proven to be highly reproducible and allowed to determine the [I]:[LexA] ratios, defined by the ϕ value, where the k0 constant is halved. As shown in Table 1 compounds 3 and 5 resulted as the most efficient in inhibiting the rate of LexA self-cleavage: only about 40 molecules of compounds 3 and 5 are able to inhibit the enzymatic activity of LexA. Our method, unfortunately, fails to determine the mechanism of inhibition for two reasons: i) in autoproteolytic enzymes it is not possible, by simple kinetic experiments, to determine if the compounds act as competitive, uncompetitive or non-competitive inhibitors. This is one of the most important consequence of the non-michaelian behaviour of autoproteolytic enzymes: the unachievable use a reporter substrate whose hydrolysis can be monitored in competition assays ii) In the molecular modelling studies we design the formation of an acylenzyme intermediate between the hydroxyl group of the active Ser-119 and the boron group of the phenylboronic compounds, on the basis of the well-known boronic acid reactivity toward serine in the presence of a stable covalent bond between the boron compound and serine, we would observe a mass increment equal to the mass of the inhibitor in the mass of the C-terminal domain, which contains Ser-119. In our experiments, however, any mass shift was detected. Anyway, even a mass shift of the CTD would not be helpful in determining which serine was actually involved in the linkage. In fact, LexA contains 12 serines, 7 located at the NTD and 5 at the CTD. The reactivity of the trigonal boron(III) compounds is responsible of their behaviour as Lewis acids, leading to covalent, tetrahedral adducts with nucleophiles, i.e. the serine. However, a role is played by the catalytic dyad present in the active site, i.e. the concomitant polarization of the β-hydroxy group of catalytic serine due to the presence of Lys-159, which enhances the serine reactivity by increasing its nucleophilicity. Therefore, it is plausible to hypothesise that the phenylboronic derivatives inhibitory activity occurs at the Ser-119 residue, supporting our in silico analysis approach. One of the most impressive results is shown in Figure 6. Since LexA is a cytoplasmatic target, the ability of the inhibitor to permeate the outer and inner membranes is crucial for the explication of their activity. One of the simplest experiments to verify the activation of the SOS response is the filamentation assay. The activation of SOS response acts on E. coli cell division by inducing filamentation as consequence of inhibition of the septum [43]. Cell division is inhibited by the expression of sulA gene, under LexA control, which product interact with tubulin-like FtsZ division protein [44]. In our experiment activation of SOS response was induced by subinhibitory concentration of levofloxacin and verified by the formation of long filaments (Figure 6C) conferring to the bacteria a “hairy” aspect which can be dramatically reduced in presence of compound 3 (Figure 6D). This result strongly supports the ability of this compound to permeate the outer and inner membranes of E. coli and to reach the cytoplasmatic target. CONCLUSIONS In this study we hypothesized and verified that small boron-containing compounds are able to efficaciously inhibit the self-cleavage of the transcriptional repressor LexA, presumably via the formation of an acyl-enzyme intermediate as also computed by in silico analysis. It also provided the basis for systematic investigations on the identified molecular scaffolds. The effect of interference of compound 3, the amino phenylboronic derivative, on induced SOS response observed in the whole cell assay in E. coli is encouraging in defining molecules capable to reach the cytoplasmatic target LexA. Methodologically, the use of LC-MS has proven to be highly accurate and reproducible for the measurement of the first-order rate constant of LexA self-cleavage and for the determination of the potency of 10

Journal Pre-proof inhibition. The proposed equation (equation 4) for the calculation of the ratio between the potential inhibitor and the autoproteolytic activity of LexA, introduces a new tool for the quantification of the inhibitory activity of compounds in self-cleaving proteins. The discovery of small molecules able to interfere with the SOS response activation might pave the way in defining a new paradigm in the antimicrobial chemotherapy. For the first time, we have the opportunity to face the antimicrobial resistance phenomenon giving us the possibility to rehabilitate antibiotics no longer effective in therapy and to extend the life of those, a few, under development. FOUNDING ACKNOWLEDGEMENTS. The project was supported by FAR2017 to DT for independent research on SOS response inhibition. CONFLICT OF INTEREST

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The authors declare that there is no conflict of interest

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Journal Pre-proof TABLES AND FIGURES Table 1. The ϕ values calculated for phenylboronic acid (1) and its derivatives included in this study. Error is expressed as ± standard error (SE) of the mean of three independent experiments.

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ϕ ± SE 181.2 ± 6.7 130.8 ± 3.7 42.7 ± 7.6 75.2 ± 9.7 43.5 ± 4.8

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R -H -OH -NH2 -COOH -(CH)2COOH

Kd ± SE (mM) 4.53 ± 0.17 3.27 ± 0.93 1.07 ± 0.19 1.88 ± 0.24 1.09 ± 0.12

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phenylboronic acid (3-hydroxyphenyl)boronic acid (3-aminophenyl)boronic acid (3-carboxyphenyl)boronic acid [3-(2-carboxyvinyl)phenyl]boronic acid

code 1 2 3 4 5

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Figure 1. SDS-PAGE of uncleaved LexA and alkaline induced LexA self-cleavage. Lane 1, uncleaved LexA; lane 2, molecular weight marker; lane 3, LexA and LexA products of hydrolysis after 30 min of incubation at 37°C.

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Figure 2. (A) LC-MS total ion current chromatogram obtained after 30 min LexA autoproteolysis without inhibitors induced by alkalinisation. (B) Deconvoluted mass spectrum from 4.7 min to 7.5 min.

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Journal Pre-proof Figure 3. Time-course plot of LexA autocleavage as fraction of uncleaved LexA. (A) Autoproteolysis of LexA in absence of inhibitors; (B) Autoproteolysis of LexA in presence of increasing concentration of compound 1 expressed as [I]:[LexA] ratio. Black (■), control; red (●), 100:1; blue (▲), 500:1; green (▼), 1000:1; lilac (♦), 1500:1. Each data is the mean ± SE of a triplicate experiment. B

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Figure 4. Plot of calculated k as function of [I]:[LexA] ratio. k0 is the first order rate constant calculated without inhibitor. The value ϕ is the [I]:[LexA] ratio that gives half of the first order rate constant k0. Data are represented as mean ± SE of a triplicate experiment.

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Journal Pre-proof Figure 5. Docking poses of the selected compounds in LexA binding site (PDB code 1jhf). Residues lining the pocket are shown as capped sticks. Ligands are lilac-coloured. Hydrogen bonds are represented by black dashed lines. Only residues forming relevant interaction with the ligands have been labelled. From (A) to (D) structures 2, 3, 4 and 5. Pictures were prepared with PyMOL v1.7.6.4. B

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Journal Pre-proof Figure 6. Fluorescence microscopy of E. coli with DAPI. (A) Control; (B) 128 µg/mL compound 3; (C) 0.0039 µg/mL levofloxacin; (D) 0.0039 µg/mL levofloxacin + 128 µg/mL compound 3. B

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Journal Pre-proof References WHO, Antibiotic resistance, (2018). https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance.

[2]

J. O’Neill, The Review on Antimicrobial Resistance, chaired by Jim O’Neill. Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations, (2014).

[3]

R.T. Cirz, J.K. Chin, D.R. Andes, V. De Crécy-Lagard, W.A. Craig, F.E. Romesberg, Inhibition of mutation and combating the evolution of antibiotic resistance, PLoS Biol. 3 (2005) 1024–1033. doi:10.1371/journal.pbio.0030176.

[4]

B. Michel, After 30 years of study, the bacterial SOS response still surprises us, PLoS Biol. 3 (2005) 1174–1176. doi:10.1371/journal.pbio.0030255.

[5]

M. Patel, Q. Jiang, R. Woodgate, M.M. Cox, M.F. Goodman, A new model for SOS-induced mutagenesis: How RecA protein activates DNA polymerase v, Crit. Rev. Biochem. Mol. Biol. 45 (2010) 171–184. doi:10.3109/10409238.2010.480968.

[6]

J.S. Lenhart, J.W. Schroeder, B.W. Walsh, L.A. Simmons, DNA Repair and Genome Maintenance in Bacillus subtilis, Microbiol. Mol. Biol. Rev. 76 (2012) 530–564. doi:10.1128/mmbr.05020-11.

[7]

G. Cambray, N. Sanchez-Alberola, S. Campoy, É. Guerin, S. Da Re, B. González-Zorn, M.C. Ploy, J. Barbé, D. Mazel, I. Erill, Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons, Mob. DNA. 2 (2011) 6. doi:10.1186/1759-8753-2-6.

[8]

R.T. Cirz, F.E. Romesberg, Controlling mutation: Intervening in evolution as a therapeutic strategy, Crit. Rev. Biochem. Mol. Biol. 42 (2007) 341–354. doi:10.1080/10409230701597741.

[9]

R.T. Cirz, M.B. Jones, N.A. Gingles, T.D. Minogue, B. Jarrahi, S.N. Peterson, F.E. Romesberg, Complete and SOSmediated response of Staphylococcus aureus to the antibiotic ciprofloxacin, J. Bacteriol. 189 (2007) 531–539. doi:10.1128/JB.01464-06.

[10]

R.T. Cirz, B.M. O’Neill, J.A. Hammond, S.R. Head, F.E. Romesberg, Defining the Pseudomonas aeruginosa SOS response and its role in the global response to the antibiotic ciprofloxacin, J. Bacteriol. 188 (2006) 7101–7110. doi:10.1128/JB.00807-06.

[11]

B. Li, P. Smith, D.J. Horvath, F.E. Romesberg, S.S. Justice, SOS regulatory elements are essential for UPEC pathogenesis, Microbes Infect. 12 (2010) 662–668. doi:10.1016/j.micinf.2010.04.009.

[12]

M. Okon, R.A. Pfuetzner, M. Vuckovic, J.W. Little, N.C. Strynadka, L.P. McIntosh, Letter to the Editor: Backbone chemical shift assignments of the LexA catalytic domain in its active conformation [6], J. Biomol. NMR. 31 (2005) 371–372. doi:10.1007/s10858-005-0944-8.

[13]

Y. Luo, R.A. Pfuetzner, S. Mosimann, M. Paetzel, E.A. Frey, M. Cherney, B. Kim, J.W. Little, N.C.J. Strynadka, Crystal Structure of LexA, Cell. 106 (2004) 585–594. doi:10.1016/s0092-8674(01)00479-2.

[14]

T. Selwood, B.J. Larsen, C.Y. Mo, M.J. Culyba, Z.M. Hostetler, R.M. Kohli, A.B. Reitz, S.D.P. Baugh, Advancement of the 5-amino-1-(carbamoylmethyl)-1H- 1,2,3-triazole-4-carboxamide scaffold to disarm the bacterial SOS response, Front. Microbiol. 9 (2018) 2961. doi:10.3389/fmicb.2018.02961.

[15]

C.Y. Mo, M.J. Culyba, T. Selwood, J.M. Kubiak, Z.M. Hostetler, A.J. Jurewicz, P.M. Keller, A.J. Pope, A. Quinn, J.L. Schneck, K.L. Widdowson, R.M. Kohli, Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership, ACS Infect. Dis. 4 (2018) 349–359. doi:10.1021/acsinfecdis.7b00122.

[16]

P. Bellio, F. Brisdelli, M. Perilli, A. Sabatini, C. Bottoni, B. Segatore, D. Setacci, G. Amicosante, G. Celenza, Curcumin inhibits the SOS response induced by levofloxacin in Escherichia coli, Phytomedicine. 21 (2014) 430– 434. doi:10.1016/j.phymed.2013.10.011.

[17]

P. Bellio, L. Di Pietro, A. Mancini, M. Piovano, M. Nicoletti, F. Brisdelli, D. Tondi, L. Cendron, N. Franceschini, G. Amicosante, M. Perilli, G. Celenza, SOS response in bacteria: Inhibitory activity of lichen secondary metabolites 17

Jo u

rn

al

Pr

e-

pr

oo

f

[1]

Journal Pre-proof against Escherichia coli RecA protein, Phytomedicine. 29 (2017) 11–18. doi:10.1016/j.phymed.2017.04.001. E.J.R. Peterson, W.P. Janzen, D. Kireev, S.F. Singleton, High-throughput screening for RecA inhibitors using a transcreener adenosine 5’-O-diphosphate assay., Assay Drug Dev. Technol. 10 (2012) 260–268. doi:10.1089/adt.2011.0409.

[19]

J.Z. Sexton, T.J. Wigle, Q. He, M.A. Hughes, G.R. Smith, S.F. Singleton, A.L. Williams, L.-A. Yeh, Novel Inhibitors of E. coli RecA ATPase Activity., Curr. Chem. Genomics. 4 (2010) 34–42. doi:10.2174/1875397301004010034.

[20]

D.J. Cline, S.L. Holt, S.F. Singleton, Inhibition of Escherichia coli RecA by rationally redesigned N-terminal helix., Org. Biomol. Chem. 5 (2007) 1525–1528. doi:10.1039/b703159a.

[21]

T.J. Wigle, S.F. Singleton, Directed molecular screening for RecA ATPase inhibitors., Bioorg. Med. Chem. Lett. 17 (2007) 3249–3253. doi:10.1016/j.bmcl.2007.04.013.

[22]

G. Celenza, M. Vicario, P. Bellio, P. Linciano, M. Perilli, A. Oliver, J. Blazquez, L. Cendron, D. Tondi, Phenylboronic Acid Derivatives as Validated Leads Active in Clinical Strains Overexpressing KPC-2: A Step against Bacterial Resistance, ChemMedChem. 13 (2018) 713–724. doi:10.1002/cmdc.201700788.

[23]

C.Y. Mo, L.D. Birdwell, R.M. Kohli, Specificity determinants for autoproteolysis of lexa, a key regulator of bacterial SOS mutagenesis, Biochemistry. 53 (2014) 3158–3168. doi:10.1021/bi500026e.

[24]

M. Baroni, G. Cruciani, S. Sciabola, F. Perruccio, J.S. Mason, A common reference framework for analyzing/comparing proteins and ligands. Fingerprints for Ligands and Proteins (FLAP): Theory and application, J. Chem. Inf. Model. 47 (2007) 279–294. doi:10.1021/ci600253e.

[25]

P.J. Goodford, A computational procedure for determining energetically favorable binding sites on biologically important macromolecules., J. Med. Chem. 28 (1985) 849–57. doi:10.1021/jm00145a002.

[26]

F. Spyrakis, P. Bellio, A. Quotadamo, P. Linciano, P. Benedetti, G. D’Arrigo, M. Baroni, L. Cendron, G. Celenza, D. Tondi, First virtual screening and experimental validation of inhibitors targeting GES-5 carbapenemase, J. Comput. Aided. Mol. Des. 33 (2019) 295–305. doi:10.1007/s10822-018-0182-2.

[27]

L. Cendron, A. Quotadamo, L. Maso, P. Bellio, M. Montanari, G. Celenza, A. Venturelli, M.P. Costi, D. Tondi, Xray Crystallography Deciphers the Activity of Broad-Spectrum Boronic Acid β-Lactamase Inhibitors, ACS Med. Chem. Lett. 10 (2019) 650–655. doi:10.1021/acsmedchemlett.8b00607.

[28]

M. Santucci, F. Spyrakis, S. Cross, A. Quotadamo, D. Farina, D. Tondi, F. De Luca, J.D. Docquier, A.I. Prieto, C. Ibacache, J. Blázquez, A. Venturelli, G. Cruciani, M.P. Costi, Computational and biological profile of boronic acids for the detection of bacterial serine- and metallo-β-lactamases, Sci. Rep. 7 (2017) 17716. doi:10.1038/s41598-017-17399-7.

[29]

J.W. Little, Autodigestion of lexA and phage lambda repressors., Proc. Natl. Acad. Sci. 81 (1984) 1375–1379. doi:10.1073/pnas.81.5.1375.

[30]

J.W. Little, Mechanism of specific LexA cleavage: Autodigestion and the role of RecA coprotease, Biochimie. 73 (1991) 411–421. doi:10.1016/0300-9084(91)90108-D.

[31]

L. Hedstrom, Serine Protease Mechanism and Specificity, Chem. Rev. 102 (2002) 4501–4524. doi:10.1021/cr000033x.

[32]

W.A. Hide, L. Chan, W.H. Li, Structure and evolution of the lipase superfamily., J. Lipid Res. 33 (1992) 167–78. http://www.ncbi.nlm.nih.gov/pubmed/1569370 (accessed July 15, 2019).

[33]

W.M. Stark, The Serine Recombinases, in: Mob. DNA III, American Society of Microbiology, 2014: pp. 73–89. doi:10.1128/microbiolspec.mdna3-0046-2014.

[34]

K. Bush, Past and Present Perspectives on β-Lactamases, Antimicrob. Agents Chemother. 62 (2018). doi:10.1128/aac.01076-18.

[35]

B. Walker, J.F. Lynas, Strategies for the inhibition of serine proteases, Cell. Mol. Life Sci. 58 (2001) 596–624.

Jo u

rn

al

Pr

e-

pr

oo

f

[18]

18

Journal Pre-proof doi:10.1007/PL00000884. E. Bialecka-Florjanczyk, A.U. Fabiszewska, J. Krzyczkowska, A. Kurylowicz, Synthetic and Natural Lipase Inhibitors, Mini-Reviews Med. Chem. 18 (2018) 672–683. doi:10.2174/1389557516666160630123356.

[37]

G.F.S. Fernandes, W.A. Denny, J.L. Dos Santos, Boron in drug design: Recent advances in the development of new therapeutic agents, Eur. J. Med. Chem. 179 (2019) 791–804. doi:10.1016/j.ejmech.2019.06.092.

[38]

S.J. Baker, C.Z. Ding, T. Akama, Y.K. Zhang, V. Hernandez, Y. Xia, Therapeutic potential of boron-containing compounds, Future Med. Chem. 1 (2009) 1275–1288. doi:10.4155/fmc.09.71.

[39]

S. Dhillon, Meropenem/Vaborbactam: A Review in Complicated Urinary Tract Infections, Drugs. 78 (2018) 1259–1270. doi:10.1007/s40265-018-0966-7.

[40]

P. Linciano, M. Vicario, I. Kekez, P. Bellio, Phenylboronic Acids Probing Molecular Recognition against Class A and Class C β -lactamases, Antibiotics. 8 (2019) 171. doi:10.3390/antibiotics8040171.

[41]

M. Bantscheff, M. Schirle, G. Sweetman, J. Rick, B. Kuster, Quantitative mass spectrometry in proteomics: A critical review, Anal. Bioanal. Chem. 389 (2007) 1017–1031. doi:10.1007/s00216-007-1486-6.

[42]

P. V. Bondarenko, D. Chelius, T.A. Shaler, Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography - Tandem mass spectrometry, Anal. Chem. 74 (2002) 4741–4749. doi:10.1021/ac0256991.

[43]

I. Erill, S. Campoy, J. Barbé, Aeons of distress: An evolutionary perspective on the bacterial SOS response, FEMS Microbiol. Rev. 31 (2007) 637–656. doi:10.1111/j.1574-6976.2007.00082.x.

[44]

D. Trusca, S. Scott, C. Thompson, D. Bramhill, Bacterial SOS checkpoint protein SulA inhibits polymerization of purified FtsZ cell division protein., J. Bacteriol. 180 (1998) 3946–53. http://www.ncbi.nlm.nih.gov/pubmed/9683493 (accessed July 29, 2019).

Jo u

rn

al

Pr

e-

pr

oo

f

[36]

19